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Journal of Biomedicine and Biotechnology bull 20054 (2005) 326ndash352 bull DOI 101155JBB2005326

RESEARCH ARTICLE

Health Considerations Regarding Horizontal Transferof Microbial Transgenes Present in Genetically

Modified Crops

Gijs A Kleter Ad A C M Peijnenburg and Henk J M Aarts

RIKILT Institute of Food Safety Wageningen University and Research Center PO Box 230 6700AE Wageningen The Netherlands

Received 18 October 2004 revised 30 May 2005 accepted 1 June 2005

The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically mod-ified organisms Horizontal gene transfer from genetically modified crops to gut microflora most likely occurs with transgenes ofmicrobial origin The characteristics of microbial transgenes other than antibiotic-resistance genes in market-approved geneticallymodified crops are reviewed These characteristics include the microbial source natural function function in genetically modifiedcrops natural prevalence geographical distribution similarity to other microbial genes known horizontal transfer activity selectiveconditions and environments for horizontally transferred genes and potential contribution to pathogenicity and virulence in hu-mans and animals The assessment of this set of data for each of the microbial genes reviewed does not give rise to health concernsWe recommend including the above-mentioned items into the premarket safety assessment of genetically modified crops carryingtransgenes other than those reviewed in the present study

INTRODUCTION

The cultivation of genetically modified (GM) cropshas rapidly increased since their large-scale commercialintroduction in 1996 The acreage of GM crops in 2004amounted to 81 millions of hectares worldwide while thenumber of nations that adopt GM crop cultivation wasalso increasing [1] Before GM crops and other geneti-cally modified organisms (GMOs) are allowed to enter themarket the law in many nations requires that these organ-isms andor derived products be assessed for their safetyTo this end the applicant which is in most cases a com-pany that has developed and produced a GMO providesa dossier to the national authorities which among oth-ers contains safety data Whereas national laws and reg-ulatory procedures may differ among each other the reg-ulatory safety assessment itself follows an internationallyharmonised approach International organisations likethe United Nationsrsquo Food and Agriculture Organisation(FAO) and World Health Organisation (WHO) as well as

Correspondence and reprint requests to Gijs A Kleter RIKILTInstitute of Food Safety Wageningen University and ResearchCenter PO Box 230 6700AE Wageningen The NetherlandsE-mail gijskleterwurnl

This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted usedistribution and reproduction in any medium provided theoriginal work is properly cited

the Organisation for Economic Cooperation and Devel-opment (OECD) and International Life Sciences Institute(ILSI) have initiated this harmonisation It has recentlyculminated into the issuance of FAOWHO Codex Ali-mentarius guidelines for the safety assessment of foodsderived from GM plants and microorganisms [2] Cen-tral in the harmonised approach is the comparative safetyassessment which entails the comparison of a GMO witha conventional counterpart that has a history of safe use[3] This comparison may include for example pheno-typic characteristics (eg field behaviour) and composi-tion (eg macronutrients micronutrients antinutrients)of a GMO and its comparator Based upon the differencesfound during the comparison between the GMO and itscomparator it can be decided which further safety testsare needed Issues that are commonly addressed duringthe safety assessment include the molecular characteri-sation (eg introduced genes) the potential for horizon-tal gene transfer potential allergenicity potential toxicitynutritional characteristics environmental effects and un-intended effects of the genetic modification (reviewed in[4])

Horizontal gene transfer

Various mechanisms exist for horizontal gene trans-fer between microorganisms such as phage transductionconjugation and transformation by free DNA (eg [5])The possible scenario for gene transfer between GM cropsand microorganisms is however limited to transforma-tion with free DNA

20054 (2005) Health Aspects of Transgene Transfer 327

A number of studies and reviews have focused onthe transfer of genes from GM plants to soil- and plant-related microorganisms (eg [6 7 8 9 10]) The results ofsome of these studies indicated that transgenes from GMcrops are most likely transferred if they contain sufficientsimilarity with the corresponding genes in the recipientbecause homologous recombination is the most probablemechanism of transfer (eg [11]) It has however recentlybeen observed that under conditions of simulated light-ning which might cause electroporation of recipient cellsDNA could be transferred to isolated soil microbes [12]

Other factors that are important for transformationwith DNA are the natural or induced competence of therecipient microorganisms such as the natural competenceof Campylobacter species Some microorganisms suchas Salmonella typhimurium have mismatch repair sys-tems that form a barrier for recombination between evenhighly similar sequences (eg reviewed for Salmonella by[13]) Some bacteria can develop naturalchemical com-petence under certain environmental conditions [6]

In addition the transgenes in plants may have beenlinked to promoters with optimal activity in the cellsof plants Sequences promoting expression in eukaryotesand prokaryotes are generally known to be different Nev-ertheless Jacob et al [14] observed that eukaryotic pro-moters from for example the cauliflower mosaic viruspotato and tobacco triggered expression of inserted re-porter genes in five eubacterial species In addition Lewinet al [15] observed that random sequences from yeast mayexhibit promoter activity in bacteria

Jonas et al [16] estimated the potential dietary in-take of transgenic DNA present in food The estimatedintake of transgenic DNA from maize soya and pota-toes amounted to approximately 038 microg per day assum-ing that only GM crops are consumed This is about000006 of the total DNA intake of 06 g per day

Still this is a ldquoworst-caserdquo scenario as DNA is prone todegradation in food matrices or during food processing(reviewed in [16]) On the other hand also the protectionof DNA against the activity of DNase I in for instancefermented sausages has been described [17]

In addition the integrity of the DNA is counteredby the activity of DNA degrading enzymes released bythe pancreas and intestinal epithelial cells during its pas-sage through the gastrointestinal tract Nevertheless it hasbeen shown that DNA can persist in the gastrointestinaltract [16 18 19] and consequently be available for up-take by intestinal competent bacteria For example thesurvival of cp4 epsps transgenes in the small intestines ofhuman volunteers who consumed a GM soy product hasrecently been demonstrated in a study by Netherwood etal [20] so there is a chance for exposure of intestinal mi-croorganisms to free transgenic DNA However the pref-erential site for transformation of competent bacteria isprobably in the colon This is because the colon containsthe largest population of bacteria within the gastrointesti-nal tract Whilst the amount of DNA reaching the colon

may only be a fraction of what is consumed DNA is lessrapidly degraded there For example ex vivo and in vivorat models simulating human gut conditions showed thatDNA is rapidly degraded in the upper part of the gastroin-testinal tract but to a lesser degree in the lower part [19]

Besides the integrity of DNA the transformabilitythat is the likelihood that this DNA will transform bac-teria in food or in the gut should be taken into accountIn foods transformation of Escherichia coli by plasmidtransfer was proven to occur in all 12 food products in-vestigated [21] In addition transfer of DNA to Strepto-coccus gordonii was also proven in homogenates of bloodsausages by marker rescue experiments [22] Kharazmi etal [23] observed the transfer of nptII kanamycin resis-tance marker gene from transgenic potatoes to Bacillussubtilis with defective nptII by homologous recombina-tion under in vitro conditions Based upon the observedfrequencies of transfer these authors calculated the prob-ability of the transfer of the intact nptII gene from con-sumed transgenic potatoes to microbes Because markerrescue by homologous recombination is the most proba-ble mechanism for gene transfer these calculations can beconsidered a ldquoworst-caserdquo scenario in view of other possi-ble mechanisms of horizontal transfer of transgenes fromGM crops

Potential health effectsCurrently the focus of the assessment of potential

transfer from GMOs is on antibiotic-resistance markergenes as for example in the previously mentioned FAOWHO Codex Alimentarius guidelines In a more gen-eral sense antibiotic resistance among microbial hu-man pathogens is currently a top priority issue inhealth care and research The horizontal gene transferof antibiotic-resistance genes between microorganismshas been important for the development of antibiotic-resistant pathogens

In modern biotechnology some antibiotic-resistancemarker genes are used for the successful molecularcloning in bacteria and plants because they enable growthon antibiotic-containing media after the genetic modi-fication process These marker genes are therefore use-ful in the development phase but have no function inthe final product An example of an antibiotic-resistancegene that is present in many commercial GM crops is thekanamycin-resistance gene nptII encoding the neomycinphosphotransferase II enzyme The use of this gene hasbeen considered to be safe based upon the widespread oc-currence of kanamycin resistance in microorganisms inthe environment the low clinical relevance of kanamycinand the low likelihood of transfer to microorganisms af-ter consumption of GM products containing nptII (eg re-viewed by [5])

For a more elaborate discussion on mechanisms ofgene transfer antibiotic-resistance genes and horizontalgene transfer from GM crops as well as a classification ofantibiotic-resistance markers based upon their risk char-acteristics we refer to a recent review by the working

328 Gijs A Kleter et al 20054 (2005)

group on horizontal gene transfer of the EU-sponsoredthematic network ENTRANSFOOD [5]

Besides the horizontal transfer of antibiotic-resistancegenes the transfer of ldquopathogenicity islandsrdquo has playedan important role in the evolution of pathogenic strainsof microorganisms such as pathogenic strains of E coliand Salmonella enterica [24 25] There are many factorsthat can influence the virulence and human pathogenicityof microorganisms These include for example the for-mation of certain adhesion molecules that bind to hostcells such as adhesins of bacterial pili In addition secre-tion systems containing multiple proteins that are trans-ferred from pathogens to the host cells help pathogensinvade these cells Pathogens may also produce enzymesand toxins that cause damage in host cells which mayfacilitate entry into tissues (eg proteinases of fungi in-fecting lungs) or suppress immune response (eg dam-age to blood cells) In addition pathogens may be self-sufficient for certain nutritional compounds or be ableto sequester them such as by producing siderophoresthat complex with iron Other common characteristics arequorum sensing by ldquoautoinducingrdquo substances the regu-lation of expression of pathogenicity-associated genes atthe appropriate stage of infection formation of capsulesand the ability of fungi to change their morphology Theseand other aspects that influence the pathogenicity of mi-croorganisms are reviewed elsewhere in more detail (eg[26 27])

The source function and characteristics of transgenesand derived products which may or may not be associ-ated with pathogenicity are commonly considered dur-ing the safety assessment of GM crops While the assess-ment in practice may also include the potential horizon-tal transfer of pathogenicity-associated transgenes suchas required by the EU [28] this issue is not explicitly men-tioned in the previously mentioned FAOWHO Codex Al-imentarius guidelines which focus solely on the transferof antibiotic-resistance genes

Scope of this study

In this article we discuss the characteristics of trans-genes of microbial origin that have been introduced intoGM crops that have received regulatory approvals for fooduse The reason for limiting the survey to transgenes ofmicrobial origin is because they are the most likely tobe transferred to microorganisms based on the follow-ing considerations As stated above homologous recom-bination between transgenes from GM crops and genespresent in microbes is the most probable mechanismfor horizontal gene transfer This implies that similar se-quences should already be present in the microorganismsbefore transfer can occur Genetic modification allows forthe introduction of DNA from unrelated species includ-ing microbes into crops Indeed a number of coding se-quences of microbial origin have been introduced intovarious commercially approved GM crops (Table 1) Theoriginal nucleotide composition of these genes may havebeen optimised in some cases for expression in plants due

to differences for example in codon preference betweenbacteria and plants In addition plant-specific promoterand terminator sequences as well as other sequences (in-trons transition peptides) may have been introduced withthe transgene to facilitate gene expression in plants

The following issues are addressed for each transgene

(i) microbial source of the gene including occur-rence and pathogenicity of the microorganism fromwhich the gene originates

(ii) natural function such as the role that the geneproduct has in its native host

(iii) natural prevalence of the gene in microorganismsother than the gene source

(iv) geographical distribution that is the geographicallocations where the gene and the microbial speciesthat harbor it occur

(v) similarity of the DNA of the transgene construct toother naturally occurring microbial genes that isa FASTA analysis has been performed to search formicrobial analogues of

(1) the gene from its microbial source

(2) the codon-modified transgene version intro-duced into GM crops

(vi) known horizontal gene transfer activity of the geneamong others the location of the native micro-bial transgene on chromosome plasmid or phageis considered since this might predispose the geneto transfer for example through conjugation (plas-mid) or transduction (phage) in addition data thatindicate that transfer might have occurred are alsoconsidered

(vii) selective conditions and environments for bacteriacarrying horizontally acquired genes

(viii) potential of the transgene to cause microbialpathogenicity or to increase virulence

(ix) conclusion based on the data considered for eachgene we conclude on whether horizontal genetransfer of the transgene in GM crops to microor-ganisms would be likely to cause or aggrevate anyadverse health effects in consumers

The FASTA analysis in search for microbial genes thatare similar to the transgenes served two purposes Firstthe occurrence of analogues in other microbes might in-dicate the extent of the dispersal of the native transgene inspecies Second the results help to identify which of theseanalogues are amenable to homologous recombinationFor homologous recombination to occur matching seg-ments should have a minimal length For example iden-tical flanking segments of at least 20 bp are required to


Table 1 Microbial transgenes in GM crops that have been approved for human food useabc

Transgene product Origin Trait Nationd

Herbicide resistanceBromoxynil nitrilase Klebsiella pneumonia ozaenae Bromoxynil resistance AUS-NZ CAN USAEnolpyruvylshikimate phosphate Agrobacterium CP4 Glyphosate resistance AUS-NZ CAN EU USAsynthaseGlyphosate oxidoreductase Achromobacter LBAA Glyphosate resistance AUS-NZ CAN EU USAPhosphinothricin acetyltransferase Streptomyces hygroscopicus Glufosinate resistance AUS-NZ CAN EU USA(bar)Phosphinothricin acetyltransferase Streptomyces viridochromogenes Glufosinate resistance AUS-NZ CAN EU USA(pat)

Male sterility and fertility restorationBarnase Bacillus amyloliquefaciens Male sterility AUS-NZ CAN EU USABarstar Bacillus amyloliquefaciens Fertility restorer AUS-NZ CAN EU USADNA adenine methylase Escherichia coli Male sterility USA

Plant hormone metabolismAminocyclopropane-carboxylate Pseudomonas 6G5 Prolonged ripening USAdeaminaseS-adenosylmethionine hydrolase Escherichia coli bacteriophage Prolonged ripening USA

T3Transformation markerBeta glucuronidase (uidA) Escherichia coli Colour reaction AUS-NZ CAN USANopaline synthase Agrobacterium tumefaciens Nopaline synthesis CAN USA

pTiC58Insecticidal proteinsCrystal protein Cry1Ab Bacillus thuringiensis kurstaki Insect resistance AUS-NZ CAN EU USACrystal protein Cry1Ac Bacillus thuringiensis kurstaki Insect resistance AUS-NZ CAN EU USACrystal protein Cry1Fa Bacillus thuringiensis aizawai Insect resistance AUS-NZ CAN USACrystal protein Cry2Aa Bacillus thuringiensis kurstaki Insect resistance AUS-NZCrystal protein Cry2Ab Bacillus thuringiensis kurstaki Insect resistance AUS-NZ CAN USACrystal protein Cry3Aa Bacillus thuringiensis tenebrionis Insect resistance AUS-NZ CAN USACrystal protein Cry3Bb Bacillus thuringiensis EG4961 Insect resistance AUS-NZ CAN USACrystal proteins Cry34Ab Cry35Ab Bacillus thuringiensis PS149B1 Insect resistance USA

aAntibiotic-resistance marker genes are not includedbSources of information [29 30 31 32]cThe American Food and Drug Administration (FDA) does not formally approve GM foods ldquoUSArdquo in the fourth column indicates that the particulartransgene is present in GM crops for which a consultation with the FDA has been completeddAUS-NZ Australia-New Zealand CAN Canada EU European Union USA United States of America

insert DNA by homologous recombination in Escherichiacoli [33 34] such that the minimal length would corre-spond to 2 times 20 bp It should be noted that this repre-sents a minimum requirement and that longer segmentsof identical nucleotides will have an increased likelihoodof recombining In addition the presence of shorter iden-tical segments (lt 20 bp) in the DNA surrounding the re-combination site facilitates complex formation with theincoming DNA thereby increasing the efficiency of thesubsequent recombination (eg [35]) Therefore the oc-currence of both a high overall similarity and identicalstretches above a particular length indicates an increasedprobability of homologous recombination with the trans-gene Given that in many cases the native sequences andnot the plant-optimised transgenic sequences have beenused for the FASTA analysis the outcomes may representa ldquoworst-caserdquo scenario

