Plasmids with a chromosome-like role in rhizobia

JOURNAL OF BACTERIOLOGY, Mar. 2011, p. 1317–1326 Vol. 193, No. 60021-9193/11/$12.00 doi:10.1128/JB.01184-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Plasmids with a Chromosome-Like Role in Rhizobia�†Cristina Landeta,1 Araceli Davalos,1 Miguel Angel Cevallos,3 Otto Geiger,2

Susana Brom,1 and David Romero1*Programas de Ingenería Genomica,1 Ecología Genomica,2 and Evolucion Genomica,3 Centro de Ciencias Genomicas,

Universidad Nacional Autonoma de Mexico, Apartado Postal 565-A, Cuernavaca, Morelos, Mexico

Received 1 October 2010/Accepted 22 December 2010

Replicon architecture in bacteria is commonly comprised of one indispensable chromosome and severaldispensable plasmids. This view has been enriched by the discovery of additional chromosomes, identifiedmainly by localization of rRNA and/or tRNA genes, and also by experimental demonstration of their require-ment for cell growth. The genome of Rhizobium etli CFN42 is constituted by one chromosome and six largeplasmids, ranging in size from 184 to 642 kb. Five of the six plasmids are dispensable for cell viability, butplasmid p42e is unusually stable. One possibility to explain this stability would be that genes on p42e carry outessential functions, thus making it a candidate for a secondary chromosome. To ascertain this, we made anin-depth functional analysis of p42e, employing bioinformatic tools, insertional mutagenesis, and programmeddeletions. Nearly 11% of the genes in p42e participate in primary metabolism, involving biosynthetic functions(cobalamin, cardiolipin, cytochrome o, NAD, and thiamine), degradation (asparagine and melibiose), andseptum formation (minCDE). Synteny analysis and incompatibility studies revealed highly stable repliconsequivalent to p42e in content and gene order in other Rhizobium species. A systematic deletion analysis of p42eallowed the identification of two genes (RHE_PE00001 and RHE_PE00024), encoding, respectively, a hypo-thetical protein with a probable winged helix-turn-helix motif and a probable two-component sensor histidinekinase/response regulator hybrid protein, which are essential for growth in rich medium. These data supportthe proposal that p42e and its homologous replicons (pA, pRL11, pRLG202, and pR132502) merit the statusof secondary chromosomes.

The classical view of replicon architecture in bacteria con-ceives of one indispensable chromosome and several dispens-able plasmids. This conception was shattered 2 decades ago bythe discovery of additional chromosomes. Based on informa-tion available in GenBank, secondary chromosomes have beendescribed in proteobacteria, including members of the Alpha-proteobacteria (Agrobacterium tumefaciens, Agrobacterium ra-diobacter, Agrobacterium vitis, all the Brucellaceae analyzed,Ochrobactrum anthropi, Paracoccus denitrificans, Rhodobactersphaeroides, and Sphingobium japonicum), Betaproteobacteria(all the Burkholderiales analyzed, Cupriavidus taiwanensis, Ral-stonia eutropha, Ralstonia picketii, and Variovorax paradoxus),and Gammaproteobacteria (Alliivibrio salmonicida, Photobacte-rium profundum, Pseudoalteromonas haloplanktis, and allthe Vibrionales analyzed). Also, secondary chromosomes werefound in species as diverse as Butyrivibrio proteoclasticus, aCyanothece sp., Deinococcus radiodurans, Leptospirales (Lepto-spira biflexa, Leptospira borgpetersenii, Leptospira interrogans),Prevotella melaninogenica, Sphaerobacter thermophilus, andThermobaculum terrenum.

