Genome Sequence of a Food Spoilage Lactic Acid Bacterium, Leuconostoc gasicomitatum LMG 18811T, in Association with Specific Spoilage Reactions

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2011, p. 4344–4351 Vol. 77, No. 130099-2240/11/$12.00 doi:10.1128/AEM.00102-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Genome Sequence of a Food Spoilage Lactic Acid Bacterium,Leuconostoc gasicomitatum LMG 18811T, in Association

with Specific Spoilage Reactions�†Per Johansson,1‡ Lars Paulin,2‡ Elina Sade,1 Noora Salovuori,2 Edward R. Alatalo,2

K. Johanna Bjorkroth,1‡* and Petri Auvinen2‡Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland,1 and

Institute of Biotechnology, University of Helsinki, Helsinki, Finland2

Received 18 January 2011/Accepted 1 May 2011

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modi-fied-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas andslime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats haveespecially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sau-sages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to studyits phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was donewithin L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostocmesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG18811T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%.It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. Asinteresting features associated with the growth and spoilage potential, LMG 18811T possesses utilizationstrategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electrontransport chain requiring only externally supplied heme for respiration. In respect of the documented specificspoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formationwere recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, buttheir functionality and role in food spoilage and processing environment contamination need further study.

Industrially manufactured food must have a reasonably longshelf life due to the production chain, involving logistics andretail sale before the domestic storage and consumption of aproduct. CO2 in modified-atmosphere packaging (MAP) andrefrigerated temperatures are two main extrinsic hurdles usedby the food industry. They create negative selective pressure toaerobic Gram-negative spoilage bacteria. Under these circum-stances, psychrotrophic, i.e., cold-tolerant, lactic acid bacteria(LAB) prevail in nutrient-rich foods, such as meat (4, 18, 21).Compared to the aerobic Gram-negative spoilage bacteria, thestationary growth phase associated with the production of sen-sory changes is reached more slowly by spoilage LAB. In ad-dition, the end products of LAB carbohydrate fermentationare not sensed as unpleasant, as in protein or amino aciddegradation. Therefore, the shift from aerobic Gram-negativebacteria to LAB is preferred.

Despite the generally moderate role of psychrotrophic LABin spoilage, they are still spoilage organisms. The growth rate

of psychrotrophic LAB can usually be predicted and the shelflife can be estimated with adequate accuracy. However, somepsychrotrophic LAB may cause considerable hardship to thefood industry. Leuconostoc gasicomitatum (3) is a LAB thatwas first encountered causing a spoilage problem of MAP,tomato-marinated, raw broiler meat strips in 1997. The pack-ages already showed clear bulging due to CO2 formation in 5days, even though the manufacturer-defined shelf life was ex-pected to be 14 days. Since the first spoilage problem, L.gasicomitatum has been shown to form slime and CO2 in aceticacid-preserved herring (24), cause greening and off odor tovalue-added MAP, raw beef steaks (42, 45), and cause slimyspoilage and bulging of cooked vegetable sausages packagedunder vacuum (45). In addition, this species has been docu-mented to prevail in MAP, marinated broiler meat strips (38)and minced meat (29). Table 1 shows reported L. gasicomita-tum spoilage with descriptive spoilage characteristics verifiedin food challenge tests. However, none of these have yet beenconfirmed through expression or other types of molecular anal-yses in food in situ.

The genomic sequencing project of L. gasicomitatum LMG18811T was prompted by a need to understand the growth andspoilage potentials of L. gasicomitatum, to study its phylogeny,and to be able to knock out and overexpress genes. This is thefirst complete genome sequence of a psychrotrophic foodspoilage LAB, and it is presented in this study with particularemphasis on the food spoilage capabilities. Comparativegenomic analysis was also carried out with the three other

* Corresponding author. Mailing address: Department of Food Hy-giene and Environmental Health, University of Helsinki, P.O. Box 6,Agnes Sjobergin Katu 2, 00014 Helsinki University, Helsinki, Finland.Phone: 358505976555. Fax: 358919157101. E-mail: [email protected].