The FASTA analysis which compared the transgenewith microbial genes was carried out using the EBI web-sitersquos FASTA facility with default settings being used Morespecifically the sequences of interest were compared withthe EBIrsquos sub-databases with nucleotide sequences de-rived from prokaryotes bacteriophages and fungi (Eu-ropean Bioinformatics Institutersquos nucleic acid databasehttpwwwebiacukfasta 33nucleotidehtml) From theresults sequences from microorganisms that showed sim-ilarity with the sequence of interest and that did not be-long to the same species as the gene source were consid-ered Of these sequences those were identified that com-plied with one or both of two criteria The first crite-rion is an expectation (E) value of 1lowast10minus30 at maximumwhich is a statistical term indicating the likelihood that analignment with the same similarity score would occur bychance within the chosen database [36] This arbitrarily


chosen E value is stringent and therefore corresponds to ahigh degree of similarity between aligned sequences Theother criterion is identical nucleotide stretches of mini-mally twice 20 nucleotides (2 times 20 bp) required for ho-mologous recombination as explained above

In a similar fashion another review that has recentlybeen published dealt with the microbial transgenes andsequences present in GM crops and the significance oftheir transfer to soil bacteria [37]

OVERVIEW OF TRANSGENES AND THEIRCHARACTERISTICS

Bromoxynil nitrilase (BXN)

Microbial source

The bxn gene used for genetic modification of cropshas been cloned from an isolate of the bacterium Kleb-siella pneumonia var ozaenae found in bromoxynil-contaminated soil This isolate was capable of growing onbromoxynil-containing media and utilising the ammoniareleased from converted bromoxynil as its sole source ofnitrogen [38 39]

Natural function

Bromoxynil nitrilase (BXN) converts the cyano (ni-trile CN)-moiety of the bromoxynil molecule to a car-boxyl (COOH)-moiety Conversion of bromoxynil by ni-trilase enzymes from other microorganisms is much lessefficient The Klebsiella BXN displays substrate speci-ficity towards aromatic molecules that have halogen sub-stituents in the meta positions with respect to the cyanomoiety [40]

A putative function of these nitrile-degrading en-zymes in conjunction with aldoxime dehydratase en-zymes is the degradation of plant-produced aldoximecompounds by soil microorganisms [41]

Function in GM crops

Genetic engineering of BXN into crop plants rendersthem resistant to application of the herbicide bromoxynil[39]

Natural prevalence

In a broader perspective nitrilases occur in a range ofmicroorganisms and plants Also other related enzymesconvert nitriles such as NHases and amidases [42 43]Phylogenetic analysis revealed that BXN is closely relatedto fungal cyanide hydratase enzymes which convert ni-triles to amides [43] The nitrile-metabolising capacity ofsome microorganisms is currently exploited in industrialprocesses like the production of acrylamide from acry-lonitrile [42 43]

Geographical distribution

A recent study reports the presence of nitrile-degrading activity in bacteria and actinomycetes fromsoil and deep-sea samples of wide-ranging geographi-cal origins For example bromoxynil-metabolising gram-negative bacteria were detected in soil samples from Ar-gentina and Namibia [44]

Similarity to other microbial genes

The native gene sequence used for FASTA analysis wasderived from K pneumoniae var ozaenae accession J03196[45] with a coding sequence size of 1050 bp No similari-ties corresponding to the threshold criteria were observed(Table 2) The codon-modified transgene sequence of bxnas has been introduced into GM crops was not availablefor FASTA analysis

Known horizontal gene transfer activity

The bxn gene is located on an 82-kDa plasmid in Kpneumoniae var ozaenae [46] After artificial transfer toE coli this plasmid was found to be stably maintained incells grown in the presence of bromoxynil However in theabsence of bromoxynil a 14-kDa deletion of the plasmidwith concurrent loss of bxn was observed This deletionwas probably recA-dependent [46] There was no infor-mation available regarding the horizontal transfer of thebxn gene

Selective conditions and environments

As stated above soil bacteria harbouring the bxn genewere able to utilize bromoxynil as the sole nitrogen sourceFurthermore after artificial transfer to E coli the nativeplasmid harbouring the bxn gene was stably maintainedin the presence of bromoxynil whereas a fragment con-taining the bxn gene was deleted in the absence of bro-moxynil In addition its putative natural function is themetabolism of plant-secreted aldoxime compounds Wetherefore conclude that in theory bacteria carrying an ac-tive bxn gene would have a selective advantage in soilssuch as crop land to which the herbicide bromoxynil isapplied or in the vicinity of plants secreting aldoximecompounds

Potential for pathogenicity or virulence

K pneumonia var ozaenae the source of the bxn geneis synonymous to Klebsiella ozaenae This bacterium isalso known as a human pathogen associated with ldquoozenardquo(atrophic rhinitis an affection of the upper respiratorytract) as well as with other affections such as bac-teremia and urinary tract infection [47] No informationwas available on the role that BXN might have in thepathogenicity of its gene source K pneumonia var ozae-nae


Table 2 Similarity of native microbial transgenes to other microbial sequences

Native genea Similarity Microorganism Gene accessionb

bxn Nocp4 epsps E lt 1lowast10minus30 and 2times 20 bp Brucella melitensis AE009625 AF326475

Brucella suis AE014291c

Mesorhizobium loti BA000012Sinorhizobium meliloti AL591783c

E lt 1lowast10minus30 Bartonella henselae BX897699c

Bartonella quintana BX897700c

Bradyrhizobium japonicum BA000040c

Caulobacter crescentus AE006017c

Gluconobacter oxydans CP000009Rhodopseudomonas palustris BX572593c

Silicibacter pomeroyi CP000032c

Zymomonas mobilis AE008692

gox Nobar E lt 1lowast10minus30 and 2times 20 bp Streptomyces X65195 M22827

viridochromogenespat E lt 1lowast10minus30 and 2times 20 bp Streptomyces hygroscopicus X05822 X17220barnase E lt 1lowast10minus30 and 2times 20 bp Bacillus circulans Z29626

E lt 1lowast10minus30 Bacillus intermedius X53697Bacillus licheniformis AE017333c CP000002c

Bacillus pumilus U068672times 20 bp B intermedius AJ006407

barstar Nodam E lt 1lowast10minus30 and 2times 20 bp Salmonella enterica AL627281 AE016847

Salmonella typhimurium AE008860 U76993Shigella flexneri AE016992

E lt 1lowast10minus30 Actinobacillus actinomycetemcomitans AF263926Erwinia carotovora BX950851Haemophilus influenzae U32705c

Legionella pneumophila AE017354Mannheimia succiniciproducens AE016827Neisseria meningitidis AF091142c

Pasteurella multocida AE006162 AF411317Photobacterium profundum CR378663c

Photorhabdus luminescens BX571859Serratia marcescens X78412Shewanella oneidensis AE015477c

Vibrio cholerae AE004329c AF274317 AY341955Vibrio parahaemolyticus BA000031c

Vibrio vulnificus BA000037 AE016801Yersinia pestis AJ414141 AE017127 AE013998Yersinia pseudotuberculosis BX936398c AF274318

ACC deaminase E lt 1lowast10minus30 and 2times 20 bp Achromobacter xylosoxidans AY604539d

Burkholderia mallei CP000011c

Burkholderia pseudomallei BX571966c

Enterobacter cloacae AF047840 AF047710Pseudomonas fluorescens U37103Pseudomonas brassicacearum AY604528d

Ralstonia solanacearum AL646080c

Variovorax paradoxus AY604531

E lt 1lowast10minus30 Acidovorax facilis AY604529d

Agrobacterium tumefaciens AF315580c

Bradyrhizobium japonicum BA000040Mesorhizobium loti AL672114c BA000012Penicillium citrinum AB038511


Table 2 Continued


Pseudomonas sp M73488

Pseudomonas putida AY604533d

Pseudomonas syringae AE016869c

Rhizobium leguminosarum AF421376 AY604535d

Rhizobium sullae AY604534d

Rhodococcus sp AY604538d AY604537d

Schizosaccharomyces pombe AL133522c

Variovorax paradoxus AY604530d AY604532d

SAMase E lt 1lowast10minus30 and 2times 20 bp Bacteriophage phiYeO3-12 AJ251805

uidA E lt 1lowast10minus30 and 2times 20 bp Shigella sp AY698518d AY698517d

Shigella boydii AY698415d AY698417d

AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d

Shigella dysenteriae AY698426d AY698427d

AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d

Shigella flexneri AE005674 AE016983

AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d

Shigella sonnei AY698418d AY698419d

AY698423d AY698513d

AY698514d AY698515d

E lt 1lowast10minus30 Penicillium canescens AY773333c AY773334

Scopulariopsis sp AY773335

nos E lt 1lowast10minus30 and 2times 20 bp Agrobacterium vitis plasmid pTiAB4 X77327

crye No

aldquoNative generdquo means the native sequence from the microbial source of the transgene without codons modified One codon-modified transgene cp4epsps that has been introduced into GM crops has been analysed by FASTA of which the results are summarised in Table 3 Coding sequences weresubmitted to a FASTA search using default settings against the EMBL nucleotide databases for prokaryotes bacteriophages and fungi The similaritythresholds applied were E lt 1lowast10minus30 andor 2times 20 bp Results for genes from the same microbial species as the gene source are not listedAbbreviations ACC deaminase 1-aminocyclopropane-1-carboxylate deaminase bar phosphinothricin acetyltransferase bxn bromoxynil nitrilasecp4 epsps CP4 3-enolpyruvylshikimate-5-phosphate synthase cry crystalline insecticidal protein dam DNA adenine methylase gox glyphosateoxidoreductase nos nopaline synthase pat phosphinothricin acetyltransferase SAMase S-adenosylmethionine hydrolase uidA β-glucuronidasebNucleotide accessions can be retrieved from the NCBI website [45]cPutative function assigned to genedPartial coding sequenceeDetails on the individual cry genes tested and their accessions are provided in Table 4 In some of the genes the coding sequences had been truncatedin analogy to the truncation of transgenes used for genetic modification of crops For cry1Ab the first 1944 nucleotides were used corresponding to aprotein sequence of 648 amino acids The truncated sequence of cry1Fa comprised the first 1815 nucleotides (605 amino acids) Full-length codingsequences were used for cry1Ac (3537 bp) cry2Aa (1902 bp) cry2Ab (1902 bp) cry3Aa (1935 bp) cry3Bb (1959 bp) cry34Ab (372 bp) and cry35Ab(1152 bp)


Table 3 Similarities of the codon-modified cp4 epsps transgene introduced into herbicide-resistant GM soybean to microbial genes

Transgenea Similarity Microorganism Gene accessionb

cp4 epsps E lt 1lowast10minus30 and 2times 20 bp Brucella melitensis AE009625 AF326475Brucella suis AE014291c






Rhodopseudomonas palustris BX572593c

abc See legend to Table 2

Conclusion

BXN activity is highly substrate-specific and the pu-tative function relates to plant compound degradationEven though the bxn gene is derived from a potential hu-man pathogen no direct impact of this gene on humanor animal health is foreseen if it were to be transferred topathogens given the apparently specific role of this genein soil environments

3-enolpyruvylshikimate-5-phosphate synthase(cp4 EPSPS)

Microbial source

The source of the cp4 epsps gene was the soil bacteriumAgrobacterium strain CP4 which was one out of a groupof glyphosate-degrading bacteria (reviewed in [48]) Bac-terial species of the genus Agrobacterium are all charac-terised by the ability to form neoplastic lesions in plants(eg [49])

Natural function

The 3-enolpyruvylshikimate-5-phosphate synthase(EPSPS) enzyme catalyses an intermediate step in theshikimate pathway for the synthesis of essential aromaticprecursor compounds of among others aromatic aminoacids and lignin which is part of lignocellulose plant fi-bres EPSPS enzymes also called AroA enzymes occur ina wide variety of organisms (eg bacteria fungi plants)EPSPS enzymes in plants are targets for the herbicide ac-tive ingredient glyphosate which binds and inhibits theplant EPSPS enzymes The EPSPS enzyme from Agrobac-terium CP4 however is not sensitive towards the actionof glyphosate (reviewed in [48])


A number of commercialised GM crops contain thecp4 epsps gene coding for the enolpyruvylshikimate-phosphate synthetase (EPSPS) enzyme from Agrobac-terium strain CP4 which confers resistance towards theotherwise lethal herbicide glyphosate [48]

Natural prevalence

The amino acid sequences of EPSPS enzymes fromvarious species present in food (soybean maize E coliB subtilis Saccharomyces cerevisiae) are divergent and theidentities that they share with the sequence of Agrobac-terium CP4 EPSPS range from 241 to 411 percent [48]Because of its relative insensitivity towards the inhibitingaction of glyphosate the Agrobacterium CP4 EPSPS en-zyme has been engineered into a number of crops to makethem glyphosate-resistant [48]


The geographical distribution of the cp4 epsps geneand its source Agrobacterium CP4 has not been specifi-cally reported in literature More generally Agrobacteriumspecies occur globally in soils for example in the rhizo-sphere of plants (eg [50])


The coding sequence of the native cp4 epsps gene(Agrobacterium CP4 accession I43998 [45] size 1368 bp)was used for FASTA analysis The search results inTable 2 show that a number of bacterial aroa genesshow a high degree of similarity to the epsps transgeneThese aroa genes are from Bradyrhizobium japonicumCaulobacter crescens Gluconobacter oxydans Mesorhi-zobium loti Rhodopseudomonas palustris Silicibacterpomeroyi Sinorhizobium meliloti and Zymomonas mo-bilis as well as of the pathogenic bacteria Bartonella quin-tana Bartonella henselae Brucella melitensis B melitensisbiovar abortus and Brucella suis The observed identitiesprobably relate to phylogenetic relationship such as ob-served between the genome of B suis and sequences of Atumefaciens B melitensis M loti and S meliloti [51 52]as well as between B henselae B melitensis and B quin-tana [53] The aroa genes of Brucella melitensis B meliten-sis biovar abortus Brucella suis Mesorhizobium loti andSinorhizobium meliloti shared identical DNA stretches ofat least twice 20 bp with the transgenic sequence whichis considered the minimum required for homologous re-combination (Table 2)


Table 4 cry transgenes present in GM crops that have been approved for food use

Gene Target pest classGene source

Genbank accessiona ReferenceB thuringiensissubspecies

cry1ab Lepidopterans Kurstaki M15271 [159]cry1ac Lepidopterans Kurstaki M11068 [160]cry1fa Lepidopterans Aizawai M63897 [161]cry2aa Lepidopterans Kurstaki M31738 [162]cry2ab Lepidopterans Kurstaki X55416 [163]cry3aa Coleopterans Tenebrionis M30503 [164]cry3bb Coleopterans EG4961 M89794 [165]cry34ab and cry35ab Coleopterans PS149B1 AY011120 [166]

aNucleotide accessions can be retrieved from [45]