Primary chromosomes usually are the largest replicons pos-sessing a dnaA-based replication system and harboring all ornearly all the essential genes that govern cellular processes,such as transcription, translation, DNA replication, and energy

metabolism (37). On the other hand, secondary chromosomesharbor some essential genes that are either absent from themain chromosome or duplicated in the secondary chromo-some. Most of the cases mentioned before possess duplicationson the secondary chromosome of genes encoding ribosomalRNAs or tRNAs, which are likely to contribute to normal cellviability. In just a few examples (A. radiobacter, Burkholderiaxenovorans, a Cyanothece sp., the Leptospirales, P. haloplanktis,and T. terrenum), secondary chromosomes lack structural RNAs,possessing instead other genes that participate in essentialfunctions. Secondary chromosomes commonly possess rep-lication systems different from the usual dnaA-based systempresent in primary chromosomes. Since these novel replicationsystems may be present in bona fide megaplasmids, a recentanalysis suggested the name “chromids” to refer to megaplas-mids that have acquired sets of essential genes, thus becomingsecondary chromosomes (26).

The most common way to identify secondary chromosomesin bacteria has been by localization of unique rRNA and/ortRNA genes through bioinformatic approaches. For instance,secondary chromosomes of A. tumefaciens and A. vitis possessrRNA operons as well as genes for prototrophic growth (24,55). Another approach to identify secondary chromosomes hasbeen by experimental demonstration of their requirement forcell growth. For instance, a segment encompassing 12.5% ofthe pSymB replicon (1.6 Mb) of Sinorhizobium meliloti is re-quired for normal growth on rich medium (9), presumably dueto the presence of an arg-tRNA gene in that sector (37). Valu-able as bioinformatic approaches are, the identification of es-sential genes based solely on sequence information remains adaunting task. Among the factors that hinder this effort are

* Corresponding author. Mailing address: Programa de IngenieríaGenomica, Centro de Ciencias Genomicas-UNAM. Apartado Postal565-A, 62210 Cuernavaca, Mor., Mexico. Phone: 52 (777) 3175867.Fax: 52 (777) 3175581. E-mail: [email protected].

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

� Published ahead of print on 7 January 2011.

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significant gaps in knowledge, variability in growth strategiesamong bacteria, nonorthologous substitutions, and reiterationof genes with important effects on cell metabolism (21, 41).

Rhizobium etli CFN42, an alphaproteobacterium and nitro-gen-fixing symbiont of the common bean (Phaseolus vulgaris),harbors a multipartite genome constituted by one chromosomeand six large plasmids (p42a to p42f), ranging in size from 184to 642 kb (23). Plasmid elimination assays demonstrated thatfive of the six replicons are dispensable for cell viability in richmedium. However, despite repeated attempts, it was not pos-sible to eliminate p42e from the wild type (4, 5). Although thisstability might be due to an unusually stable partition system,another interesting possibility is that genes on p42e performessential functions, thus making it a candidate for a secondarychromosome. To ascertain if this is the case, we decided toperform an in-depth functional analysis of p42e, employingbioinformatic tools, insertional mutagenesis, and programmeddeletions.

In this work, we demonstrate the active participation of p42ein the primary metabolism of R. etli. Synteny analysis andincompatibility studies revealed highly stable replicons equiv-alent to p42e in content and gene order in other Rhizobiumspecies. Also, it was not possible to eliminate p42e or homol-ogous plasmids by incompatibility; recA-independent cointe-gration of p42e with other replicons of the strain was observedinstead. Finally, two genes of p42e were identified as essentialfor growth in rich medium. These data suggest that p42eand its homologous replicons (pA, pRL11, pRLG202, andpR132502) merit the status of secondary chromosomes.

(This research was conducted by C. Landeta in partial ful-fillment of the requirements for a Ph.D. in Ciencias Biomedi-cas from the Universidad Nacional Autonoma de Mexico, Cu-ernavaca, Mexico, 2011.)

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains and plasmids used in this study arelisted in Table S1 in the supplemental material.