‡ These authors share authorships for the first and last authorpositions.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 13 May 2011.

4344

Leuconostoc species with completely assembled genomes,L. citreum KM20 (20), L. mesenteroides ATCC 8293T (25), andL. kimchii IMSNU11154 (30). L. gasicomitatum diverges fromthe three other leuconostocs, while it has not been used as astarter culture in food fermentations.

MATERIALS AND METHODS

Origin, culturing, and maintenance of LMG 18811T. L. gasicomitatum LMG18811T originates from a study identifying a spoilage LAB population associatedwith the rapid gaseous spoilage of MAP, marinated broiler meat (3). In afollow-up study (44) dealing with isolates recovered from foods from 1997 to2007, strain LMG 18811T was shown to belong to genotype 4, which has repeat-edly been detected in the products of two large-scale poultry processing plants.

L. mesenteroides type strain DSM 20343T (ATCC 8293T) was also used in thegrowth test. Unless otherwise stated, cultures were grown aerobically in MRSbroth (Difco Laboratories, Detroit, MI) or anaerobically on MRS agar (Oxoid,Basingstoke, United Kingdom) at 25°C and were maintained at �72°C in MRSbroth.

Phenotypic analyses and growth in hemin-containing medium. Table 1 showsthe main carbon sources associated with the foods affected by L. gasicomitatumspoilage. To depict the variety of food-associated carbon and nitrogen sources inmore detail and complement the previous analyses (3), phenotype MicroArraysPM1 to PM20 were purchased from Biolog Inc. (Hayward, CA). Table S1 in thesupplemental material lists the most relevant substrates for this study.

L. gasicomitatum LMG 18811T and L. mesenteroides DSM 20343T (ATCC8293T) were grown (three experimental replicates) in MRS broth with andwithout hemin (in dimethyl sulfoxide [DMSO]; Sigma-Aldrich) supplementation(2 �g/ml). In medium without hemin, an equivalent amount of DMSO wasadded. Growth was tested under aerobic, anaerobic, and high-oxygen MAP (20%CO2 and 80% O2) atmospheres. Cultures grown under aerobic and high-oxygenMAP atmospheres were shaken at 250 rpm, and cultures grown under anaerobicconditions were left stationary. All cultures were grown at 25°C for 48 h, andgrowth was measured as the optical density (OD) at 600 nm. Microscopy wasdone to check cell size and culture density.

DNA isolation, sequencing, and assembly. DNA was isolated using a modified(2) method of Pitcher et al. (33), and the genomic DNA was mechanicallysheared with a needle. Fosmid libraries were constructed in Copy ControlpCC1FOS vector (Epicentre). Plasmid libraries of several insert sizes (2, 4, 6, and10 kb) were constructed in the pZEro 2 vector (Invitrogen). Fosmid ends andplasmids were sequenced using an ABI 3730 sequencer and BigDye chemistry(Applied Biosystems). In total, 41,549 reads (ca. 8 times) were obtained. Contigorder and gaps were filled by PCR from genomic DNA and direct sequencing ofthe fragments, linker PCR, and using in vitro Mu transposition of appropriateclones. Genome sequences were quality checked using the Phred program andassembled with the Phrap program, followed by editing by the Gap4 program inthe Staden package (36).

Prediction of genes and annotation. The completed L. gasicomitatum se-quence was annotated using the Manatee program (http://manatee.sourceforge.net). Open reading frame (ORF) sequences were determined using the Easy-Gene (28) and Glimmer (11) programs, and the predicted ORFs were alsomanually reviewed and alterations were made when appropriate on the basis ofthe presence of potential ribosomal binding sites, sequence alignments, andavailable literature data. Details about bioinformatic annotations are presentedin File S1 in the supplemental material.