The sequence of the epsps transgene in GM soybeanhas been described in literature [54] The coding sequenceof this transgene was used for FASTA analysis (accessionAY125353 [45] size 1368 bp) The results were largelysimilar to those with the native gene (see above) ex-cept for the fact that the aroa genes from G oxydans Spomeroyi and Z mobilis did not score sufficiently with re-spect to the threshold values (E lt 1lowast10minus30 2times20 bp Table3)


With regard to the location of the native gene withinthe genome the cp4 epsps gene has been isolated fromchromosomal DNA of Agrobacterium CP4 [55] No in-formation was found on the natural horizontal transferof aroa or epsps genes Netherwood et al reported thedetection of the cp4 epsps gene in bacteria isolated fromsmall intestines of ileostomic patients who had consumedtransgenic soy but not in bacteria from feces of healthysubjects These authors were however unable to cultivatethe bacteria with the transferred transgenes preempting afurther confirmation of their results [20]


No specific information was available on the selectiveadvantage of the cp4 epsps transgene to microorganismsAs previously mentioned this gene was isolated from asoil bacterium that was able to degrade glyphosate It istherefore conceivable in our view that the transfer of thecp4 epsps sequence would convey a selective advantageto microorganisms in glyphosate-treated soil that is theability to sustain glyphosate toxicity and to utilise it as asubstrate


The aroa gene which codes for EPSPS is consid-ered a factor that influences the virulence of a number ofpathogenic microorganisms Pathogenic bacteria with ei-ther defective or without aroa genes (ie aroaminus mutants)

are unable to produce aromatic intermediates and there-fore are auxotrophic that is dependent upon the supplyof aromatic substrates such para-aminobenzoic acid Be-cause humans and animals do not produce aromatic pre-cursors the aroaminus mutants of pathogens are unable tomultiply in their bodies

Aroaminus mutants of a number of pathogenic microor-ganisms have been developed as candidates for live ldquoat-tenuatedrdquo avirulent vaccines For example aroaminus mutantsof Salmonella typhimurium and other Salmonella speciesare well described in literature also in combination withother mutations (such as for adenine nucleotides) thatimpact on virulence While these mutants have been suc-cessfully tested as oral vaccines against S typhimurium forexample in laboratory and domestic animals they mayalso serve as vehicle for transgenic protein antigens in re-combinant vaccines or for transgenic DNA in DNA vac-cines These vaccines exploit the mutantsrsquo retained capac-ity of S typhimurium to enter the hostrsquos immune systemfrom the intestines and thereby prime this system againstthe antigens of interest (see for review [56 57])

In addition the aroa genes of Pasteurella haemolyt-ica Pasteurella multocida Haemophilus somnus andAeromonas salmonicida have been mutated in pre-commercial attenuated live vaccines for cattle poultryand fish as reported in scientific literature [58] and sec-ondary information sources [59 60 61]

Reversion of auxotrophy in mutants by restoration ofaroa by horizontal transfer of transgenes would in theoryconfer a selective advantage to the recipient

In the FASTA analysis with the cp4 epsps genes presentin Agrobacterium CP4 and GM soybean aroa genes fromBrucella showed a high similarity including identical nu-cleotide stretches of at least 2times 20 bp Because Brucella isan intracellular pathogen like Salmonella it may be suit-able for development as attenuated live vaccine or vaccinecarrier Defective aromatic amino acid biosynthesis hasbeen associated with attenuation of Brucella such as inan aroC mutant of B suis [62] and an auxotrophic strainof Brucella abortus [63] There are currently no reportshowever of specific aroaminus mutants of B suis or B meliten-sis as candidate attenuated oral vaccines


Conclusion

There is a widespread occurrence of EPSPS in naturewhich relates to its role as a ldquohouseholdrdquo enzyme in manyorganisms With regard to mutated aroa genes of aroaminus

oral vaccines repair by homologous recombination ofthese genes with the cp4 epsps transgene appears unlikelygiven the lack of sufficient similarity between them In ad-dition glyphosate towards which CP4 EPSPS is insensi-tive does not have a role in treatment of human or animaldisease Therefore we conclude that there is no indicationthat the potential transfer of the cp4 epsps gene from GMcrops to microorganisms would alter the pathogenicity ofthe latter

Glyphosate oxidoreductase (GOX)Microbial source

The source organism Achromobacter LBAA was one ofthe bacteria isolated from activated industrial and domes-tic sludge that were capable of degrading glyphosate [64]

Natural function

The enzyme glyphosate oxidoreductase (GOX) hy-drolyzes the C-N bond of glyphosate yielding amino-methylphosphonic acid (AMPA) and glyoxylic acid Thesequence of GOX has been reported to be unique Oxy-gen serves as a cosubstrate in the enzymatic reaction anda putative flavin binding site for the FAD cofactor has beenidentified at the N-terminus of GOX (reviewed in [48])


GOX obtained from Achromobacter LBAA has beenintroduced into some GM-crops together with glypho-sate-resistant EPSPS (see above) in order to make thesecrops glyphosate-resistant [48]

Natural prevalence

It has been widely observed that the soil microfloraconverts glyphosate to AMPA A limited number of stud-ies address the GOX activity by which glyphosate is con-verted to AMPA and glyoxylic acid through lysis of theC-N bond within specific bacteria such as from indus-trial activated sludge that has been exposed to glyphosateand byproducts of its production (eg [65 66] and ref-erences cited herein) However Forlani et al [67] ob-served that bacteria isolated from soil were not capableof utilising glyphosate as sole C or N source and con-cluded that formation of AMPA should therefore be dueto non-culturable bacteria In addition Dick and Quinn[68] observed that unlike the lysis of the C-N bond byGOX isolated glyphosate-degrading soil microorganismscleaved the C-P bond of glyphosate While GOX-activityhas been predominantly been observed in environmentscontaining glyphosate it has recently been demonstratedin a thermophilic Geobacillus which unlikely had been ex-posed to glyphosate The function of this GOX activity inabsence of glyphosate is unknown [69]


We are not aware of reports describing the geograph-ical dispersion of the gox genes However the occurrenceof Achromobacter species in the environment has been re-ported to be widespread for example in Europe Mid-dle East and Central America [70 71 72] In additionthe formation of AMPA from glyphosate in glyphosate-treated soils has been reported in various regions includ-ing among others Europe North and South America[73 74 75]


The sequence used for FASTA analysis was the cod-ing sequence of the native gox gene from AchromobacterLBAA (sequence number 3 US patent 5 776 760 [64] size1296 bp) No similarities that complied with the thresholdcriteria were found (Table 2) The sequence of the trans-genic gox transgene introduced into GM crops was notavailable however and no FASTA analysis could thus beperformed on this sequence


No specific data about a possible selective advantageof the transfer of the gox gene for recipients were retrievedfrom literature As stated above the gox gene was obtainedfrom a glyphosate-degrading bacterium that had likelybeen exposed to glyphosate The transfer of the gox genemight in our view enable recipient microorganisms intheory to sustain the toxicity of glyphosate and to utiliseit as a substrate


Chromosomal DNA of Achromobacter LBAA hasserved as source for the gox transgene indicating that thelatter has a chromosomal location [64] No informationwas found on the natural horizontal transfer of aroa orepsps genes


The gene source belonged to the Achromobacterspecies which can in rare cases cause human diseasesuch as bacteremia due to A xylosoxidans [76 77] No datawere available on the possible role of gox in pathogenicityor virulence of Achromobacter

Conclusion

There is still uncertainty about the precise functionof GOX in its natural environment in the absence ofglyphosate As discussed above there is a background ofwidespread microbial GOX-like activity in soil In addi-tion glyphosate which is converted by GOX has no rolein the treatment of human and animal disease Thereforewe consider it unlikely that the potential transfer of GOXfrom transgenic plants would exert a significant effect onthe pathogenicity of recipient microorganisms


Phosphinothricin acetyltransferase (PAT)Microbial source

Streptomyces hygroscopicus (bar gene) and Strepto-myces viridochromogenes (pat gene) are streptomycetesthat occur in soil and that produce the natural herbi-cide bialaphos (phosphinothricin-alanine-alanine) Afterits release from bialaphos phosphinothricin inhibits theenzyme glutamine synthase which is important in ni-trogen metabolism in plants and microorganisms (eg[78]) Another phosphinothricin-containing peptide isphosalacine produced by the streptomycete Kitasatospo-ria phosalacinea [79] In a more general sense strepto-mycetes are soil microorganisms The production of an-tibiotics for example streptomycin and extracellular en-zymes by streptomycetes is exploited on an industrialscale

Natural function

Phosphinothricin N-acetyl transferase (PAT encodedby bar and pat) inactivates phosphinothricin by acety-lating the NH2 group Besides the target substrate phos-phinothricin PAT also acetylates to a lesser extentdemethyl-phosphinothricin (DMPT) methionine sulfox-imine (MSO) hydroxylysine and glutamate [80 81 82]


PAT has been engineered into a number of crops con-veying resistance against the herbicide glufosinate a syn-thetic analogue of phosphinothricin It serves either asa marker of genetic transformation or for the purposeof weed management in crops (see [80] and referencesherein)

Natural prevalence

The ability to detoxify phosphinothricin has been ob-served to be a prerequisite for its biosynthesis so that thiscompound cannot become toxic to its producer S hygro-scopicus [83] Wehrmann et al [80] mention that vari-ous acetyltransferases from Streptomyces griseus Strepto-myces coelicolor and Alcaligenes faecalis are also capableof acetylating phosphinothricin with however compara-tively weak affinity In addition Bedford et al [84] men-tion that such activity was also present in Streptomyceslividans


To our knowledge there are no reports describing thegeographical distribution of the bar and pat genes Thesources of the bar and pat genes S hygroscopicus and Sviridochromogenes belong to the streptomycetes whichare ubiquitously occurring soil microorganisms


For the FASTA analysis the coding sequences of thenative genes of bar (S hygroscopicus accession X05822[45] size 552 bp) and pat (S viridochromogenes M22827[45] 552 bp) were used These genes only shared with

each other similarities that complied with the thresholdcriteria (Table 2) No FASTA analysis could be done on thecodon-modified bar and pat transgene sequences presentin GM crops because they were unavailable


The native genes of bar and pat isolated from S hygro-scopicus and S viridochromogenes respectively are chro-mosomally located [85 86] No accounts are known ofthe horizontal transfer of the bar and pat genes from GMplants to microorganisms


No information was available on the selective advan-tage that the introduction of PAT by horizontal trans-fer may have on recipient microorganisms As mentionedabove the enzyme glutamine synthase which is inhibitedby phosphinothricin analogues like glufosinate has an es-sential role in microorganisms It is therefore conceivablein our view that microorganisms in environments con-taining glufosinate or other phosphinothricin analoguessuch as in herbicide-treated soils would benefit from PAT-induced resistance against these compounds


With regard to the potential role of PAT in humanpathogenicity and virulence of microorganisms no infor-mation could be found

Conclusion

PAT shows substrate specificity for phosphinothricinand similar compounds (see above) Whereas phos-phinothricin is considered a natural antibiotic it has noknown application in the treatment of human and ani-mal disease We therefore conclude that the transfer of thePAT enzyme is unlikely to confer increased pathogenicityto pathogens

Barnase and BarstarMicrobial source

The genes encoding Barnase and Barstar have beencloned from Bacillus amyloliquefaciens The first iso-lates of B amyloliquefaciens obtained from soil producedhigh levels of extracellular α-amylase which distinguishedthese bacteria from Bacillus subtilis (Fukumoto 1943cited by [87]) This characteristic is exploited for indus-trial production of the α-amylase enzyme

Natural function

Barnase is a ribonuclease which cleaves RNA yield-ing 3prime nucleotides through a 2prime 3prime-cyclic intermediate Itsstructure displays a characteristic fold formed by an α-helix and an antiparallel β-sheet Barstar is the inhibitorof Barnase and both proteins form a one-to-one complexThe structures of both proteins and their complex havebeen the subject of study in many peer-reviewed articles[88 89]


It has been hypothesised that Barnase may either servethe utilisation of extracellular ribonucleotides or as a toxinfor other microorganisms By binding to Barnase Barstarprevents B amyloliquefaciens from damage before it se-cretes Barnase [89]


The ribonuclease Barnase from B amyloliquefacienshas been cloned into some crops under control of atapetum-specific promoter The expression of this con-struct switches on specifically during anther developmentsuch that it impairs pollen formation and makes the cropmale sterile Male sterility is a useful trait for hybrid breed-ing and has also been obtained by non-GM breeding prac-tices Similar to GM crops expressing Barnase ldquorestorerrdquocrop lines with tapetum-specific expression of Barstarhave been developed Crop fertility can be restored bycrossing a male-sterile crop line transgenic for Barnasewith a restorer line transgenic for Barstar [90]

Natural prevalence

Bacterial- streptomycete- and fungal-homologues ofthe Barnase ribonuclease from B amyloliquefaciens havebeen identified including Binase (Bacillus intermedius)St (Saccharopolyspora erythrea) T1 (Aspergillus oryzae)C2 (Aspergillus clavatus) Ms (Aspergillus saitoi) U1 andU2 (Ustilago sphaerogena [88]) Several of the homol-ogous fungal ribonucleases for example α-sarcin arepart of a group of the so-called ldquoribotoxinsrdquo The func-tion of these ribotoxins as well as that of other Barnase-homologues is not known These ribonucleases all sharethe same three-dimensional ldquobarnase-foldrdquo structure aspreviously mentioned with three conserved amino acidresidues (Glu Arg His) that are involved in the catalyticreaction [88 91]

In addition homologues of Barstar have been foundin the streptomycetes Streptomyces aureofaciens (Sai14)and S erythrea (Sti) which inhibit the Sa- and St-ribonucleases respectively produced by these organisms[92 93]


No specific data are available on the geographical dis-tribution of the occurrence of barnase and barstar genesand their microbial source B amyloliquefaciens


The coding sequences of the native genes of barnase(B amyloliquefaciens accession M14442 [45] size 450 bp)and barstar (B amyloliquefaciens accession X15545 [45]size 273 bp) were used for FASTA analysis Barnaseshowed high similarity to ribonuclease genes from otherBacillus species (Table 2) Barstar did not show similaritiesbelow the threshold E value (E lt 1lowast10minus30) The sequencesof the codon-modified versions of these transgenes that

are present in GM crops were not known and thereforecould not be analysed


No information is provided on the location of thegenes that is chromosomal or plasmid-bound by theoriginal reports that describe the isolation and cloning ofthe native barnase and barstar genes from B amyloliquefa-ciens [94 95] Another report describes the PCR amplifi-cation of the barnase sequence located on a chromosomalfragment of B amyloliquefaciens [96]

It has been suggested that the occurrence of ribonucle-ases with the characteristic barnase-fold in both prokary-otes and eukaryotes is indicative of either common ances-try or horizontal gene transfer [97] The occurrence in arestricted number of organisms would indicate that theseribonucleases have recently evolved [91]


There were no observations reported of a possible se-lective advantage of the horizontal acquisition of the bar-nase and barstar genes However we estimate that if therole of barnase were to function as a toxin to other mi-croorganisms the barstar gene could convey a selectiveadvantage to its recipients


Unlike Bacillus cereus which can cause food poison-ing B amyloliquefaciens the source of the barnase andbarstar transgenes neither exerts toxicity on cultured cellsnor produces enterotoxins as reported in peer-reviewedliterature and in a regulatory product evaluation [98 99]