Growth conditions. R. etli, R. leguminosarum bv. viciae, and Agrobacteriumtumefaciens were grown in PY medium (peptone-yeast extract medium supple-mented with CaCl2 at a final concentration of 4.5 mM) at 30°C (42). Escherichiacoli strains were grown in Luria-Bertani (LB) broth at 37°C. When needed,antibiotics were added at the following concentrations (in �g ml�1): kanamycin(Kan), 30 (E. coli) or 15 (R. etli); nalidixic acid (Nal), 20; gentamicin (Gm), 30;tetracycline (Tc), 10 (E. coli) or 3 (R. etli); and spectinomycin (Sp), 100.

All the R. etli strains were also grown at 30°C in Y minimal medium (MMY)with 10 mM succinic acid and 10 mM ammonium chloride (3) as carbon andnitrogen sources, respectively. To prevent growth arrest by metabolic imbalancein subsequent cultures, MMY was also supplemented with biotin at 1 mg liter�1

(19). When needed, amino acids or vitamins were added to MMY at the follow-ing concentrations (in �g ml�1): cobalamin, 0.5; methionine, 100; and nicotinicacid, 12. For determinations of growth kinetics, the corresponding strains weregrown overnight at 30°C in liquid PY medium. Cells were collected by centrif-ugation at 13,000 rpm in an Eppendorf microcentrifuge and washed twice withfresh MMY, and appropriate volumes were used to inoculate 50-ml MMYcultures at an initial A620 of 0.05. The cultures were incubated for up to 24 h withorbital shaking (200 rpm) at 30°C; culture samples (1 ml) were taken every 3 h,and growth was measured by determining the total cell protein concentration bythe Lowry method. For determination of nicotinate auxotrophy, it was necessaryto starve the desired strains from nicotinate by previous growth for 24 h in MMYmedium lacking nicotinic acid. Cells obtained by this procedure were used toinoculate fresh MMY. The CE3 cyoA mutant was grown in MMY plates andincubated at 30°C in a sealed chamber, where microaerobic conditions were setup by repeated flushing with a gaseous mixture of argon-oxygen (99:1, vol/vol).The CE3 actP mutant was grown in PY medium with copper sulfate at a 1 mMfinal concentration.

Recombinant DNA procedures. Total and plasmid DNA isolation, digestionwith restriction enzymes, cloning, agarose gel electrophoresis, and E. coli trans-formation were performed by standard procedures (51).

Specific PCR primers were designed using the Oligo 6.0 software and werepurchased from Unidad de Síntesis Química (Instituto Biotecnología, Univer-sidad Nacional Autonoma de Mexico [UNAM]). Oligonucleotides used in thiswork are listed in Table S2 in the supplementary material. PCR amplificationswere done in a TC-312 thermocycler (Techgene, Burlington, NJ). Taq DNApolymerase (Altaenzymes, Alberta, Canada) was used in most PCRs, with acycling regime that includes a denaturing step at 94°C for 3 min, followed by 30cycles of 94°C for 1 min and 68°C for 1 min, followed by a final elongation stepat 72°C for 5 min. To reduce synthesis errors in amplifications involving therepABC region, Platinum Taq DNA polymerase high fidelity (Invitrogen, Carls-bad, CA) was used under the same cycling regime as before. For some primerpairs, a three-step cycling regime, consisting of a denaturing step at 94°C for 3min, followed by 30 cycles of 94°C for 1 min, 50 to 56°C (depending on the primerannealing temperature) for 1 min, and 72°C for 1 min, followed by a finalelongation step at 72°C for 5 min, was used.

For Southern hybridizations, DNA was digested with appropriate restrictionenzymes, electrophoresed in 1% (wt/vol) agarose gels, and blotted onto nylon(Hybond N�). Hybridization was carried out under high-stringency conditions,using Amersham’s Rapid-hyb buffer (GE Healthcare, United Kingdom). Specificprobes were labeled with [32P]CTP by random priming using Amersham’s Re-diprime system.