RESULTS

Genome and general aspects related to growth in food. Ta-ble 2 shows the main properties of the genome of L. gasicomi-tatum LMG 18811T (GenBank accession no. FN822744). Thegenome is plasmid free and contains a 1,954,080-bp circularchromosome with an average GC content of 36.7%. Two pro-phages which are not located within the operons of interest inrespect to spoilage reactions were detected. Figure 1 shows thegenome map of L. gasicomitatum LMG 18811T colored accord-ing to the Automated Resource Classifier (ARC) classificationbased on the gene annotation. The proteome is presented

TA

BL

E1.

Reported

Leuconostoc

gasicomitatum

spoilagew

ithspoilage

characteristicsand

challengetest

outcomes

Type

offood

Food

productPackage

type

Spoilagecharacteristics

Challenge

outcome

aR

eferenceSensory

defectsM

aincarbohydrate(s)

Spoilage-associated

endproducts

Fresh

meat

Marinated

broilerm

eatM

odifiedatm

osphere(80%

CO

2 ,20%N

2 )G

aseousspoilage,pungent

offodor

Glucose

fromm

arinade,m

eat-derivedglucose

andribose

CO

2 ,aceticacid

Not

challengedB

jorkrothet

al.(3)

Moisture

enhancedand

marinated

beefM

odifiedatm

osphere(70%

O2 ,30%

CO

2 )G

reening,butteryoff

odorG

lucosefrom

marinade,

meat-derived

glucoseand

ribose

H2 O

2 ,diacetylB

eefchallenged,m

anifestedgreening

andbuttery

offodor

Vihavainen

andB

jorkroth(42

)

Marinated

andunprocessed

beef,pork,and

broiler

Modified

atmosphere

(0-70%

O2 ,20-80%

CO

2 ,20-80%

N2 )

Some

packagesw

ithbuttery

andother

offodors

Glucose

fromm

arinade,m

eat-derivedglucose

andribose

Diacetyl,othervolatiles

Not

challengedV

ihavainenand

Bjorkroth

(44)

Fish

productA

ceticacid-preserved

herringPlastic

containerSlim

e,gaseousspoilage

Sucrosefrom

marinade

Dextran,C

O2

Herring

challenged,showed

gasform

ationverified

tobe

CO

2

Lyhs

etal.(24)

Other

Cooked

vegetablesausages

Vacuum

Slime,gaseous

spoilageand

souroff

odorG

lucose,sucroseD

extran,CO

2 ,lactic

acid,acetic

acid

Vegetable

sausageschallenged,

showed

gasand

slime

formation

Vihavainen

etal.(45)

aIn

achallenge

test,thefood

inquestion

was

spikedw

ithL

.gasicomitatum

ina

controlledexperim

entpublished

inthe

indicatedreference.

VOL. 77, 2011 GENOME SEQUENCE OF LEUCONOSTOC GASICOMITATUM LMG 18811T 4345

according to clusters of orthologous groups categories in TableS2 in the supplemental material.

Figure 2 shows a Venn diagram comparing the genome of L.gasicomitatum LMG 18811T to the three other publicly avail-able Leuconostoc genomes: those of L. citreum KM20 (20), L.mesenteroides ATCC 8293T (25), and L. kimchii IMSNU11154(30). The unique genes for each of the four species are listedin Table S3 in the supplemental material. Like the other leu-conostocs, LMG 18811T has a wide set of genes involved in theuptake of sugars, citrate, and amino acids. The genome in-cludes the genes for the phosphoketolase pathway and threealternative pathways for pyruvate utilization by lactate dehy-drogenase, pyruvate dehydrogenase, and �-acetolactate syn-thase. Compared to L. mesenteroides ATCC 8293T, it has fewerpathways involved in the biosynthesis of amino acids, vitamins,and cofactors (see Table S4 in the supplemental material).Pyruvate utilization is a hub for many of the spoilage reactions,and Table 3 summarizes the enzymes and coding genes attrib-uted to the formation of spoilage compounds.