Extracellular ribonucleases other than Barnase areknown to exert toxicity after cellular uptake such asby binding to receptors on the surface of prokaryotic-and human cells [100 101] Ribonuclease(ribotoxin)-deficient mutants of Aspergillus fumigatus have been cre-ated by gene disruption through homologous recombina-tion Both wildtype and mutant strains were administeredto mice through the inhalatory route The animals wereobserved for mortality during the experiment and for fun-gal growth in lungs by postmortem histopathology It wasthus observed that ribotoxin-deficient fungi were no lesspathogenic than wildtype strains in invasive Aspergillus-mediated pulmonary infections (aspergillosis) [102] An-other study reported similar results [103] Fungal ribonu-cleases therefore do not appear to have an important rolein the pathogenicity of their hosts

Conclusion

As stated above B amyloliquefaciens the source of thebarnase and barstar genes is not known to be a pathogenunlike some other Bacillus species However the actualfunction of Barnase in its native host including its po-tential role in pathogenicity and virulence remains un-clear Barnase-related fungal ribonucleases do not appear


to have a role in the pathogenicity of moulds notwith-standing their toxicity to cells Based on this circumstan-tial evidence we conclude provisionally that the potentialtransfer of the barnase and barstar transgenes is unlikelyto influence the pathogenicity of recipient microorgan-isms

DNA adenine methylase (DAM)Microbial source

The dam gene has been isolated from Escherichia coli[104] This bacterium is a common commensal intesti-nal microorganism while pathogenic strains may occurPathogenic E coli strains can cause both intestinal andextraintestinal disease For example intestinal symptomsare caused by enteropathogenic (EPEC) enterotoxigenic(ETEC) enterohaemorrhegic (EHEC) or enteroinvasive(EIEC) E coli Extraintestinal symptoms are caused forexample by uropathogenic (UPEC) E coli Meningitis isanother example of an extraintestinal affection causedby particular strains of E coli Genes involved with vir-ulence of pathogenic E coli such as those encoding ad-hesins siderophores and toxins are linked with mobilegenetic elements These elements are not present in non-pathogenic strains and probably have been introduced byhorizontal gene transfer [105]

Natural function

The DNA adenine methylase (DAM) enzyme reg-ulates gene expression by methylation of adenine-N6

within the DNA sequence GATC [106]By methylation of the transcription initiation site

where RNA polymerase binds the expression of genes caneither be stimulated or inhibited In addition methyla-tion can also affect the binding of regulatory proteins toDNA [106] More generally DAM has also a role in DNAreplication initiation and mismatch repair [107] DAM-activity is associated with protection of bacteria againstDNA damage probably due to increased DNA breakageby intrinsic enzymes in the absence of dam which pre-disposes DNA to further damage [108] In addition damprotects again membrane damage by bile acids in the in-testinal environment which probably relates to the role ofdam in remodelling peptidoglycan which can be part ofthe bacterial envelope [108 109]


DAM has been introduced into GM crops in order torender them male-sterile such as in maize approved forcommercialisation in the USA [29] and in experimentalmaize [110] In the commercialised maize dam is said tobe expressed only in the anthers [29]

Natural prevalence

DAM activity has been reported for other γ-proteo-bacteria besides E coli and also among others in cyano-bacteria archaebacteria and spirochetes [104 111 112]

In addition adenine-N6-methylating enzymes are en-coded by bacteriophages such as phage T4 which infectsE coli [113]


No specific data were available on the geographicaldistribution of the native dam transgene E coli has beenisolated from a wide variety of geographical backgroundsfor example from human and animal samples from dif-ferent continents (eg [114])


The FASTA analysis was performed with the codingsequence of the native dam gene (E coli accession J01600[45] size 837 bp) The results showed that this sequencewas present in the nonpathogenic E coli strain K-12 aswell as in the enterohaemorrhagic strain O157H7 and theuropathogenic strain CFT073 (results not shown) DAMgenes occur in other bacteria with high sequence similar-ity to the native E coli gene indicating widespread occur-rence of this essential gene (Table 2) The occurrence ofmany pathogenic bacteria among these results likely is ac-counted for by the fact that the genomes of a wide array ofother γ-proteobacteria besides E coli have been sequenced

The sequence of the codon-optimised DNA of thedam transgene introduced into GM crops was not avail-able Therefore no FASTA analysis could be performedon this sequence


The dam transgene is located on the chromosome of Ecoli [104] The DNA sequences of the dam genes of E coliand phage T4 have different AT-contents and are thereforenot similar Based on the fact that a number of amino acidresidues appeared to have been conserved in the derivedprotein sequences of both genes a common evolutionaryorigin was postulated [115]

Horizontal transfer for example by phage transduc-tion might restore the DAM activity within cells of dam-deficient recipients This has been shown with phage damgenes artificially cloned into damminus E coli which repairedthe methylation of plasmids by this bacterium (describedeg by [116 117])


As previously mentioned DAM has a role in the pro-tection of bacteria against damage to DNA and mem-branes in the intestinal environment [108] The transferof dam to intestinal bacteria deficient in this gene mighttherefore in our opinion confer a selective advantagesuch as increased survivability


In pathogens like S typhimurium DAM has beenshown to have an essential role in their virulence Tar-get sequences of DAM include multiple genes that are


involved with the virulence of bacterial species such asthe gene encoding the toxin-coregulated pilus (tcp) inuropathogenic E coli and DAM is therefore named aldquomasterswitchrdquo of bacterial virulence (reviewed in [106])

Avirulent DAM-deficient mutants of pathogenic bac-teria have been described While these avirulent mutantsare able to induce an immune response they are not asinvasive as the wildtype pathogens and occur in muchlower numbers in host tissues after oral administrationyet are able to prime an immune response against subse-quent challenges with the wildtype pathogen Reversion tovirulence of damminus mutants by their transformation withfunctional dam genes has been observed in animal exper-iments [106]

The use of this technology for creating avirulentpathogens as live vaccines may be commercialised in thenear future since the website of a biotechnology companyoffers DAM-deficient mutants for development of vac-cines and vaccine carriers [118]

Conclusion

The dam transgene is derived from E coli whichis a common resident of human intestines (eg 80ndash87 log cfug in fecal samples from positive infants [119])In addition native dam occurs in both pathogenic andnonpathogenic strains of E coli while counterparts withhighly similar sequences occur widely in closely relatedγ-proteobacteria as described above To our knowledgedamminus live attenuated oral vaccines have not been com-mercialised yet Therefore we assume that given the con-tinuous background presence of natural counterparts thepotential transfer of the dam transgene would not impacton the pathogenicity of recipient microorganisms

1-aminocyclopropane-1-carboxylate (ACC)deaminase

Microbial source

The gene encoding the 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been isolated fromPseudomonas 6G5 Out of 600 bacteria isolated from soilthis and another Pseudomonas bacterium showed abil-ity to grow on minimal media containing ACC [120]Members of the genus Pseudomonas belong to the γ-proteobacteria and are ubiquitous and diverse compris-ing strains that can be nonpathogenic or pathogenic toplants animals and humans The pathogenic traits havebeen linked to the presence of genes that are absent fromnonpathogenic Pseudomonas [121]

Natural function

The enzyme ACC deaminase from Pseudomonas 6G5diverts ACC into ammonia and ketobutyric acid [120]

The plant hormone ethylene is formed from ACC inplants Soil bacteria associated with roots of crops andplants have been found to express ACC deaminase ac-tivity This activity suppresses the ethylene synthesis by

plants and causes increased root formation by these crops(eg [122])


Introduction of the enzyme ACC deaminase into GMtomatoes prevents ethylene formation in fruits which inturn delays fruit ripening [120]

Natural prevalence

The occurrence of ACC deaminase activity has beendescribed in plant growth promoting soil bacteria in-cluding strains of Alcaligenes Bacillus Enterobacter Pseu-domonas Rhodococcus Rhizobium and Variovorax as wellas from the yeast Hansenula saturnus and the mould Peni-cillium citrinum [122 123 124 125] Comparison of theamino acid sequences of ACC deaminase enzymes frombacteria yeast and mould shows a high degree of similar-ity and the conservation of residues that are essential foractivity [126]


As stated above the occurrence of bacteria of thegenus Pseudomonas is ubiquitous [121] ACC deaminase-containing microbes can be found in soil samples froma wide range of origins such as for example USA Rus-sia a number of European countries and Bhutan in Asia[122 124 127] No data were available on the distributionof the ACC deaminase gene


The coding sequence of the native ACC deaminasegene from Pseudomonas 6G5 was used for FASTA analy-sis (accession M80882 [45] size 1017 bp) This sequenceshowed high similarity with the corresponding genesfrom many soil bacteria species as well as from theyeast Schizosaccharomyces pombe and the fungus Penicil-lium citrinum (Table 2) These similarities are in generalagreement with the similarities reported in literature (seeabove) It was not possible to carry out the same analy-sis on the sequence of the codon-modified version of theACC deaminase transgene that had been introduced intoGM crops due to unavailability of this sequence


The ACC deaminase gene was isolated from chromo-somal DNA of Pseudomonas 6G5 indicating that it has achromosomal position [120] There were no accounts ofthe horizontal transfer of the ACC deaminase gene avail-able in literature


As previously mentioned ACC deaminase allows itsmicrobial hosts to utilise ACC as a sole N-source whichin our view would convey a selective advantage to mi-croorganisms grown in the vicinity of plants which pro-duce ACC as an ethylene precursor


Conclusion

ACC deaminase is directed towards a plant hormonewhich is not present in humans and animals In additionits gene from the gene source Pseudomonas 6G5 has simi-lar counterparts in many related soil bacteria from a widerange of geographic origins as well as in some eukaryoticmicroorganisms We therefore conclude that the potentialtransfer of the ACC deaminase transgene from GM cropsis unlikely to contribute to pathogenicity of recipient mi-croorganisms

S-adenosylmethionine hydrolase (SAMase)

Microbial source

The SAMase gene encoding the enzyme S-adeno-sylmethionine hydrolase (SAMase) as used for geneticmodification is derived from the E coli bacteriophage T3[128]

Natural function

Hydrolysis of SAM by T3 SAMase yields 5prime-methyl-thioadenosine and homoserine [128]

The supposed function of native T3 SAMase is to in-activate the hostrsquos type I restriction endonuclease and todeplete its cofactor S-adenosylmethionine (SAM) in E colicells infected by T3 [129] This would protect the phagefrom being degraded by the hostrsquos DNA restriction activ-ity

In addition artificial cloning of SAM hydrolase intobacteria like E coli has been shown to reduce among oth-ers DNA methylation and biosynthesis of quorum sens-ing signaling molecules [130 131 132]


ACC is a precursor to the plant hormone ethylenewhile the formation of ACC involves reactions with SAMHydrolysis of SAM by the transgenic enzyme SAMase ofwhich the gene is under the control of a ripening stage-specific promoter inhibits ethylene formation and delaysfruit ripening in GM tomatoes [128]

Natural prevalence

The E coli bacteriophage T7 which is closely relatedand highly similar to T3 does not contain the SAMasegene In addition the gene and amino acid sequencesof the 03 protein from T7 phage which also show anti-restriction activity do not show much similarity to thoseof T3 SAMase [129]


No specific data were available on the geographicaldistribution of the SAMase gene and its source bacterio-phage T3


The native sequence of SAMase from bacteriophageT3 was used for FASTA analysis (accession X04791 [45]size 459 bp) This sequence shared a high degree of sim-ilarity with the 03 gene which fulfils the same functionin the bacteriophage phiYeO3-12 (Table 2) This ldquoyersin-iophagerdquo infects Yersinia enterolytica serovar O3 and itsgenome sequence shows a high overall similarity to that ofT3 [133] Interestingly deletion of 03 gene from phiYe03-12 did not impair the phagersquos efficiency in vitro [134]

No data were available on the sequence of the codon-modified SAMase transgene introduced into GM cropsTherefore it was not possible to carry out a FASTA analy-sis with this sequence


As previously mentioned the native SAMase gene islocated on a bacteriophage The similarities and differ-ences of the DNA including the SAMase gene betweenbacteriophages T3 T7 and phiYe03-12 has led to the hy-pothesis that T3 might have originated from a recombina-tion event between T7 and a yersiniophage In a model ex-periment recombination between T7 and phiYe03-12 wasindeed observed in E coli that had been genetically mod-ified with the O3 receptor of Y enterolytica in order to fa-cilitate coinfection with both phages The recombinationof the SAMase-like gene 03 of phiYe03-12 was not ob-served in this case The 03 gene was flanked upstream anddownstream by stretches of identical nucleotides whichcould be used for recombination that is horizontal trans-fer [135]


Mutant phages with T3 SAMase deleted are not lessefficient and SAMase therefore appears not to be essentialfor lytic activity (eg [134]) We infer from this data thattransfer of SAMase would not convey a selective advantageto recipient phages


As mentioned above the cloning of SAMase into bac-teria suppresses among others DNA methylation andbiosynthesis of quorum sensing signaling molecules bothof which are known to stimulate virulence of microorgan-isms

Conclusion

As mentioned above the function of native SAMase isto prevent bacteriophages from degradation by infectedbacterial hosts In addition expression of SAMase thathas been cloned into bacteria may indirectly suppresspathogenicity of microorganisms by decreasing DNAmethylation and the biosynthesis of quorum sensing sig-naling compounds We conclude therefore that introduc-tion of SAMase into mico-organisms by horizontal trans-fer is unlikely to contribute to pathogenicity


β-glucuronidase (GUS)

Microbial source

The uidA gene which codes for the β-glucuronidase(GUS) enzyme has been isolated from E coli (reviewed in[136]) E coli is described in more detail in the section onthe dam gene

Natural function

GUS hydrolyzes glucuronide-conjugated compoundsintracellularly in E coli releasing the glucuronide [136]

The glucuronide released by GUS activity is used by Ecoli as a carbon substrate for its metabolism [136]


The ability of GUS to convert chromogenic or fluoro-genic substrates yielding color or fluorescence develop-ment by the reaction has also been exploited in biotech-nology by using GUS from E coli as a marker gene (re-viewed in [137]) In addition the use of transgenic maizeplants expressing GUS for commercial production of thisenzyme in purified form has also been reported [138]

Natural prevalence

E coli and other coliforms exhibit GUS activity Be-sides coliform bacteria a limited number of other mi-croorganisms including Shigella also display such activ-ity Bacteroides and Clostridium are among the gut res-idents showing β-glucuronidase Whereas their activitywas weaker than for E coli these bacteria are generallymore numerous in the gut [136] GUS activity of E colihas been exploited for rapid tests to detect coliform bac-teria in environmental food water and clinical samples[139] indicating ubiquitous presence of GUS-activity

The allele frequency within a 587 bp fragment ofthe uida gene has been studied in environmental GUS-positive isolates of E coli In this study 114 alleles wereidentified in 941 isolates of which 60 alleles occurred intwo or more isolates [140]


As mentioned above GUS and E coli are ubiquitouslypresent in a range of environments


For the FASTA analysis the coding sequence of thenative uidA gene coding for GUS from E coli (accessionS69414 [45] size 1812 bp) was used Besides E coli the 100most similar sequences (E le 47e-153) in the results of theFASTA analysis within the prokaryote nucleotide databasewere from Shigella species Within the database for fungalnucleotides highly similar genes coding for GUS occurredin Penicillium canescens and Scopulariopsis (Table 2)

No data were available on the sequence of the codon-modified version of the uidA transgene present in GM

crops Due to this lack of data no FASTA analysis couldbe carried out on the codon-modified uidA transgene