Vector insertion mutagenesis (VIMS). All the mutations in this work wereintroduced into the R. etli CE3 genetic background. The mutagenesis designemployed entails the cloning of an intragenic segment for the gene of interestinto a small, conjugative plasmid that is unable to replicate in Rhizobium (suicideplasmid). Upon transfer to Rhizobium, single-crossover recombination with thehomologous target introduces the whole plasmid as a cointegrate, producing aknockout of the gene of interest. Additionally, if the gene is located at thebeginning of an operon, loss of expression of the whole operon will ensue, dueto polarity effects. To this end, intragenic PCR products (300 to 800 bp) of thedesired genes, derived with the appropriate primers, were digested with BamHIand ligated, in most cases, into BamHI-restricted pK18mob (53). Ligated prod-ucts were transformed into E. coli DH5� (25). After verification of the clones,each plasmid was transformed in E. coli S17-1 (54), and these were used asdonors in plate matings with R. etli CE3. R. etli transconjugants were selected onPY medium with kanamycin and nalidixic acid. PCR amplifications of selectedNalr Kanr transconjugants were done to verify that the expected single-crossoverevent had occurred, using a combination of universal oligonucleotides (M13forward or reverse primers; Invitrogen) and oligonucleotides corresponding tothe insert. Mutants were also verified by analyzing BamHI-restricted genomicDNA by Southern blot hybridization, using the appropriate 32P-labeledpK18mob recombinant plasmids as probes, as described previously (20). For theconstruction of cobG and mmuM mutants, appropriate PCR products weredigested with BamHI and XbaI and cloned into similarly restricted pVEX1311and pIC20RDA, respectively. Ligated products were transformed into E. coliEK0610 (2) (for pVEX1311) or DH5�. Transfer of pIC20RDA derivatives wasdone by conventional biparental matings using E. coli S17-1 as the donor; trans-fer of pVEX1311 derivatives required a triparental mating, employing the ap-propriate R. etli strain, E. coli EK0610/pVEX1311 cobG, and HB101/pRK2013 asthe conjugation helper.

Incompatibility curing. To explore the incompatibility relationships of p42ewith diverse rhizobial replicons, a plasmid harboring the putative incompatibilitydeterminants of p42e was built. To that end, the repABC region of plasmid p42ewas amplified by PCR with appropriate primers (see Table S2 in the supplemen-tal material). The amplified segment included a sector encompassing from 500bp upstream of the start codon of repA to the middle of repC (codon 238), thusincluding the two proposed incompatibility regions for plasmids of this family (8).The PCR product was then cloned into pCR2.1-TOPO by T-A ligation andtransformed into E. coli DH5�. From the resulting plasmid, the wholerepABCp42e region was excised by digestion with KpnI and XbaI and cloned intosimilarly restricted pBBR1MCS5 (33). The resulting plasmid (pBBRrep1) wasintroduced into E. coli S17-1, and this strain was used as a donor in plate matingswith different Rhizobium or Agrobacterium strains. The plasmid transfer fre-quency was obtained by dividing the number of Nalr Gmr transconjugants by thetotal number of output recipient cells.

Selected transconjugants from these matings were verified for plasmid contentby a variant of the Eckhardt technique (see below). Transconjugants that pre-sented rearrangements (see Results) were additionally verified by PCR amplifi-cation of different p42e sectors (nadB, cobF, minC, cyoA, actP, selA, cls), using

1318 LANDETA ET AL. J. BACTERIOL.

specific primers and by Southern blot hybridization of the plasmid profiles withappropriate probes.