Utilization of ribose. The majority of LAB does not utilizeribose, which is a pentose of interest in plant- and meat-de-rived foods. Within the genus Leuconostoc, the species L. car-nosum, L. gasicomitatum, L. gelidum, L. inhae, and L. kimchii,which belong to the same 16S rRNA gene-based phylogeneticbranch, can utilize ribose (14). Most ribose-utilizing LABtransport it via an H� symporter, e.g., RbsU of L. sakei (37). L.gasicomitatum LMG 18811T and L. kimchii IMSNU11154 havethe ribose ABC transporter RbsDACB (46) (LEGAS_0026 toLEGAS_0029 in L. gasicomitatum) generally more common inthe bacterial domain. In the genomes of L. mesenteroidesATCC 8293T and L. citreum KM20, genes do not exist eitherfor the ribose ABC transporter or for the RbsU symporter. Inthe pentose phosphate pathway, the enzyme ribose 5-phos-phate isomerase A (RpiA, EC 5.3.1.6) plays an important rolein the branching between the pentose phosphate pathwayand the nucleotide pathways. All four sequenced Leucono-stoc genomes contain multiple rpiA genes (LEGAS_0278,LEGAS_0031, and LEGAS_1232 in L. gasicomitatum), butthe function and expression of them are unknown.

Salvage and utilization of nucleotides, nucleosides, andnucleobases. All four fully assembled Leuconostoc genomesshow the ability for de novo synthesis of purines and pyrimi-dines. They do not have the genes encoding nucleotide phos-phorylases or phosphopentomutase (deoB) for nucleotide

salvage, nor do they have deoxyriboaldolase (deoC) for deg-radation of the pentose moiety of the nucleosides. They do,however, have ntd (LEGAS_0737), encoding N-deoxyribosyl-transferase, which catalyzes the exchange reaction of thenucleobase of deoxyribonucleoside to salvage the deoxyribosemoiety and convert between the different deoxyribonucleo-sides (8). L. gasicomitatum LMG 18811T can use external nu-cleotides and nucleosides as both a carbon and an energysource; and the amino group of nucleotides and free nucleo-bases can be utilized as a nitrogen source (see Table S1 in thesupplemental material), or they can be salvaged and rescuedfor nucleotide synthesis. Both guanosine and xanthine, butnot adenosine or cytidine, can be used as nitrogen sources,whereas inosine, uridine, and adenosine, but not thymidine,can be utilized as carbon sources (see Table S1 in the supple-mental material). 2�-Deoxyadenosine and 2-deoxyribose canalso be utilized as carbon sources (see Table S1 in the supple-mental material), although it is not clear how. The deoQKPXgenes, for the uptake and utilization of 2-deoxyribose in otherspecies (9), are not found in Leuconostoc genomes. Instead,genes for nucleotidases dephosphorylating the nucleotides tothe corresponding nucleoside exist (L. gasicomitatum LEGAS_1431and LEGAS_0848), as do genes for a nucleoside permease,nupC (LEGAS_0024), and rnsACD, encoding a nucleosideABC transporter (LEGAS_1844 to LEGAS_1847) with twoseparate nucleoside-binding subunits, rnsB1 and rnsB2(LEGAS_1460 and LEGAS_1179), enabling transportation ofnucleosides into the cell. Genes for three transporters ofnucleobases exist: a uracil transporter, pyrP (LEGAS_1771),and two guanine/hypoxanthine transporters, pbuG1 and pbuG2(LEGAS_1320 and LEGAS_0450). Ribonucleosides can alsobe hydrolased by the ribonucleoside hydrolases (rihA1, rihA2,rihB, and rihC, corresponding to LEGAS_0022, LEGAS_1534,LEGAS_0456, and LEGAS_0023, respectively), to generatefree nucleobases and ribose. The ribose formed can subse-quently be fed into the pentose phosphate pathway. Figure 3shows salvage and catabolic pathways for nucleosides inL. gasicomitatum LMG 18811T.