The uida gene is located on the chromosome of E coliand is part of the GUS-operon [136] The presence ofhighly similar sequences coding for GUS in soil isolatesof P canescens and Scopulariopsis fungi and Arthrobacterbacteria has recently been described In this study the hy-pothesis was tested that gus genes would be amenable tohorizontal gene transfer from bacteria to fungi This hy-pothesis was based on the assumption that gus would con-vey to fungi in soil the capacity to utilise glucuronides ex-creted by animals as a source of carbon Based on charac-teristics of the gus genes and their products in these fungiand Arthrobacter the authors concluded that these genesmust have been derived from a common ancestor [141]


As mentioned previously the horizontally transferreduida gene is assumed by other authors to convey a se-lective advantage to recipient soil microorganisms sinceGUS enables the utilisation of glucuronides derived fromanimal excretions shed onto land [141]


GUS activity is generally considered to be absent froma minority of all E coli strains including the pathogenicenterohaemorrhagic E coli O157H7 The gus gene is stillpresent in this and other E coli strains lacking GUS ac-tivity the inactivity probably being caused by mutationsin this gene [142 143 144] Recent reports however de-scribe incidences of E coli O157H7 showing GUS activity(eg [145])

GUS activity contributes to the so-called enterohep-atic circulation of hydrophobic compounds in humansand animals During this process compounds are glu-curonidated in the liver excreted through the bile into thegut deglucuronidated by the gut flora and subsequentlyabsorbed from the gut [136]

In addition bacterial beta-glucuronidase activity hasbeen considered to be one of the factors that contribute tothe formation of gallstones in the liver This is thought tobe due to the deglucuronidation of bilirubin glucuronidespresent in the gall which would facilitate the formation ofcalcium bilirubinate a component of gallstones [146]

Conclusion

As described above there is a ubiquitous backgroundpresence of GUS in a range of environments includ-ing the intestinal microflora In addition no link is evi-dent between GUS and the pathogenicity of particular Ecoli strains We conclude therefore that it is unlikely that


the horizontal transfer of GUS if it would occur wouldhave a noticeable impact on intestinal GUS activity or onpathogenicity of recipient organisms

Nopaline synthase (NOS)

Microbial source

The native tumor-inducing plasmid pTiC58 ofAgrobacterium tumefaciens strain C58 harbours amongothers the gene nos encoding nopaline synthase This plas-mid is transferred to plants infected by A tumefacienscausing the formation of calli that is ldquocrown gall tumorsrdquo[147]

Natural function

Nopaline synthase (NOS) which is also known asnopaline dehydrogenase catalyzes the formation of nopa-line [N2-(1 3-dicarboxypropyl)arginine] from the pre-cursors α-ketoglutaric acid and arginine in an NADPH-dependent reaction It also catalyzes the formation ofornaline [N2-(1 3-dicarboxypropyl)ornithine] from α-ketoglutaric acid and ornithine [147]

The transfer of the nos gene to infected plant cells trig-gers the synthesis by these cells of nopaline which is oneof the ldquoopinesrdquo that can be utilised as substrate by A tume-faciens [148] For example both ldquooctopinerdquo and ldquonopa-linerdquo types of crown galls can be discerned while the latterhas been associated with nopaline synthase activity [147]


Nopaline synthase has been engineered into trans-genic flax in order to serve as a transformation markerthat facilitated detection of transformed plant embryosby the presence of nopaline (see the Canadian regulatoryevaluation document [149])

Natural prevalence

The amino acid sequences of NOS and octopine syn-thase share domains of comparatively high sequence sim-ilarity with each other and to a lesser degree with dehy-drogenase enzymes from other organisms [150] BesidesAgrobacterium related opine dehydrogenase enzymes alsooccur in the bacterium Arthrobacter and aquatic inverte-brates [151 152] In the latter these enzymes have a rolein the anaerobic glycolysis that is in energy metabolism[152]


No data were available on the geographical distribu-tion of the nos gene The microbial host of the pTiC58plasmid A tumefaciens C58 is able to grow in associa-tion with plants around the globe [153] More generallyAgrobacterium species occur widely in soils from differentgeographical origins as described above for the cp4 epspsgene


The coding sequence of the native nos gene fromAgrobacterium tumefaciens plasmid pTiC58 was usedfor the FASTA analysis (accession AJ237588 [45] size1242 bp) The results of this analysis showed that the nosgene shared a high degree of similarity with the corre-sponding gene on plasmid pTiAB4 of the related Agrobac-terium vitis (Table 2) The actual sequence of the nostransgene introduced into GM crops was not availableand therefore no analysis could be carried out on this se-quence


The microbial native nos transgene is located on plas-mid pTiC58 as previously mentioned In a model exper-iment with nonsterile soil the conjugative transfer of amodified pTiC58 plasmid from Agrobacterium to otherbacteria was observed [154] While this study did notspecifically analyze for the transfer of the nos gene itshowed that the native plasmid carrying nos could betransferred horizontally by conjugation

The A vitis plasmid pTiAB4 shows similarity to otherplant ldquotumor-inducingrdquo (Ti) plasmids such as for exam-ple a fragment containing the nos gene that was very sim-ilar to the pTiC58 (source of the transgene) These simi-larities were considered by Otten and De Ruffray [155] tooriginate from horizontal transfer between Ti plasmids


No data were available that in our opinion would in-dicate a particular selective advantage for the nos gene torecipients after its potential horizontal transfer


The nos gene has a role in the pathogenicity of its na-tive host A tumefaciens in plants as described above whileneither one is known to have a role in pathogenicity orvirulence of human or animal pathogens

Conclusion

As mentioned above the nos gene has a specific rolein plant pathogenesis by A tumefaciens We therefore con-clude that its potential horizontal transfer to microorgan-isms would unlikely contribute to the latterrsquos human andanimal pathogenicity

Cry proteins

Microbial source

The source of the cry genes used for genetic modifica-tion of crops is Bacillus thuringiensis This bacterium wasdescribed for the first time in 1901 following its isolationfrom diseased silkworm larvae It was observed later thatthese bacteria produced spores containing crystals that are


toxic to insects These crystals contain among others theCry proteins encoded by cry genes located on plasmidsand on the chromosome The insecticidal properties of Bthuringiensis and its crystal inclusions have been exploitedfor the production as biological pesticides since 1938 (re-viewed in [156])

The classification of subspecies of B thuringiensis isbased upon serological reactions of the H-flagellae [157]The various cry genes that have been introduced in theGM crops and their sources are mentioned in Table 4 Inaddition a website dedicated to the nomenclature of Cryproteins provides the database accession and host strainfor each protein [158]

B thuringiensis including the subspecies from whichthe cry transgenes are derived occurs ubiquitously in theenvironment including soil water sediment plant leavesand food (eg [167 168 169 170])

These Cry proteins are subdivided into various groupsbased on the degree of similarity between their amino acidsequences [171]

Natural function

The active subsequence of Cry proteins which is re-leased by enzymes in the insect gut is composed of threedomains Two of these domains participate in the bind-ing of the Cry protein to aminopeptidase N receptormolecules on the surface of epithelial cells lining the in-sect gut The third domain subsequently forms pores inthe cell membrane leading to leaching and finally in-sect lethality Distinct classes of Cry proteins show ac-tivities against specific insects such as Cry1Ab againstLepidoptera and Cry3Aa against Coleoptera (reviewed in[172])

The insecticidal mechanism is very specific as the Cryproteins are not bound by intestinal tissues of humans andexperimental rodents for which these proteins are non-toxic (eg [173])


A number of insecticidal Cry proteins originatingfrom various strains of Bacillus thuringiensis have beenengineered into GM crops in order to protect these cropsfrom phytophagous (plant-feeding) insects (reviewed in[174])

Natural prevalence

Whereas the ubiquitous presence of B thuringiensisstrains has been acknowledged for a long time studiesscreening for the occurrence of cry genes are of a compar-atively recent date These studies were reviewed by Por-car and Juarez-Perez [175] The frequency of detectionmay differ from one particular type of cry gene to an-other For example within the group of cry1 genes whichare frequently observed cry1F appears to be less commonthan for example cry1A [175 176] In addition combi-nations of cry genes can be detected in single isolates of B

thuringiensis which may be specific for certain strains ge-ographical origins or ecological systems (eg [177 178])

Cry sequences have also been identified in variousbacteria other than Bacillus thuringiensis For examplethe cry16Aa and cry17Aa genes have been identified in astrain of Clostridium bifermentans with insecticidal activ-ity against dipterans [179]

In addition cry genes have been identified in Paeni-bacillus including cry43Aa cry43Ba and cry43-like fromP lentimorbus [180] as well as cry18Aa from P popil-liae [181] Given that P popilliae acts differently from Bthuringiensis that is as a parasite of beetles (coleopterans)rather than an insecticide Zhang et al [181] argued thatthe Cry18Aa protein should have a different role in insectpathology than that of the Cry proteins from B thuringien-sis

The amino acid sequence of the cry35ab gene prod-uct from B thuringiensis PS149B1 shows similarity to the419-kilodalton protein from Bacillus sphaericus Inter-estingly both proteins are only toxic to target insects incombination with a coexpressed protein that is Cry34Aband Cry35Ab in corn rootworm and the 419- and 514-kilodalton proteins in mosquitoes [182 183]


Similar to the occurrence of cry genes described abovestudies on the geographical distribution of these geneshave been carried out recently These studies were re-viewed by Porcar and Juarez-Perez [175] while additionaldata have been published since then [176 184 185 186]The results of these studies indicate that in general manycry genes are present in isolates of B thuringiensis from awide range of geographical origins including Latin Amer-ica Asia and Europe


FASTA analysis of the native versions of the cry genesthat have been introduced into GM crops showed no sim-ilarities of E lt 1lowast10minus30 or minimally 2 times 20 bp otherthan with other accessions for sequences from Bacillusthuringiensis (Table 2) In some cases that is for cry1Aband cry1Ac the highest E-value of the 100 best scoringalignments (maximum output) with the prokaryote nu-cleotide sequence database was still below the thresholdof E lt 1lowast10minus30 The codon-modified versions of the crytransgenes introduced into GM crops have not been anal-ysed by FASTA because their sequences were not avail-able


With regard to the presence of cry genes in Clostrid-ium (see above) Barloy et al [179] suggested that mobileelements such as transposons might have contributed tothe dissemination of these genes

The native microbial cry transgenes occur both onplasmids and in the chromosome of Bacillus thuringiensis


(eg [187]) Transfer of these genes by exchange of trans-missible plasmids through conjugation with other strainsof B thuringiensis and Bacillus species has been observedunder laboratory conditions [188 189]

Based on his review of structural similarities of thevarious Cry proteins de Maagd et al [172] postulatedthat ldquodomain swappingrdquo might have occurred between crygenes through homologous recombination Such an ex-change of domains can be accomplished under laboratoryconditions and can change the activity spectrum of the re-sultant mutant Cry protein [172]


Cry proteins may constitute an important virulencefactor of strains of B thuringiensis and B cereus that are op-portunistic insect pathogens [190] We therefore speculatethat horizontally transferred cry genes would in theoryconvey a selective advantage to recipient microorganismslacking these genes within the insect environment


B thuringiensis is genetically related to Bacillus an-thracis and B cereus which are known pathogens Banthracis causing anthrax and B cereus causing oppor-tunistic infections The specific phenotypic characteris-tics of B thuringiensis and B anthracis are associated withextrachromosomal elements [191] Based upon exten-sive review of the safety data on B thuringiensis to ani-mals and humans various sources have concluded that Bthuringiensis preparations are safe (eg [157 192])

Conclusion

As described above native cry genes display ubiqui-tous presence and have also been detected in foods Over-all reviews of safety data indicate that there is no toxicityof Cry proteins to humans [157 174 192] We thereforeconclude that the horizontal gene transfer of cry genesfrom plants to microorganisms if it would occur is un-likely to contribute to pathogenicity of recipient microor-ganisms in humans and domestic animals

DISCUSSION

The microbial genes that have been introduced intomarket-approved GM crops constitute a fairly heteroge-nous group with regard to source and function in mi-croorganisms In the safety assessment of these crops sofar the focus has been on antibiotic resistance genes Inthis work the function and characteristics of microbialtransgenes other than antibiotic resistance genes as wellas the potential health aspects of their horizontal transferhave been discussed In the survey we took into accountcharacteristics of the transgenes that might be relevantto health These characteristics included the microbial

source of the native transgene the function of the trans-gene in its natural environment and in genetically modi-fied crops the natural prevalence and geographical distri-bution of the native and similar transgenes the homologyto genes of other microbial species which is also indicativefor the background presence and the likelihood of trans-fer that is by homologous recombination known hori-zontal transfer activity of the transgene and the poten-tial contribution of the transgene to pathogenicity or vir-ulence of human and animal microbial pathogens Eachsingle item may not be totally predictive of gene transferand associated health effects and therefore the ldquoweighedevidencerdquo of the items in combination should be consid-ered In many cases it was noted that there was a wide-ranging background presence a specific function or anapparent lack of relationship with pathogenicity of thegene considered We concluded for each gene that its po-tential horizontal transfer to microorganisms would un-likely raise health concerns

In addition we noted some conspicuous items forthe genes considered For example the presence in soilfungi of analogues of the bacterial gene coding for β-glucuronidase might originate from a horizontal transferbetween bacteria and fungi as reported recently in litera-ture [141] Interestingly this gene was considered by theauthor of the study to convey a selective advantage to therecipient fungi since it would allow for utilisation of glu-curonides from excretions (feces urine) of animals In amore general sense we may extend this to survival andcompetitive advantage of microorganisms in the environ-ment including the soil In this respect also the trans-fer of herbicide resistance genes may in theory providea selective advantage to soil microorganisms sensitive toherbicide action as may the transfer of the ACC deami-nase gene to microorganisms colonising the rhizosphereof plants It may be speculated that an increased surviv-ability of pathogens in the environment may indirectly in-crease the likelihood of exposure to these pathogens

Another conspicuous item was the presence in live at-tenuated oral vaccines of mutated aroa genes which arefunctional analogs of the cp4 epsps transgene in herbicide-resistant crops We consider the likelihood of repair ofthe mutant genes by homologous recombination with thetransgene to be comparatively low or absent given thelack of similarity to aroa genes in precommercial vaccinesand the background presence of aroa genes in other mi-croorganisms Some experimental GM crops have beenmodified with bacterial aroa genes other than cp4 epspssuch as aroa from S typhimurium of which mutants havealso been used as attenuated live vaccines A discussion onexperimental GM crops is however beyond the scope ofthis paper

Some of these experimental GM crops may enterthe market in the near future and contain novel traitsand transgenes which should also be assessed for theirsafety by a comparative safety assessment [3] The sec-tion on gene transfer of the FAOWHO Codex Alimenta-rius guidelines for the safety assessment of foods derived


from GM crops focuses on antibiotic resistance [2] Thepotential impact of gene transfer on the pathogenicity ofrecipient microorganisms is also an important item inpractice however We therefore recommend consideringthe abovementioned items including the characteristicsof transgenes and their native counterparts in the safetyassessment of GMOs carrying transgenes other than thosereviewed in this paper

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial supportfrom the Dutch Ministry of Agriculture Nature and FoodQuality In addition the authors wish to thank Ms Ir EJ Kok for her comments on the manuscript

REFERENCES

[1] James C Global Status of Commercialized BiotechGM Crops 2004 Ithaca International Service forthe Acquisition of Agri-Biotech Applications 2004ISAAA Briefs No 32-2004 httpwwwisaaaorgkcCBTNewspress releasebriefs32ESummaryExecutive20Summary20 (English)pdf

[2] Codex Alimentarius Commission Codex Principlesand Guidelines on Foods Derived from Biotechnol-ogy Rome ItalyFood and Agriculture Organi-sation 2003 Codex Alimentarius CommissionJoint FAOWHO Food Standards ProgrammeftpftpfaoorgcodexstandardenCodexTextsBiotechFoodspdf