Generation of site-specific deletions with the Cre-loxP system. All the dele-tions constructed here were generated using a modification of the vector-medi-ated excision system (2). In this system, the region to be eliminated is flanked byloxP sites, the target of the site-specific Cre recombinase. To introduce thesesites, a PCR product (ranging from 600 bp to 1.8 kb), delimiting the left end ofthe planned deletion, was cloned into the loxP Spr suicide plasmid pVEX1311.Upon transfer to Rhizobium, single-crossover recombination with the homolo-gous target introduces the whole plasmid as a cointegrate, introducing the loxPsite as well. To delimit the right end of the deletion, a second PCR product wascloned into the loxP Tcr suicide plasmid pIC20R loxP oriT. This plasmid lacksappreciable sequence identity, other than the oriT sequence, with pVEX1311.Integration of the pIC20R loxP oriT derivative into the strain harboring theprevious pVEX1311 derivative generates a double-cointegrate strain in whichthe region to be deleted is flanked by loxP sites in a direct orientation. Theorientation of the insertion of these PCR products has to be planned to ensurea direct orientation of the loxP sites in the final, double-cointegrate strain. Togenerate an in vivo deletion of the desired region, plasmid pBBRCre, whichexpresses the site-specific recombinase Cre, was introduced by plate matings intothe strain harboring the appropriate double cointegrate, selecting for Nalr Gmr

transconjugants. Excision of the desired region by Cre-mediated popout recom-bination occurred in these strains at frequencies approaching 90%. The design ofthe deletion system permits selection of strains carrying the planned deletion byits Spr Tcs phenotype, since the Tcr determinant lies within the loxP-flankedregion and the Spr determinant lies outside it. Elimination of pBBRCre from thedeleted derivatives was done by growing them in PY medium lacking any anti-biotic, looking for Spr Gms derivatives. To ensure that the desired deletion hadtaken place, each strain was checked for the size of p42e, the absence ofpBBRCre in Eckhardt-type gels (17) (see below), and the absence of specificsegments lying within the deleted region by PCR amplification with appropriateprimers (see Table S2 in the supplemental material).

Plasmid and chromosome visualization. For visualization of the large plasmidsin Rhizobiales, the Eckhardt technique (17), as modified by Hynes and McGregor(27), was used. In this technique, a mild, in-gel lysis of bacterial cells, followed byelectrophoresis in 0.75% (wt/vol) agarose gels for 20 h, allows the easy separationof circular plasmid molecules from 10 kb up to 2 Mb in length.

Chromosome visualization was achieved by pulsed-field gel electrophoresis(PFGE), as described previously (38), with the following modifications. Plugswere incubated overnight at 37°C in a cell lysis solution (10 mM Tris-HCl [pH7.2], 50 mM NaCl, 100 mM EDTA, 0.2% deoxycholate, 0.5% N-lauroyl-sar-cosine, 1 �g ml�1 lysozyme, and 20 �g of RNase) and washed twice in washingbuffer (20 mM Tris-HCl [pH 7.5], 50 mM EDTA) and once with water. Afterincubation for 1 day at 50°C in proteinase K buffer (100 mM EDTA [pH 8], 0.2%deoxycholate, 0.1% N-lauroyl-sarcosine, and 50 �g ml�1 of proteinase K), theplugs were washed three times for 20 min each time in washing buffer. Gelelectrophoresis was done in a Bio-Rad CHEF-DRIII system under the followingconditions: one-sixth of the plug; initial switch time (IS), 800 s; final switch time(FS), 800 s; temperature, 13.5°C; field angle,106°; run time, 64 h at 2.2 V cm�1.

Lipid analysis by TLC. Cultures (1 ml) of R. etli were inoculated from freshovernight cultures to an A620 of 0.1. The cultures were labeled with 1 �Ci of[1-14C]acetate (58 mCi mmol�1; Amersham Biosciences) for 24 h. Lipids wereextracted, and the chloroform phase was subjected to lipid analysis in one-dimensional thin-layer chromatography (TLC) plates (high-performance TLCaluminum sheets, Silica Gel 60; Merck) using a chloroform-methanol-glacialacetic acid solvent system as described previously (52).