Citrate metabolism and buttery off odor. L. gasicomitatum18811T can utilize citrate. It has a citIMCDEFGXRP citratelocus (LEGAS_0211 to LEGAS_0219), which encodes the en-zymes necessary for the uptake and conversion of citrate topyruvate. The genes for the diacetyl/acetoin pathway are pres-ent. This pathway consumes pyruvate, forming �-acetolactateby the catabolic �-acetolactate synthase (LEGAS_0526).Under aerobic circumstances, �-acetolactate may be decar-boxylated to acetoin, either via diacetyl, by a nonenzymaticdecarboxylative oxidation followed by an NAD(P)H-depen-dent reduction to acetoin by diacetyl reductase (LEGAS_0209,LEGAS_1299), or directly to acetoin by acetolactate decarbox-ylase (LEGAS_1346) (Fig. 1). Acetoin and diacetyl can also beformed from the amino acid aspartate in the presence of �-ke-toglutarate (22), but leuconostocs do not have the glutamatedehydrogenase, which can convert glutamate to �-ketoglutarateas in a few other LAB species (41). Instead, L. gasicomitatum18811T has transporters for both aspartate (LEGAS_1791) and�-ketoglutarate (LEGAS_1138), and in the presence of �-ke-toglutarate, the aspartate aminotransferase (LEGAS_1168)may convert aspartate to oxaloacetate and glutamate. The ox-aloacetate can enter the diacetyl/acetoin pathway via the last

TABLE 2. Main properties of the genome of Leuconostocgasicomitatum 18811T

Main property Value

Genome size (bp) ............................................................ 1,954,080No. of protein-encoding genes ....................................... 1,913No. (%) of genes with a functional annotationa.......... 1,566 (81)No. of putative pseudogenes .......................................... 12No. of plasmids ................................................................ 0No. of rRNA operons ..................................................... 4No. of tRNA genes..........................................................67 (�1 pseudo)No. of tmRNAs................................................................ 1% GC content .................................................................. 37% coding efficiency .......................................................... 87Avg gene size (bp) ........................................................... 890

a Genes not containing the word “hypothetical” in the annotation.

4346 JOHANSSON ET AL. APPL. ENVIRON. MICROBIOL.

enzyme of the citrate pathway oxaloacetate decarboxylase(LEGAS_0212). Acetoin can in turn be converted to 2,3-bu-tanediol by 2,3-butanediol dehydrogenase (LEGAS_1018).

Enhanced growth when heme and O2 are available and thegenes associated with the electron transport chain. All fourLeuconostoc genomes possess genes encoding cytochrome bdterminal oxidase and for synthesizing menaquinone. However,unlike L. mesenteroides DSM 20343T, L. gasicomitatum LMG18811T has a functional electron transport requiring only ex-ternally supplied heme for respiration. The presence of heme

increased the produced biomass by �50% under aerobic andhigh-oxygen MAP cultivation, while the addition of hemehad no effect on anaerobically growing cells (Fig. 4). Cellsize did not alter between the experiments. The nonfunc-tional cytochromes of L. mesenteroides ATCC 8293T werealso reported by Brooijmans et al. (5). The L. gasicomitatumLMG 18811T genome encodes a cytochrome bd terminaloxidase (LEGAS_1333 and LEGAS_1334), an NADH de-hydrogenase (LEGAS_1702), and the entity related to theability to synthesize menaquinone in eight enzymatic steps

FIG. 1. Genome map of Leuconostoc gasicomitatum LMG 18811T with the genes colored according to the ARC classification on the basis ofthe gene annotation. The two outer rings denote genes on the forward and reverse strands, respectively. The following four rings inwards representthe positions of complete and partial prophages (red), the positions of rRNA (blue) and tRNA (green) genes, percent GC plot (gray), and GCskew ([G � C]/[G � C]), respectively.

VOL. 77, 2011 GENOME SEQUENCE OF LEUCONOSTOC GASICOMITATUM LMG 18811T 4347

(LEGAS_0426, LEGAS_0912 to LEGAS_0915, LEGAS_1879and LEGAS_1880, and LEGAS_1895).