[3] Kok EJ Kuiper HA Comparative safety assessmentfor biotech crops Trends Biotechnol 200321(10)439ndash444

[4] Kuiper HA Kleter GA Noteborn HP Kok EJ As-sessment of the food safety issues related to geneti-cally modified foods Plant J 200127(6)503ndash528

[5] van den Eede G Aarts H Buhk HJ et al The rele-vance of gene transfer to the safety of food and feedderived from genetically modified (GM) plantsFood Chem Toxicol 200442(7)1127ndash1156

[6] Lorenz MG Wackernagel W Bacterial gene trans-fer by natural genetic transformation in the envi-ronment Microbiol Rev 199458(3)563ndash602

[7] Smalla K Gebhard F Van Elsas JD Matzk A Schie-mann J Bacterial communities influenced by trans-genic plants In Jones DD ed Proceedings of the3rd Symposium on the Biosafety Results of FieldTests of Genetically Modified Plants and Microorgan-isms Monterey OaklandUniversity of California1994157ndash167

[8] Schluter K Futterer J Potrykus I ldquoHorizontalrdquogene transfer from a transgenic potato line to a bac-terial pathogen (Erwinia chrysanthemi) occursmdashifat allmdashat an extremely low frequency Biotechnol-ogy (NY) 199513(10)1094ndash1098

[9] Droge M Puhler A Selbitschka W Horizontal genetransfer as a biosafety issue a natural phenomenonof public concern J Biotechnol 199864(1)75ndash90

[10] Nielsen KM Gebhard F Smalla K Bones AM VanElsas JD Evaluation of possible horizontal genetransfer from transgenic plants to the soil bac-terium Acinetobacter calcoaceticus BD413 Theoret-ical and applied genetics 199795815ndash821

[11] Tepfer D Garcia-Gonzales R Mansouri H et alHomology-dependent DNA transfer from plants toa soil bacterium under laboratory conditions im-plications in evolution and horizontal gene trans-fer Transgenic Res 200312(4)425ndash437

[12] Ceremonie H Buret F Simonet P Vogel TM Isola-tion of lightning-competent soil bacteria Appl En-viron Microbiol 200470(10)6342ndash6346

[13] Edwards RA Olsen GJ Maloy SR Comparative ge-nomics of closely related salmonellae Trends Mi-crobiol 200210(2)94ndash99

[14] Jacob D Lewin A Meister B Appel B Plant-specificpromoter sequences carry elements that are recog-nised by the eubacterial transcription machineryTransgenic Res 200211(3)291ndash303

[15] Lewin A Tran TT Jacob D Mayer M Frey-tag B Appel B Yeast DNA sequences initiatinggene expression in Escherichia coli Microbiol Res2004159(1)19ndash28

[16] Jonas DA Elmadfa I Engel KH et al Safety con-siderations of DNA in food Ann Nutr Metab200145(6)235ndash254

[17] Straub JA Hertel C Hammes WP The fate of re-combinant DNA in thermally treated fermentedsausages Eur Food Res Technol 199921062-67

[18] van der Vossen JMBM Havekes WALM Koster DSet al Development and application of in vitro in-testinal tract model for safety evaluation of genet-ically modified foods In Food Safety Evaluation ofGenetically Modified Foods as a Basis for Market In-troduction Market Introduction Genetically Modi-fied Foods The Hague The NetherlandsMinistry ofEconomic Affairs 199881ndash98

[19] Wilcks A van Hoek AH Joosten RG Jacobsen BBAarts HJ Persistence of DNA studied in different exvivo and in vivo rat models simulating the humangut situation Food Chem Toxicol 200442(3)493ndash502

[20] Netherwood T Martın-Orue SM OrsquoDonnell AGet al Assessing the survival of transgenic plant DNAin the human gastrointestinal tract Nat Biotechnol200422(2)204ndash209

[21] Bauer F Hertel C Hammes WP Transformation ofEscherichia coli in foodstuffs Syst Appl Microbiol199922(2)161ndash168

[22] Kharazmi M Sczesny S Blaut M Hammes WPHertel C Marker rescue studies of the transfer ofrecombinant DNA to Streptococcus gordonii in vitroin foods and gnotobiotic rats Appl Environ Micro-biol 200369(10)6121ndash6127

[23] Kharazmi M Bauer T Hammes WP Hertel CEffect of food processing on the fate of DNAwith regard to degradation and transformation


capability in Bacillus subtilis Syst Appl Microbiol200326(4)495ndash501

[24] Hacker J Kaper JB Pathogenicity islands andthe evolution of microbes Annu Rev Microbiol200054641ndash679

[25] Wain J House D Pickard D Dougan G Frankel GAcquisition of virulence-associated factors by theenteric pathogens Escherichia coli and Salmonellaenterica Philos Trans Roy Soc London Ser B Biol Sci20013561027ndash1034

[26] Cao H Baldini RL Rahme LG Common mecha-nisms for pathogens of plants and animals AnnuRev Phytopathol 200139259ndash284

[27] van Burik JA Magee PT Aspects of fungalpathogenesis in humans Annu Rev Microbiol200155743ndash772

[28] European Commission 94211EC CommissionDecision of 15 April 1994 amending Council De-cision 91596EEC concerning the summary notifi-cation information format referred to in Article 9 ofCouncil Directive 90220EEC Off J Eur Commun199410526ndash44

[29] FDA List of Completed Consultations on Bioengi-neered Foods Washington DCOffice of Food Addi-tive Safety Center for Food Safety and Applied Nu-trition US Food and Drug Administration 2005httpwwwcfsanfdagovsimlrdbioconhtml

[30] CFIA Status of Regulated Plants with Novel Traitsin Canada Unconfined Environmental ReleaseNovel Livestock Feed Use Variety Registration andNovel Food Use NepeanPlant Biosafety OfficePlant Products Directorate Canadian Food Inspec-tion Agency 2005 httpwwwinspectiongccaenglishplavegbiopntvcneshtml

[31] European Commission Lists of AuthorisedGMOs BrusselsDirectorate General Health andConsumer Affairs European Commission 2005httpeuropaeuintcommfoodfoodbiotechnologygmfoodindex enhtm

[32] FSANZ GM FoodsmdashCurrent Applications andApprovals (Updated February 2005) Canberra andWellingtonFood Standards Australia NewZealand 2005 httpwwwfoodstandardsgovauwhatsinfoodgmfoodsgmcurrentapplication1030cfm

[33] Watt VM Ingles CJ Urdea MS Rutter WJ Homol-ogy requirements for recombination in Escherichiacoli Proc Natl Acad Sci USA 1985824768ndash4772

[34] WHO Health Aspects of Marker Genes in GeneticallyModified Plants Geneva SwitzerlandWorld HealthOrganisation 1993 Report of a WHO WorkshopWHOFNUFOS936

[35] Biet E Sun JS Dutreix M Stimulation of D-loop formation by polypurinepolypyrimidine se-quences Nucleic Acids Res 200331(3)1006ndash1012

[36] Pearson WR Wood TC Statistical significance inbiological sequence comparison In Balding DJ

Bishop M Cannings C eds Handbook of Statisti-cal Genetics LondonUKWiley 200139ndash65

[37] Nielsen KM An assessment of factors affecting thelikelihood of horizontal transfer of recombinantplant DNA to bacterial recipients in the soil andphytosphere In Collection of Biosafety Reviews 1sted ItalyICGEB Italian Ministry of Environment200396ndash149

[38] McBride KE Kenny JW Stalker DM Metabolismof the herbicide bromoxynil by Klebsiella pneu-moniae subsp ozaenae Appl Environ Microbiol198652(2)325ndash330

[39] Stalker DM McBride KE Malyj LD Herbicide re-sistance in transgenic plants expressing a bacterialdetoxification gene Science 1988242419ndash423

[40] Stalker DM Malyj LD McBride KE Purificationand properties of a nitrilase specific for the her-bicide bromoxynil and corresponding nucleotidesequence analysis of the bxn gene J Biol Chem1988263(13)6310ndash6314

[41] Kato Y Ooi R Asano Y Distribution of aldoximedehydratase in microorganisms Appl Environ Mi-crobiol 200066(6)2290ndash2296

[42] Banerjee A Sharma R Banerjee UC The nitrile-degrading enzymes current status and futureprospects Appl Microbiol Biotechnol 200260(1-2)33ndash44

[43] OrsquoReilly C Turner PD The nitrilase family of CNhydrolysing enzymesmdasha comparative study J ApplMicrobiol 200395(6)1161ndash1174

[44] Brandao PF Clapp JP Bull AT Discriminationand taxonomy of geographically diverse strains ofnitrile-metabolizing actinomycetes using chemo-metric and molecular sequencing techniques En-viron Microbiol 20024(5)262ndash276

[45] NCBI Nucleotide Databank httpwwwncbinlmnihgoventrezqueryfcgidb=Nucleotide

[46] Stalker DM McBride KE Cloning and expressionin Escherichia coli of a Klebsiella ozaenae plasmid-borne gene encoding a nitrilase specific for the her-bicide bromoxynil J Bacteriol 1987169(3)955ndash960

[47] Goldstein EJC Lewis RP Martin WJ Edelstein PHInfections caused by Klebsiella ozaenae a changingdisease spectrum J Clin Microbiol 19788413ndash418

[48] Padgette SR Re DB Barry GF et al New weedcontrol opportunities development of soybeanswith a Roundup ReadyTM gene In Duke SOed Herbicide-Resistant Crops Agricultural Envi-ronmental Economic Regulatory and Technical As-pects Boca Raton Fla CRC Lewis 199653ndash84

[49] Farrand SK Van Berkum PB Oger P Agrobac-terium is a definable genus of the family Rhizobi-aceae Int J Syst Evol Microbiol 200353(pt 5)1681ndash1687

[50] Bala A Murphy P Giller KE Distribution and di-versity of rhizobia nodulating agroforestry legumes


in soils from three continents in the tropics MolEcol 200312(4)917ndash929

[51] Paulsen IT Seshadri R Nelson KE et al The Bru-cella suis genome reveals fundamental similaritiesbetween animal and plant pathogens and sym-bionts Proc Natl Acad Sci USA 200299(20)13148ndash13153

[52] Tsolis RM Comparative genome analysis of thealpha-proteobacteria relationships between plantand animal pathogens and host specificity ProcNatl Acad Sci USA 200299(20)12503ndash12505

[53] Alsmark CM Frank AC Karlberg EO et al Thelouse-borne human pathogen Bartonella quintanais a genomic derivative of the zoonotic agent Bar-tonella henselae Proc Natl Acad Sci USA 2004101(26)9716ndash9721

[54] Son DY Sequencing cloning and expression ofCP4EPSPS roundup ready soybean insert Food SciBiotechnol 200312133ndash136

[55] Barry GF Kishore GM Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases USpatent 5 804 425 1998151 pp

[56] Schodel F Curtiss R Salmonellae as oral vaccinecarriers Dev Biol Stand 199584245ndash253

[57] Stocker BAD Aromatic-dependent salmonella asanti-bacterial vaccines and as presenters of het-erologous antigens or of DNA encoding them JBiotechnol 200083(1-2)45ndash50

[58] Marsden MJ Vaughan LM Fitzpatrick RM Fos-ter TJ Secombes CJ Potency testing of a live ge-netically attenuated vaccine for salmonids Vaccine199816(11-12)1087ndash1094

[59] Cooke McGraw L First genetically engineered vac-cine for shipping fever Agricultural Research Maga-zine 199846(12)20

[60] Australian Poultry CRC Towards Rapid Regis-tration of a Live Attenuated Pasteurella multocidaVaccine Armidale AustraliaAustralian Poultry Co-operative Research Center 2004 Tech Rep 03012httpwww1poultrycrccomaupagesprojectaspxprojectid=87

[61] Aquaflow Development of a Multi-specific Vac-cine Oostende BelgiumEuropean Network forthe Dissemination of Aquaculture RTD Informa-tion European Aquaculture Society 1998 Techni-cal Leaflet TL1998-025 httpwwwaquafloworghomedefaultasp

[62] Foulongne V Walravens K Bourg G et al Aro-matic compound-dependent Brucella suis is atten-uated in both cultured cells and mouse models In-fect Immun 200169(1)547ndash550

[63] Hong PC Tsolis RM Ficht TA Identification ofgenes required for chronic persistence of Brucellaabortus in mice Infect Immun 200068(7)4102ndash4107

[64] Barry GF Kishore GM Glyphosate tolerant plantsUS patent 5 776 760199873 pp

[65] Carson DB Heitkamp MA Hallas LE Biodegrada-tion of N-phosphonomethyliminodiacetic acid bymicroorganisms from industrial activated sludgeCan J Microbiol 199743(1)97ndash101

[66] Jacob GS Garbow JR Hallas LE Kimack NMKishore GM Schaefer J Metabolism of glyphosatein Pseudomonas sp strain LBr Appl Environ Micro-biol 198854(12)2953ndash2958

[67] Forlani G Mangiagalli A Nielsen E SuardiCM Degradation of the phosphonate herbicideglyphosate in soil evidence for a possible involve-ment of unculturable microorganisms Soil BiolBiochem 199931(7)991ndash997

[68] Dick RE Quinn JP Glyphosate-degrading isolatesfrom environmental samples occurrence and path-ways of degradation Appl Microbiol Biotechnol199543(3)545ndash550

[69] Obojska A Ternan NG Lejczak B Kafarski P Mc-Mullan G Organophosphonate utilization by thethermophile Geobacillus caldoxylosilyticus T20Appl Environ Microbiol 200268(4)2081ndash2084

[70] Bertrand H Plassard C Pinochet X Touraine BNormand P Cleyet-Marel JC Stimulation of theionic transport system in Brassica napus by a plantgrowth-promoting rhizobacterium (Achromobac-ter sp) Can J Microbiol 200046(3)229ndash236

[71] Mayak S Tirosh T Glick BR Plant growth-promoting bacteria confer resistance in tomatoplants to salt stress Plant Physiol Biochem200442(6)565ndash572

[72] Rojas Avelizapa NG Rodriguez Vasquez R En-riquez Villanueva F Martinez Cruz J Poggi VaraldoHM Transformer oil degradation by an indigenousmicroflora isolated from a contaminated soil Re-sources Conservat Recycl 19992715ndash26

[73] Kjaeligr J Olsen P Ullum M Grant R Leachingof glyphosate and amino-methylphosphonic acidfrom Danish agricultural field sites J Environ Qual200534(2)608ndash620

[74] Major WW Grue CE Gardner SC GrassleyJM Concentrations of Glyphosate and AMPAin sediment following operational applications ofRodeo rcopy to control smooth cordgrass in WillapaBay Washington USA Bull Environ Contam Tox-icol 200371912ndash918

[75] Araujo AS Monteiro RT Abarkeli RB Effect ofglyphosate on the microbial activity of two Brazil-ian soils Chemosphere 200352(5)799ndash804

[76] Aisenberg G Rolston KV Safdar A Bacteremiacaused by Achromobacter and Alcaligenes speciesin 46 patients with cancer (1989ndash2003) Cancer2004101(9)2134ndash2140

[77] Gomez-Cerezo J Suarez I Rios JJ et al Achro-mobacter xylosoxidans bacteremia a 10-year anal-ysis of 54 cases Eur J Clin Microbiol Infect Dis200322(6)360ndash363


[78] Lea PJ Joy KW Ramos JL Guerrero MG Theaction of 2-amino-4-(methylphosphinyl)-butanoicacid (phosphinothricin) and its 2-oxo-derivativeon the metabolism of cyanobacteria and higherplants Phytochemistry 1984231ndash6