Transmission electron microscopy (TEM) and cell-length analysis. Electronmicroscopy images were obtained from negatively stained preparations on car-bon-coated grids. Samples of the desired strains were obtained from culturesgrown either in PY medium or in MMY for 9 h, centrifuged, and washed with 10mM HEPES buffer (pH 8.0). Washed cells were placed over a grid and stainedwith a 1% uranyl acetate solution for 8 min, dried at room temperature, andobserved at 80 kV with a JEM-1200EXII electron microscope (JEOL Ltd.,Japan). The length of at least 100 cells for each strain and condition was mea-sured from photographs. Cell length data were ranked every 0.5 �m, and thechi-square test was applied using the CE3 data as the expected values.

Bioinformatic analyses. To locate genes on p42e that possibly participate inmetabolic pathways, the sequence of p42e was subjected to functional automaticannotation of genes and assignation to pathways using the KEGG automaticannotation server (KAAS) (40; http://www.genome.jp/tools/kaas/) running in bi-directional best-hit mode. Results of this analysis were compared to the predic-tion of metabolic pathways for the complete R. etli CFN42 genome (available inthe Metacyc database [7]; http://metacyc.org/) retaining those genes with pre-

dictable function, present in p42e and not repeated elsewhere in the genome; thegenes chosen were mainly those that participate in central metabolism andcellular functions.

For determination of syntenic relationships, alignments were generated usingthe predicted protein sequences in each replicon, using the program PROmer,part of the MUMmer program suite (34). Conservation and context analysis ofrelevant regions was done using the MicrobesOnline database (13) (available athttp://www.microbesonline.org/). Searching for distant homologs of predictedproteins was done by PSI-BLAST (1) (available at http://blast.ncbi.nlm.nih.gov/Blast.cgi). Prediction of protein secondary structure and comparison with pub-lished protein structures were done at the PSIPRED server (6) (http://bioinf.cs.ucl.ac.uk/psipred/), using pGenTHREADER (35).

RESULTS

A significant fraction of the genes in p42e are involved inprimary metabolic functions. To ascertain which genes in p42emay be involved in primary metabolic functions, genes in thisreplicon were assigned to metabolic pathways using the KAASserver (40) as described in Materials and Methods. The resultsof this prediction were compared to the prediction of meta-bolic pathways for the complete genome of R. etli CFN42, withthe aim of identifying those genes in p42e that participate inknown pathways and that are not present elsewhere in thegenome. Those are shown in Fig. 1.

As befits its plasmid origin, p42e has a replication region(repABC) typical of Rhizobium plasmids (23). Besides the pre-viously recognized minCDE operon, involved in proper septumlocalization, genes participating in a variety of biosynthetic,degradative, and transport functions were also found. Amongthese, there is a cobFGHIJKLM operon involved in generationof cobalamin (vitamin B12) through the aerobic pathway, anadABC operon responsible for the early steps of NAD bio-synthesis, a thiMED operon participating in a newly describedthiamine salvage pathway (30), a cls gene responsible for car-diolipin synthesis, and the glnT gene, encoding glutamine syn-thetase III (10). We also found the cyoABCDE operon, encod-ing the cytochrome O terminal oxidase, one of the threerespiratory terminal oxidases present in R. etli.

Regarding degradative functions, p42e harbors an asnRPABoperon, which encodes aspartase and a thermolabile aspa-raginase (43), part of the pathway for histidine degradation(the hutUGHI operon), genes for melibiose utilization(agpA, agaL1, and agaL2) and two sets of genes that mightparticipate in protocatechuic acid degradation (pcaDCHGBand pcaIJF). Transport functions include a kdpABCD operon,encoding one of the four predicted potassium uptake systems(14), as well as the actP and hmrR genes, involved in copperexpulsion. A gene that might be involved in selenocysteine-tRNA formation (selA) was also found.

It is interesting to note that in some of these cases, part ofthe corresponding pathway is located in p42e, but the rest ofthe pathway is in another replicon. Examples of this are thecobFGHIJKLM operon, involved in generation of cobalamin,where the rest of the pathway is encoded on the chromosome,the nadABC operon responsible for the early steps of NADbiosynthesis, where the late steps and the salvage pathway forNAD are encoded on the chromosome, as well as the thiMEDoperon, participating in the thiamine salvage pathway, wherethe de novo thiamine pathway (thiCOSGE) is encoded on plas-mid p42b (39).