Genes associated with H2O2 production and meat greening.Of the enzymes known to generate hydrogen peroxide in LAB,L. gasicomitatum LMG 18811T has only the genes for pyruvateoxidase (poxB, LEGAS_1053), NADH oxidase (nox, LEGAS_0926),and two unknown NADH:flavin oxidoreductase/NADH oxi-dases (LEGAS_0056 and LEGAS_1753). The NADH oxidaseslikely produce water and not hydrogen peroxide as an endproduct. For protection against peroxide, a capability forthree different peroxidases, thioredoxin peroxidase (tpx,LEGAS_0306), glutathione peroxidase (bsaA, LEGAS_1017),and heme-containing Dyp-type peroxidase (LEGAS_1694), ex-ists, whereas no potential for catalase production was detected.

Genes encoding EPSs, adhesion, and mucus binding. L.gasicomitatum LMG 18811T has genes for two dextransucrases:epsA (LEGAS_0699) is part of a large exopolysaccharide(EPS) cluster, while dsrA (LEGAS_1012) is located as a singlegene in the chromosome. Three genes encoding proteins con-taining putative LPXTG anchors were detected. LEGAS_0414

encodes a putative mucus binding protein with unknown func-tion. Orthologs were also detected in the genomes of L. mes-enteroides and L. citreum (plasmid encoded). LEGAS_0537encodes Srr-2, a serine-rich protein. The serine-rich domainshows homology to the platelet-binding protein GspB of strep-tococci (34), but since it is missing a large portion of thenonrepeat region, the function cannot be predicted. The genessecY2, asp1 to asp3, secA2, nss, and gftAB were located in thesame locus with LEGAS_0537. They all are required for thesecretion and glycosylation of Srr-2 (39). LEGAS_1063 is partof an intercellular adhesion locus (ica) ABC, where it encodesthe putative collagen adhesion protein IcaC. The ica locus hasbeen shown to be required for biofilm formation in Staphylo-coccus aureus (10) and among LAB is otherwise found in onlya few Lactococcus species (35). No orthologs of LEGAS_0537or LEGAS_1063 are found in other Leuconostoc genomes (seeTable S3 in the supplemental material).

DISCUSSION

An unexpected story related to a novel food spoilage organ-ism in Finland has been seen over the last 14 years. The firstincident was considered to be related to the use of a specifictomato-based marinade (3), but the following years haveshown that this species is persisting (44) and causing spoilagein many types of cold-stored MAP foods of several manufac-turers in Finland.

An ability to grow well on MAP meat with no added carbo-hydrates (29, 44) is interesting, while this species is not able toobtain energy from proteinaceous substrates, lactate, or fattyacids. It has the genes required for energetic catabolism ofnucleosides, and it also grows well on adenosine and inosine.Nucleosides, particularly inosine, are abundant in meat, and ifglucose is exhausted, they provide an alternative source ofenergy. Differing from Lactobacillus sakei 23K, which is con-sidered a meat ecosystem-adapted LAB (6, 7), L. gasicomita-tum LMG 18811T cannot release amino acids from meat pro-teins or utilize arginine as an energy source. Bearing foodsafety aspects in mind, the genome analysis confirms the high-pressure liquid chromatography determinations (27) that themeat-derived amino acids are not decarboxylated as biogenic

FIG. 2. Venn diagram showing the distribution of orthologous re-lationships of genes between four Leuconostoc species.

TABLE 3. Enzymes and coding genes in genome of L. gasicomitatum LMG 18811T involved in formation of spoilage compounds

Spoilage compound Enzyme Gene(s) Locus tag

Acetate Acetate kinase ackA1, ackA2 LEGAS_1085 LEGAS_1559Citrate lyase complex citCDEF LEGAS_0213 to LEGAS_0216N-Acetylglucosamine-6-phosphate deacetylase nagA LEGAS_0472

CO2 Acetolactate decarboxylase alsD LEGAS_1346Oxaloacetate decarboxylase citM LEGAS_02126-Phosphogluconate dehydrogenase gnd1, gnd2 LEGAS_1343 LEGAS_0931Pyruvate dehydrogenase complex pdhABCD LEGAS_1381 to LEGAS_1378Pyruvate oxidase poxB LEGAS_1053

Diacetyl Acetolactate synthase alsS LEGAS_0526H2O2 Pyruvate oxidase poxB LEGAS_1053Slime Dextransucrase dsrA LEGAS_1012

Protein cluster related to formation of an EPS withunknown structure

eps gene cluster LEGAS_0699 to LEGAS_0710

4348 JOHANSSON ET AL. APPL. ENVIRON. MICROBIOL.

amines. Few leuconostocs (17, 32) are capable of formingbiogenic amine from tyrosine.