[79] Omura S Murata M Hanaki H Hinotozawa KOiwa R Tanaka H Phosalacine a new herbicidalantibiotic containing phosphinothricin Fermenta-tion isolation biological activity and mechanismof action J Antibiot (Tokyo) 198437(8)829ndash835

[80] Wehrmann A Van Vliet A Opsomer C Botter-man J Schulz A Thesimilarities of bar and pat geneproducts make them equally applicable for plantengineers Nat Biotechnol 199614(10)1274ndash1278

[81] Thompson CJ Movva NR Tizard R et alCharacterization of the herbicide-resistance genebar from Streptomyces hygroscopicus EMBO J198762519ndash2523

[82] Vinnemeier J Droege-Laser W Pistorius EKBroer I Purification and partial characterizationof the Streptomyces viridochromogenes Tu494phosphinothricin-N-acetyltransferase mediatingresistance to the herbicide phosphinothricin intransgenic plants Z Naturforsch 199550C796ndash805

[83] Kumada Y Anzai H Takano E et al The bialaphosresistance gene (bar) plays a role in both self-defense and bialaphos biosynthesis in Streptomyceshygroscopicus J Antibiot (Tokyo) 198841(12)1838ndash1845

[84] Bedford DJ Lewis CG Buttner MJ Charac-terization of a gene conferring bialaphos resis-tance in Streptomyces coelicolor A3(2) Gene1991104(1)39ndash45

[85] Anzai H Murakami T Imai S Satoh A NagaokaK Thompson CJ Transcriptional regulation ofbialaphos biosynthesis in Streptomyces hygroscop-icus J Bacteriol 1987169(8)3482ndash3488

[86] Strauch E Wohlleben W Puhler A Cloning ofa phosphinothricin N-acetyltransferase gene fromStreptomyces viridochromogenes Tu494 and its ex-pression in Streptomyces lividans and Escherichiacoli Gene 198863(1)65ndash74

[87] Welker NE Campbell LL Unrelatedness of Bacillusamyloliquefaciens and Bacillus subtilis J Bacteriol196794(4)1124ndash1130

[88] Hill C Dodson G Heinemann U et al The struc-tural and sequence homology of a family of micro-bial ribonucleases Trends Biochem Sci 19838364ndash369

[89] Hartley RW Barnase and barstar two small pro-teins to fold and fit together Trends Biochem Sci198914(11)450ndash454

[90] Mariani C Gossele V De Beuckeleer M et al A chi-maeric ribonuclease-inhibitor gene restores fertilityto male sterile plants Nature 1992357384ndash387

[91] Aravind L Koonin EV A natural classification ofribonucleases Methods Enzymol 20013413ndash28

[92] Krajcikova D Hartley RW Sevcik J Isolation andpurification of two novel streptomycete RNase in-hibitors SaI14 and SaI20 and cloning sequencingand expression in Escherichia coli of the gene cod-ing for SaI14 J Bacteriol 1998180(6)1582ndash1585

[93] Krajcikova D Hartley RW A new member of thebacterial ribonuclease inhibitor family from Sac-charopolyspora erythraea FEBS Lett 2004557(1ndash3)164ndash168

[94] Hartley RW Barnase and barstar Expression of itscloned inhibitor permits expression of a cloned ri-bonuclease J Mol Biol 1988202(4)913ndash915

[95] Paddon CJ Hartley RW Cloning sequencing andtranscription of an inactivated copy of Bacillusamyloliquefaciens extracellular ribonuclease (bar-nase) Gene 198540(2-3)231ndash239

[96] Burgess DG Ralston EJ Hanson WG et al A noveltwo-component system for cell lethality and itsuse in engineering nuclear male-sterility in plantsPlant J 200231(1)113ndash125

[97] Hartley RW Homology between prokary-otic and eukaryotic ribonucleases J Mol Evol198015(4)355ndash358

[98] Pedersen PB Bjoslashrnvad ME Rasmussen MD Pe-tersen JN Cytotoxic potential of industrial strainsof Bacillus sp Regul Toxicol Pharmacol 200236(2)155ndash161

[99] EFSA Opinion of the FEEDAP Panel on the Safety ofthe Product ldquoKemzyme W Dryrdquo Regarding the Abilityof Bacillus Strains Involved in the Manufacture toProduce Toxins BrusselsParmaEuropean FoodSafety Authority 2004 Question No EFSA-Q-2004-111 Adopted on 19 November 2004 httpwwwefsaeuintsciencefeedapfeedap opinions701 enhtml

[100] Lazdunski CJ Bouveret E Rigal A Journet LLloubes R Benedetti H Colicin import into Es-cherichia coli cells J Bacteriol 1998180(19)4993ndash5002

[101] Makarov AA Ilinskaya ON Cytotoxic ribonucle-ases molecular weapons and their targets FEBSLett 2003540(1ndash3)15ndash20

[102] Paris S Monod M Diaquin M et al A trans-formant of Aspergillus fumigatus deficient in theantigenic cytotoxin ASPFI FEMS Microbiol Lett1993111(1)31ndash36

[103] Smith JM Davies JE Holden DW Constructionand pathogenicity of Aspergillus fumigatus mu-tants that do not produce the ribotoxin restrictocinMol Microbiol 19939(5)1071ndash1077

[104] Brooks JE Blumenthal RM Gingeras TR The iso-lation and characterization of the Escherichia coliDNA adenine methylase (dam) gene Nucleic AcidsRes 198311(3)837ndash851

[105] Kaper JB Nataro JP Mobley HL Pathogenic Es-cherichia coli Nat Rev Microbiol 20042(2)123ndash140


[106] Low DA Weyand NJ Mahan MJ Roles of DNAadenine methylation in regulating bacterial geneexpression and virulence Infect Immun 200169(12)7197ndash7204

[107] Barras F Marinus MG The great GATC DNAmethylation in E coli Trends Genet 19895(5)139ndash143

[108] Prieto AI Ramos-Morales F Casadesus J Bile-induced DNA damage in Salmonella enterica Ge-netics 2004168(4)1787ndash1794

[109] Pucciarelli MG Prieto AI Casadesus J Garcıa-delPortillo F Envelope instability in DNA adeninemethylase mutants of Salmonella enterica Micro-biology 2002148(pt 4)1171ndash1182

[110] Unger E Betz S Xu R Cigan AM Selection andorientation of adjacent genes influences DAM-mediated male sterility in transformed maizeTransgenic Res 200110(5)409ndash422

[111] Barbeyron T Kean K Forterre P DNA ade-nine methylation of GATC sequences appeared re-cently in the Escherichia coli lineage J Bacteriol1984160(2)586ndash590

[112] Stamm LV Greene SR Barnes NY Bergen HLHardham JM Identification and characterizationof a Treponema pallidum subsp pallidum gene en-coding a DNA adenine methyltransferase FEMSMicrobiol Lett 1997155(1)115ndash119

[113] Schlagman SL Miner Z Feher Z Hattman SThe DNA [adenine-N6]methyltransferase (Dam)of bacteriophage T4 Gene 198873(2)517ndash530

[114] Yang Z Kovar J Kim J et al Identification of com-mon subpopulations of non-sorbitol-fermentingbeta-glucuronidase-negative Escherichia coli O157H7 from bovine production environments andhuman clinical samples Appl Environ Microbiol200470(11)6846ndash6854

[115] Hattman S Wilkinson J Swinton D Schlagman SMacdonald PM Mosig G Common evolutionaryorigin of the phage T4 dam and host Escherichiacoli dam DNA-adenine methyltransferase genes JBacteriol 1985164(2)932ndash937

[116] Allison GE Angeles D Tran-Dinh N Verma NKComplete genomic sequence of SfV a serotype-converting temperate bacteriophage of Shigellaflexneri J Bacteriol 2002184(7)1974ndash1987

[117] Kapfhammer D Blass J Evers S Reidl J Vibriocholerae phage K139 complete genome sequenceand comparative genomics of related phages J Bac-teriol 2002184(23)6592ndash6601

[118] Remedyne Founders of Remedynersquos DAM Antimi-crobial Technology Featured in American Societyof Microbiology News Santa Barbara Calif Rem-edyne 2001 httpwwwremedynecomreleasesrelase7 10 01html

[119] Nowrouzian F Hesselmar B Saalman R et al Es-cherichia coli in infantsrsquo intestinal microflora col-onization rate strain turnover and virulence genecarriage Pediatr Res 200354(1)8ndash14

[120] Klee HJ Hayford MB Kretzmer KA Barry GFKishore GM Control of ethylene synthesis by ex-pression of a bacterial enzyme in transgenic tomatoplants Plant Cell 19913(11)1187ndash1193

[121] Nelson KE Weinel C Paulsen IT et al Com-plete genome sequence and comparative analysisof the metabolically versatile Pseudomonas putidaKT2440 Environ Microbiol 20024(12)799ndash808

[122] Belimov AA Safronova VI Sergeyeva TA et alCharacterization of plant growth promoting rhi-zobacteria isolated from polluted soils and con-taining 1-aminocyclopropane-1-carboxylate deam-inase Can J Microbiol 200147(7)642ndash652

[123] Jia YJ Kakuta Y Sugawara M et al Synthesis anddegradation of 1-aminocyclopropane-1-carboxylicacid by Penicillium citrinum Biosci BiotechnolBiochem 199963(3)542ndash549

[124] Ghosh S Penterman JN Little RD Chavez RGlick BR Three newly isolated plant growth-promoting bacilli facilitate the seedling growth ofcanola Brassica campestris Plant Physiol Biochem200341277ndash281

[125] Ma W Sebestianova SB Sebestian J Burd GIGuinel FC Glick BR Prevalence of 1-aminocyclo-propane-1-carboxylate deaminase in Rhizobiumspp Antonie Van Leeuwenhoek 200383(3)285ndash291

[126] Hontzeas N Zoidakis J Glick BR Abu-OmarMM Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase fromthe rhizobacterium Pseudomonas putida UW4 akey enzyme in bacterial plant growth promotionBiochim Biophys Acta 20041703(1)11ndash19

[127] Wanga C Ramettea A Punjasamarnwong P et alCosmopolitan distribution of phlD-containing di-cotyledonous crop-associated biocontrol pseu-domonads of worldwide origin FEMS MicrobiologyEcology 200137(2)105ndash116

[128] Good X Kellogg JA Wagoner W Langhoff DMatsumura W Bestwick RK Reduced ethylenesynthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase Plant Mol Biol199426(3)781ndash790

[129] Hughes JA Brown LR Ferro AJ Nucleotide se-quence and analysis of the coliphage T3 S-adeno-sylmethionine hydrolase gene and its surroundingribonuclease III processing sites Nucleic Acids Res198715(2)717ndash729

[130] Macintyre G Atwood CV Cupples CG Lower-ing S-adenosylmethionine levels in Escherichia colimodulates C-to-T transition mutations J Bacteriol2001183(3)921ndash927

[131] Posnick LM Samson LD Influence of S-adenosylmethionine pool size on spontaneousmutation dam methylation and cell growth of Es-cherichia coli J Bacteriol 1999181(21)6756ndash6762

[132] Val DL Cronan JE Jr In vivo evidence thatS-adenosylmethionine and fatty acid synthesis


intermediates are the substrates for the LuxI familyof autoinducer synthases J Bacteriol 1998180(10)2644ndash2651

[133] Pajunen MI Kiljunen SJ Soderholm ME SkurnikM Complete genomic sequence of the lytic bac-teriophage φYeO3-12 of Yersinia enterocoliticaserotype O3 J Bacteriol 2001183(6)1928ndash1937

[134] Kiljunen S Vilen H Pajunen M Savilahti HSkurnik M Nonessential genes of phage φYeO3-12 include genes involved in adaptation to growthon Yersinia enterocolitica serotype O3 J Bacteriol2005187(4)1405ndash1414

[135] Pajunen MI Elizondo MR Skurnik M KieleczawaJ Molineux IJ Complete nucleotide sequence andlikely recombinatorial origin of bacteriophage T3 JMol Biol 2002319(5)1115ndash1132

[136] Wilson KJ Hughes SG Jefferson RA The Es-cherichia coli gus operon induction and expressionof the gus operon in E coli and the occurrence anduse of GUS in other bacteria In Gallagher SR edGUS Protocols Using the GUS Gene as a Reporter ofGene Expression San Diego Calif Academic Press19927ndash22

[137] Martin T Wohner R-V Hummel S Willmitzer LFrommer WB The GUS reporter system as a toolto study plant gene expression In Gallagher SR edGUS Protocols Using the GUS Gene as a Reporter ofGene Expression San Diego CalifAcademic Press199223ndash43

[138] Witcher DR Hood EE Peterson D et al Commer-cial production of β-glucuronidase (GUS) a modelsystem for the production of proteins in plants MolBreed 19984301ndash312

[139] Frampton EW Restaino L Methods for Escherichiacoli identification in food water and clinical sam-ples based on beta-glucuronidase detection J ApplBacteriol 199374(3)223ndash233

[140] Ram JL Ritchie RP Fang J Gonzales FS SelegeanJP Sequence-based source tracking of Escherichiacoli based on genetic diversity of β-glucuronidase JEnviron Qual 200433(3)1024ndash1032

[141] Wenzl P Wong L Kwang-won K Jefferson RA Afunctional screen identifies lateral transfer of β-glucuronidase (gus) from bacteria to fungi MolBiol Evol 200522(2)308ndash316

[142] Feng P Lum R Chang GW Identification of uidAgene sequences in β-D-glucuronidase-negative Es-cherichia coli Appl Environ Microbiol 199157(1)320ndash323

[143] Feng P Lampel KA Genetic analysis of uidAexpression in enterohaemorrhagic Escherichiacoli serotype O157H7 Microbiology 1994140(pt8)2101ndash2107

[144] Monday SR Whittam TS Feng PC Genetic andevolutionary analysis of mutations in the gusA genethat cause the absence of beta-glucuronidase ac-tivity in Escherichia coli O157H7 J Infect Dis2001184(7)918ndash921

[145] Dontorou A Papadopoulou C Filioussis G et alIsolation of a rare Escherichia coli O157H7 strainfrom farm animals in Greece Comp Immunol Mi-crobiol Infect Dis 200427(3)201ndash207

[146] Stewart L Oesterle AL Erdan I Griffiss JM WayLW Pathogenesis of pigment gallstones in Westernsocieties the central role of bacteria J GastrointestSurg 20026(6)891ndash904

[147] Kemp JD Sutton DW Hack E Purificationand characterization of the crown gall spe-cific enzyme nopaline synthase Biochemistry197918(17)3755ndash3760

[148] Zhu J Oger PM Schrammeijer B Hooykaas PJFarrand SK Winans SC The bases of crown gall tu-morigenesis J Bacteriol 2000182(14)3885ndash3895

[149] CFIA Determination of the Safety of the CropDevelopment Centrersquos ldquoCDC Triffidrdquo a Flax(Linum usitatissimum L) Variety Tolerant toSoil Residues of Triasulfuron and Metsulfuron-methyl Nepean Plant Biosafety Office PlantProducts Directorate Canadian Food Inspec-tion Agency 1996 Decision Document 98-24httpwwwinspectiongccaenglishplavegbiodddd9824eshtml

[150] Monneuse MO Rouze P Sequence compar-isons between Agrobacterium tumefaciens T-DNA-encoded octopine and nopaline dehydrogenasesand other nucleotide-requiring enzymes struc-tural and evolutionary implications J Mol Evol19872546ndash57

[151] Dairi T Asano Y Cloning nucleotide sequencingand expression of an opine dehydrogenase genefrom Arthrobacter sp strain 1C Appl Environ Mi-crobiol 199561(8)3169ndash3171