Based solely on gene annotation, a significant fraction of the

VOL. 193, 2011 SECONDARY CHROMOSOMES IN RHIZOBIA 1319

genes in p42e (51 out of 459, 11%) appear to be devoted toprimary metabolic functions. Other than the chromosome, thisis the second largest genomic element in R. etli containinggenes involved in primary metabolism, suggesting that p42emerits the status of secondary chromosome.

Plasmids equivalent to p42e are present in other Rhizobiumspecies. To merit the status of secondary chromosome, repli-cons equivalent to p42e should be conserved among relatedRhizobium species. In order to demonstrate if this is the case,we made protein sequence alignments of the complete repli-cons of other sequenced Rhizobium species against p42e, asdescribed in Materials and Methods. As shown in Fig. 2A,there is an extensive similarity and conservation of gene orderbetween R. etli CFN42 p42e and R. etli CIAT652 pA (22).Synteny between these replicons is broken only in the intervalspanning coordinates 120 kb to 200 kb in the p42e sequence.Interestingly, the only spontaneous deletion of p42e (obtainedpreviously during attempts to eliminate p42e) covers this in-terval (Fig. 1, �185). Remarkably, synteny analysis between allthe replicons in these two strains revealed extensive similarity

only between the respective chromosomes or for the p42e-pApair and significant, albeit lower similarities, between p42f-pCand p42d-pB (22).

Extensive similarity of p42e was also found with similar-sized replicons in other Rhizobium species. As shown in Fig.2B to D, equivalence in order and conservation to p42e wasfound with pRL11 of R. leguminosarum bv. viciae 3841 (29,61), pRLG202 of R. leguminosarum bv. trifolii WSM2304 (45),and pRLG132502 of R. leguminosarum bv. trifolii WSM1325(44). Interestingly, in all these cases, synteny was missing inroughly the same sector (coordinates 120 kb to 200 kb) as inthe comparisons between p42e and pA, indicating that thisregion is dispensable in the corresponding genomes.

Although no extensive synteny was detected between p42eand other replicons in the Rhizobiales, it was previously notedthat several gene clusters are shared between p42e and thechromosomes II of A. tumefaciens C58, A. vitis S4, and A.radiobacter K84, as well as with pSymB of Sinorhizobium me-liloti (26, 55). Notably, all the genes involved in primary me-tabolism described in the previous section are located in p42e

FIG. 1. Map of plasmid p42e. Relevant genes are indicated inside the circle. Deletions employed in this work are indicated on the outsidearcs.


or equivalent replicons in R. etli and R. leguminosarum biovarsviciae and trifolii but in chromosomes or putative secondarychromosomes in other Rhizobiales (see Table S3 in the supple-mental material). Taken together, the finding of repliconsequivalent in content and gene order to p42e in other Rhizo-bium species supports our contention that these should beconsidered secondary chromosomes.

Plasmid p42e evades its elimination through recA-indepen-dent cointegration. Previous attempts to eliminate p42e reliedon a positive selection system, based on the use of the sacBgene (4, 5). Although this system has been successful toachieve elimination of the rest of the plasmids in R. etli CFN42(4, 5), an alternative approach would be to force the loss ofp42e through incompatibility. To this end, we cloned, in aconjugative vector replicable in R. etli (pBBR1MCS), the rep-lication sector of p42e (repABCp42e); the sector chosen in-cludes the two regions known to function as incompatibilityfactors (8) but lacks a complete repC gene, hence precludingreplication from this region. The resulting plasmid should rep-licate using only the replication system from pBBR1MCS butshould exert incompatibility toward p42e. Thus, if eliminationof p42e were possible, introduction of this plasmid by conju-gation into R. etli would suffice to achieve its dislodgment fromthe strain.