Related to the metabolism of pentoses or citrate, L. gasi-comitatum LMG 18811T has the central genes involved in thepyruvate-dissipating routes leading to the formation of acetate

and diacetyl. Like the other leuconostocs (1, 47), L. gasicomi-tatum is likely to metabolize pyruvate to acetate or diacetylwhen intracellular pyruvate accumulates, for example, whenoxygen, citrate, or fructose is available. Presence of oxygen hasbeen reflected in the type of off odor (Table 1). The buttery off

FIG. 3. Salvage and catabolic pathways for nucleosides in L. gasicomitatum. External nucleotides are dephosphorylated by extracellularnucleotidases (step 1) (LEGAS_1431, LEGAS_0848) and are then transported into the cell by a nucleoside permease or nucleoside ABCtransporter (step 2) (LEGAS_0024, LEGAS_1844–1846, LEGAS_1460, LEGAS_1179); there are also separate nucleobase transporters (step3) (LEGAS_1771, LEGAS_1320, LEGAS_0450). Nucleosides entering the cell can be directly phosphorylated by the corresponding kinase(step 7) (LEGAS_0367, LEGAS_0687, LEGAS_0374, LEGAS_0393, LEGAS_0602, LEGAS_1125, LEGAS_1345, LEGAS_1390, LEGAS_1662,LEGAS_1710), or deoxyribonucleosides can first exchange nucleobases by a N-deoxyribosyltransferase (step 4) (LEGAS_0737), ribonucleosidescan be cleaved to free nucleobase and ribose by ribonucleoside hydrolases (step 5) (LEGAS_0022, LEGAS_1534, LEGAS_0456, LEGAS_0023),and the free nucleobase can be salvaged either by the N-deoxyribosyltransferase (step 4) or by one of the phosphoribosyltransferases (step 6)(LEGAS_0433, LEGAS_1197, LEGAS_0720, LEGAS_0874, LEGAS_1261, LEGAS_1902). The ribose formed by the hydrolysis of ribonucleo-sides can be either salvaged by the phosphoribosyltransferase (step 6) or used as an energy source by feeding it to the pentose phosphate pathway.NMP, nucleoside monophosphate; N1dR, a deoxyribonucleoside; N2dR, another deoxyribonucleoside; N1R, a nucleoside; PRPP, phosphoribosylpyrophosphate.

FIG. 4. Growth of Leuconostoc gasicomitatum LMG 18811T and Leuconostoc mesenteroides DSM 20343T in MRS broth with and without heminsupplementation (2 �g/ml) under aerobic and anaerobic conditions. Error bars show the differences obtained between three tests. Under amodified atmosphere containing 20% CO2 and 80% oxygen, growth was similar to growth under aerobic conditions.

VOL. 77, 2011 GENOME SEQUENCE OF LEUCONOSTOC GASICOMITATUM LMG 18811T 4349

odor marking diacetyl has been associated with products pack-aged under an oxygen-containing modified atmosphere (MA)or containers with an aerobic atmosphere (24, 42), whereas thepungent acidic odor has occurred in foods packaged underoxygen-deprived atmospheres (3, 45). Notably, some Lactoba-cillus and Lactococcus strains can also form diacetyl via catab-olism of aspartate (19, 22), an amino acid present in meat. Thegenes required for aspartate catabolism are present in L. gasi-comitatum 18811T, but no gene exists for glutamate dehydro-genase, considered important for the formation of �-ketoglu-tarate, the amino group acceptor essential for the pathway(40). Instead, L. gasicomitatum LMG 18811T encodes an �-ke-toglutarate transporter, suggesting that exogenous �-ketoglu-tarate may be exploited. However, whether L. gasicomitatumLMG 18811T produces diacetyl via aspartate catabolism and ifthis occurs in the meat ecosystem must be further studied.