[152] Kimura T Nakano T Yamaguchi T et al Com-plementary DNA cloning and molecular evolutionof opine dehydrogenases in some marine inverte-brates Mar Biotechnol (NY) 20046(5)493ndash502

[153] Nester E Wood D Piu L Global analysis ofAgrobacterium-plant interactions In Tsuyumu SLeach JE Shiraishi T Wolpert T eds Genomic andGenetic Analysis of Plant Parasitism and DefenseProceedings of the 9th Japan US Science Seminar onPlant Pathogens Shizuoka Japan November 2003Saint Paul MinnAmerican Phytopathological So-ciety 20051ndash10

[154] Teyssier-Cuvelle S Mougel C Nesme X Direct con-jugal transfers of Ti plasmid to soil microflora MolEcol 19998(8)1273ndash1284

[155] Otten L De Ruffray P Agrobacterium vitis nopa-line Ti plasmid pTiAB4 relationship to other Tiplasmids and T-DNA structure Mol Gen Genet1994245(4)493ndash505

[156] Beegle CC Yamamoto T History of Bacillusthuringiensis Berliner research and developmentCan Entomol 1992124587ndash616

[157] IPCS Bacillus thuringiensis Environmental HealthCriteria 217 Geneva SwitzerlandInternational


Program on Chemical Safety World Health Orga-nization 1999

[158] Crickmore N Zeigler DR Schnepf E et alBacillus thuringiensis Toxin Nomenclature FalmerBrightonUniversity of Sussex 2005 httpwwwbiolssusxacukhomeNeil Crickmore

[159] Geiser M Schweitzer S Grimm C The hypervari-able region in the genes coding for entomopatho-genic crystal proteins of Bacillus thuringiensis nu-cleotide sequence of the kurhd1 gene of subspkurstaki HD1 Gene 198648(1)109ndash118

[160] Adang MJ Staver MJ Rocheleau TA LeightonJ Barker RF Thompson DV Characterized full-length and truncated plasmid clones of the crys-tal protein of Bacillus thuringiensis subsp kurstakiHD-73 and their toxicity to Manduca sexta Gene198536(3)289ndash300

[161] Chambers JA Jelen A Gilbert MP Jany CS John-son TB Gawron-Burke C Isolation and character-ization of a novel insecticidal crystal protein genefrom Bacillus thuringiensis subsp aizawai J Bacte-riol 1991173(13)3966ndash3976

[162] Donovan WP Dankocsik CC Gilbert MP Gawron-Burke MC Groat RG Carlton BC Amino acid se-quence and entomocidal activity of the P2 crystalprotein An insect toxin from Bacillus thuringien-sis var kurstaki [Erratum in J Biol Chem1989264(8)4740] J Biol Chem 1988263(1)5617

[163] Dankocsik C Donovan WP Jany CS Activa-tion of a cryptic crystal protein gene of Bacillusthuringiensis subspecies kurstaki by gene fusionand determination of the crystal protein insecti-cidal specificity Mol Microbiol 19904(12)2087ndash2094

[164] McPherson S Perlak F Fuchs R Marrone P LavrikP Fischhoff D Characterization of the coleopteran-specific protein gene of Bacillus thuringiensis vartenebrionis Biotechnology 1988661ndash66

[165] Donovan WP Rupar MJ Slaney AC Malvar TGawron-Burke MC Johnson TB Characterizationof two genes encoding Bacillus thuringiensis insec-ticidal crystal proteins toxic to Coleoptera speciesAppl Environ Microbiol 199258(12)3921ndash3927

[166] Moellenbeck DJ Peters ML Bing JW et al In-secticidal proteins from Bacillus thuringiensis pro-tect corn from corn rootworms Nat Biotechnol200119(7)668ndash672

[167] Damgaard PH Larsen HD Hansen BM BrescianiJ Jorgensen K Enterotoxin-producing strains ofBacillus thuringiensis isolated from food Lett ApplMicrobiol 199623(3)146ndash150

[168] Ichimatsu T Mizuki E Nishimura K et al Occur-rence of Bacillus thuringiensis in fresh waters ofJapan Curr Microbiol 200040(4)217ndash220

[169] Kaelin P Gadani F Occurrence of Bacillusthuringiensis on cured tobacco leaves Curr Micro-biol 200040(3)205ndash209

[170] Maeda M Mizuki E Nakamura Y Hatano T OhbaM Recovery of Bacillus thuringiensis from ma-rine sediments of Japan Curr Microbiol 200040(6)418ndash422

[171] Crickmore N Zeigler DR Feitelson J et al Revisionof the nomenclature for the Bacillus thuringiensispesticidal crystal proteins Microbiol Mol Biol Rev199862(3)807ndash813

[172] de Maagd RA Bravo A Crickmore N How Bacillusthuringiensis has evolved specific toxins to colonizethe insect world Trends Genet 200117(4)193ndash199

[173] Noteborn HPJM Bienenmann-Ploum ME vanden Berg JHJ et al Safety assessment of the Bacillusthuringiensis insecticidal crystal protein CRYIA(b)expressed in transgenic tomatoes In Engel K-HTakeoka GR Teranishi R eds Genetically ModifiedFoodsmdashSafety Aspects vol 605 of ACS SymposiumSeries Washington DC American Chemical Soci-ety 1995134ndash147

[174] Betz FS Hammond BG Fuchs RL Safety and ad-vantages of Bacillus thuringiensis-protected plantsto control insect pests Regul Toxicol Pharmacol200032(2)156ndash173

[175] Porcar M Juarez-Perez V PCR-based identificationof Bacillus thuringiensis pesticidal crystal genesFEMS Microbiol Rev 200326(5)419ndash432

[176] Wang J Boets A Van Rie J Ren G Characterizationof cry1 cry2 and cry9 genes in Bacillus thuringien-sis isolates from China J Invertebr Pathol 200382(1)63ndash71

[177] Chen FC Tsai MC Peng CH Chak KF Dissectionof cry gene profiles of Bacillus thuringiensis isolatesin Taiwan Curr Microbiol 200448(4)270ndash275

[178] Uribe D Martinez W Ceron J Distribution anddiversity of cry genes in native strains of Bacillusthuringiensis obtained from different ecosystemsfrom Colombia J Invertebr Pathol 200382(2)119ndash127

[179] Barloy F Lecadet MM Delecluse A Distribu-tion of clostridial cry-like genes among Bacillusthuringiensis and Clostridium strains Curr Micro-biol 199836(4)232ndash237

[180] Yokoyama T Tanaka M Hasegawa M Novel crygene from Paenibacillus lentimorbus strain Se-madara inhibits ingestion and promotes insectici-dal activity in Anomala cuprea larvae J InvertebrPathol 200485(1)25ndash32

[181] Zhang J Hodgman TC Krieger L SchnetterW Schairer HU Cloning and analysis of thefirst cry gene from Bacillus popilliae J Bacteriol1997179(13)4336ndash4341

[182] Baumann L Broadwell AH Baumann P Sequenceanalysis of the mosquitocidal toxin genes en-coding 514- and 419-kilodalton proteins fromBacillus sphaericus 2362 and 2297 J Bacteriol1988170(5)2045ndash2050


[183] Ellis RT Stockhoff BA Stamp L et al Novel Bacil-lus thuringiensis binary insecticidal crystal proteinsactive on western corn rootworm Diabrotica vir-gifera virgifera LeConte Appl Environ Microbiol200268(3)1137ndash1145

[184] Hernandez CS Andrew R Bel Y Ferre J Isola-tion and toxicity of Bacillus thuringiensis frompotato-growing areas in Bolivia J Invertebr Pathol200588(1)8ndash16

[185] Swiecicka I Mahillon J The clonal structureof Bacillus thuringiensis isolates from north-eastPoland does not correlate with their cry gene di-versity Environ Microbiol 20057(1)34ndash39

[186] Vilas-Boas GT Lemos MVF Diversity of crygenes and genetic characterization of Bacillusthuringiensis isolated from Brazil Can J Microbiol200450(8)605ndash613

[187] Carlson CR Kolsto AB A complete physical mapof a Bacillus thuringiensis chromosome J Bacteriol1993175(4)1053ndash1060

[188] Hu X Hansen BM Eilenberg J et al Conjuga-tive transfer stability and expression of a plasmidencoding a cry1Ac gene in Bacillus cereus groupstrains FEMS Microbiol Lett 2004231(1)45ndash52

[189] Vilas-Boas GFLT Vilas-Boas LA Lereclus DArantes OMN Bacillus thuringiensis conjugationunder environmental conditions FEMS MicrobiolEcol 199825369ndash374

[190] Schnepf E Crickmore N Van Rie J et al Bacillusthuringiensis and its pesticidal crystal proteins Mi-crobiol Mol Biol Rev 199862(3)775ndash806

[191] Helgason E Oslashkstad OA Caugant DA et alBacillus anthracis Bacillus cereus and Bacillusthuringiensismdashone species on the basis of geneticevidence Appl Environ Microbiol 200066(6)2627ndash2630

[192] Siegel JP The mammalian safety of Bacillusthuringiensis-based insecticides J Invertebr Pathol200177(1)13ndash21

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

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Enzyme Research



Microbiology


A number of studies and reviews have focused onthe transfer of genes from GM plants to soil- and plant-related microorganisms (eg [6 7 8 9 10]) The results ofsome of these studies indicated that transgenes from GMcrops are most likely transferred if they contain sufficientsimilarity with the corresponding genes in the recipientbecause homologous recombination is the most probablemechanism of transfer (eg [11]) It has however recentlybeen observed that under conditions of simulated light-ning which might cause electroporation of recipient cellsDNA could be transferred to isolated soil microbes [12]

Other factors that are important for transformationwith DNA are the natural or induced competence of therecipient microorganisms such as the natural competenceof Campylobacter species Some microorganisms suchas Salmonella typhimurium have mismatch repair sys-tems that form a barrier for recombination between evenhighly similar sequences (eg reviewed for Salmonella by[13]) Some bacteria can develop naturalchemical com-petence under certain environmental conditions [6]

In addition the transgenes in plants may have beenlinked to promoters with optimal activity in the cellsof plants Sequences promoting expression in eukaryotesand prokaryotes are generally known to be different Nev-ertheless Jacob et al [14] observed that eukaryotic pro-moters from for example the cauliflower mosaic viruspotato and tobacco triggered expression of inserted re-porter genes in five eubacterial species In addition Lewinet al [15] observed that random sequences from yeast mayexhibit promoter activity in bacteria

Jonas et al [16] estimated the potential dietary in-take of transgenic DNA present in food The estimatedintake of transgenic DNA from maize soya and pota-toes amounted to approximately 038 microg per day assum-ing that only GM crops are consumed This is about000006 of the total DNA intake of 06 g per day

Still this is a ldquoworst-caserdquo scenario as DNA is prone todegradation in food matrices or during food processing(reviewed in [16]) On the other hand also the protectionof DNA against the activity of DNase I in for instancefermented sausages has been described [17]

In addition the integrity of the DNA is counteredby the activity of DNA degrading enzymes released bythe pancreas and intestinal epithelial cells during its pas-sage through the gastrointestinal tract Nevertheless it hasbeen shown that DNA can persist in the gastrointestinaltract [16 18 19] and consequently be available for up-take by intestinal competent bacteria For example thesurvival of cp4 epsps transgenes in the small intestines ofhuman volunteers who consumed a GM soy product hasrecently been demonstrated in a study by Netherwood etal [20] so there is a chance for exposure of intestinal mi-croorganisms to free transgenic DNA However the pref-erential site for transformation of competent bacteria isprobably in the colon This is because the colon containsthe largest population of bacteria within the gastrointesti-nal tract Whilst the amount of DNA reaching the colon

may only be a fraction of what is consumed DNA is lessrapidly degraded there For example ex vivo and in vivorat models simulating human gut conditions showed thatDNA is rapidly degraded in the upper part of the gastroin-testinal tract but to a lesser degree in the lower part [19]

Besides the integrity of DNA the transformabilitythat is the likelihood that this DNA will transform bac-teria in food or in the gut should be taken into accountIn foods transformation of Escherichia coli by plasmidtransfer was proven to occur in all 12 food products in-vestigated [21] In addition transfer of DNA to Strepto-coccus gordonii was also proven in homogenates of bloodsausages by marker rescue experiments [22] Kharazmi etal [23] observed the transfer of nptII kanamycin resis-tance marker gene from transgenic potatoes to Bacillussubtilis with defective nptII by homologous recombina-tion under in vitro conditions Based upon the observedfrequencies of transfer these authors calculated the prob-ability of the transfer of the intact nptII gene from con-sumed transgenic potatoes to microbes Because markerrescue by homologous recombination is the most proba-ble mechanism for gene transfer these calculations can beconsidered a ldquoworst-caserdquo scenario in view of other possi-ble mechanisms of horizontal transfer of transgenes fromGM crops

Potential health effectsCurrently the focus of the assessment of potential

transfer from GMOs is on antibiotic-resistance markergenes as for example in the previously mentioned FAOWHO Codex Alimentarius guidelines In a more gen-eral sense antibiotic resistance among microbial hu-man pathogens is currently a top priority issue inhealth care and research The horizontal gene transferof antibiotic-resistance genes between microorganismshas been important for the development of antibiotic-resistant pathogens

In modern biotechnology some antibiotic-resistancemarker genes are used for the successful molecularcloning in bacteria and plants because they enable growthon antibiotic-containing media after the genetic modi-fication process These marker genes are therefore use-ful in the development phase but have no function inthe final product An example of an antibiotic-resistancegene that is present in many commercial GM crops is thekanamycin-resistance gene nptII encoding the neomycinphosphotransferase II enzyme The use of this gene hasbeen considered to be safe based upon the widespread oc-currence of kanamycin resistance in microorganisms inthe environment the low clinical relevance of kanamycinand the low likelihood of transfer to microorganisms af-ter consumption of GM products containing nptII (eg re-viewed by [5])

For a more elaborate discussion on mechanisms ofgene transfer antibiotic-resistance genes and horizontalgene transfer from GM crops as well as a classification ofantibiotic-resistance markers based upon their risk char-acteristics we refer to a recent review by the working





Scope of this study
































Microbial source


Natural function





Natural prevalence















































Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Scope of this study
































Microbial source


Natural function





Natural prevalence















































Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Microbial source


Natural function





Natural prevalence















































Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Microbial source


Natural function





Natural prevalence















































Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued













AY698420d AY698422d

AY698424d AY698425d

AY698502d AY698504d

AY698506d AY698509d

AY698510d AY698511d


AY698428d AY698430d

AY698431d AY698434d

AY698435d AY698473d

AY698480d


AY698414d AY698416d

AY698432d AY698433d

AY698449d AY698450d

AY698451d AY698452d

AY698484d AY698485d

AY698486d AY698487d

AY698488d AY698489d

AY698490d AY698492d

AY698493d


AY698423d AY698513d

AY698514d AY698515d




crye No













Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Conclusion



Microbial source


Natural function




Natural prevalence

























Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Conclusion





Natural function




Natural prevalence












Conclusion





Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Natural function




Natural prevalence













Conclusion




Natural function






Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Natural prevalence
















Conclusion






Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Natural function






Natural prevalence



















Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Conclusion



Microbial source


Natural function






Natural prevalence











Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Conclusion



Microbial source


Natural function






Natural prevalence













Conclusion




Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Microbial source


Natural function





Natural prevalence

















Conclusion





Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Microbial source


Natural function





Natural prevalence













Conclusion


Cry proteins

Microbial source







Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Natural function





Natural prevalence




















Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Conclusion


DISCUSSION








ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



ACKNOWLEDGMENTS


REFERENCES




















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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