Vihavainen and Bjorkroth proposed (42) that H2O2 pro-duced by an NADH oxidase in L. gasicomitatum strains causedgreen discoloration on beef steaks. Analysis of the genome ofL. gasicomitatum LMG 18811T revealed that pyruvate oxidaseis the only enzyme with a known ability to generate H2O2.Slime formation on vegetable sausages (45) and in a herringproduct (24) was proposed to be due to sucrose-derived ho-mopolysaccharide dextran, since L. gasicomitatum producesslime from sucrose in vitro (3). Consistent with this, L. gasi-comitatum LMG 18811T encodes a dextransucrase, a cell wall-associated glycosyltransferase catalyzing the formation of dex-tran from sucrose. In addition, a gene cluster homologous tothe heteropolysaccharide EPS gene cluster present in Strepto-coccus thermophilus (26) was detected. Heteropolysaccharideformation in leuconostocs has not been reported. Compared tohomopolysaccharides, their biosynthesis is more complex (12,26), ruling out prediction of the EPS structure, physical prop-erties, and possible role in food spoilage.

Addition of heme to aerated MRS medium increased thebiomass formation of L. gasicomitatum considerably, whereasaddition of CO2 (20%) to the oxygen-containing atmosphereto mimic the atmosphere used to create the high-oxygen MAfor red meats did not limit the biomass increase. Since neitherthe present study nor that of Brooijmans et al. (5) showedfunctional respiration in L. mesenteroides ATCC 8293T, we didnot anticipate this finding. Heme-induced respiration dramat-ically alters the phenotype of Lactococcus lactis, as it improvesnot only growth efficiency but also robustness as improvedstress resistance (13, 16). No heme uptake transporters havebeen characterized in any LAB, despite numerous efforts (15,31). Nevertheless, meat contains heme, and since the CO2

added in the atmosphere did not have any effect, L. gasicomi-tatum may respire while growing on high-oxygen MAP meats,leading to succession in the spoilage LAB population due toeffective growth and improved stress resistance.

L. gasicomitatum has not been detected on skin or mucousmembranes of broiler chickens (43) or pigs (23). The precisehabitat of this species is not known, but on the basis of itsgrowth temperatures and carbon source utilization (plant-de-rived pentoses), we have considered it an environmental LAB.Thus, it was interesting to detect unique genes (see Table S3 inthe supplemental material) associated with adhesion and plate-let binding. For genes encoding the putative mucus bindingprotein LEGAS_0414, orthologs were detected in the genomes

of L. mesenteroides and L. citreum (plasmid encoded), but noneof the other genomes contain orthologs for the putative colla-gen adhesion protein enabling biofilm formation in staphylo-cocci (10). The collagen binding capabilities might enable bet-ter survival in the meat environment. In preliminary analyses,L. gasicomitatum strain KG1-16, isolated in the spoiled vege-table sausages (47), did not harbor these genes. The role andexpression of these genes in association with meat spoilage willbe an interesting target of further studies.

With the help of the genome sequence, many of the spoilagereactions found their rationale. In addition, interesting newhypotheses arose, such as the potential increase of growth andstress resistance capabilities through respiratory capacity inhigh-oxygen meat products. L. gasicomitatum 18811T providesan interesting model of psychrotrophic spoilage LAB for fu-ture studies.

ACKNOWLEDGMENTS

The financial support of the Finnish Funding Agency for Technol-ogy and Innovation projects (TEKES 440472/03 and 4069/05) and theAcademy of Finland support for the Centre of Excellence of MicrobialFood Safety Research are gratefully acknowledged.

Markku Ala-Pantti, Janne Backman, Henna Niinivirta, Erja Meriv-irta, Riikka Raty, Eeva-Marja Turkki, and Hannu Vaananen arethanked for their excellent technical assistance.

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