Introduction by conjugation of both vector pBBR1MCS5and its derivative harboring repABCp42e (pBBRrep1) occurredreadily with E. coli (Table 1). Interestingly, although introduc-tion of pBBR1MCS5 into either wild-type R. etli CE3 or itsrecA derivative occurred at frequencies comparable to the onesseen for E. coli, introduction of pBBRrep1 occurred only atvastly lower frequencies (10,000-fold lower than the ones seenwith pBBR1MCS5). Since both plasmids share the same con-jugative and replicative systems, the restriction in the fre-

quency of transconjugants obtained with pBBRrep1 should bedue to difficulties in the maintenance of the incoming plasmid,perhaps caused by a strong selection against the elimination ofp42e.

Verification of the transconjugants of both R. etli CE3 andits recA derivative harboring pBBRrep1 by plasmid profilesrevealed that, in all cases, a normally sized band correspondingto p42e was absent (Fig. 3A). The absence of p42e was accom-panied by the loss of another plasmid (depending on thetransconjugant analyzed), coupled with the finding of new,larger plasmid bands. These changes should be readily ex-plained by invoking cointegration of p42e with other plasmidsin the strain, rather than simple losses of p42e. In support ofthis explanation, Southern blots of the plasmid profiles, hybrid-ized against probes corresponding to each plasmid, revealed

FIG. 2. Plasmids equivalent to p42e are present in other Rhizobium species. Alignments of R. etli CFN42 plasmid p42e with R. etli CIAT652plasmid pA (A), R. leguminosarum bv. viciae plasmid pRL11 (B), R. leguminosarum bv. trifolii WSM2304 plasmid pRLG202 (C), and R.leguminosarum bv. trifolii WSM1325 plasmid pR132502 (D) were done using PROmer. The scale is in Mbp.

TABLE 1. Reduced transfer frequency of a plasmid harboringrepABCp42e in Rhizobiales

Recipient strain

Plasmid transfer frequency (10�7)(mean � SD)a

pBBR1MCS5b pBBRrep1c

E. coli DH5� 6,600 � 3,100 5,800 � 12,000R. etli CE3 240 � 250 0.98 � 1R. etli CE3 recA 8,500 � 1,300 0.1 � 0.26R etli CIAT652 51,600 � 15,000 0.075 � 0.15R. leguminosarum bv.

viciae 3841358,000 � 430,000 1.1 � 0.81

A. tumefaciens GMI9023 68,000 � 113,000 0.58 � 0.57

a Plasmid transfer frequency is expressed as the number of transconjugant cellswith the desired plasmid divided by the total number of output recipient cells.Each datum is the mean of at least three independent determinations.

b Control plasmid.c Plasmid carrying the repABCp42e operon.


the recombinant nature of the new larger plasmid bands (datanot shown). Moreover, the presence of p42e in the transcon-jugants was ascertained by PCR amplification of different sec-tors scattered over p42e (data not shown).

This analysis allowed us to conclude that, rather than beinglost, p42e had been cointegrated with either plasmid p42b,p42c, p42f (Fig. 3A), or p42d (data not shown). Only oneisolate appeared to have lost p42e (Fig. 3A, rightmost lane);this isolate, however, still retains most of p42e, as suggested byPCR amplification of different sectors scattered over p42e(data not shown). To evaluate whether cointegration betweenp42e and the chromosome occurred in this isolate, chromo-some size was analyzed by PFGE. As shown in Fig. 3B, chro-mosome size in this isolate was increased from 4.3 Mb to 4.8Mb, consistent with the insertion of p42e in the R. etli CE3chromosome. Thus, p42e evades its elimination by recA-inde-pendent cointegration with other replicons.

To ascertain whether plasmids equivalent in order andcontent to p42e belong to the same incompatibility group,pBBRrep1 was introduced into R. etli CIAT652 and R. legumino-sarum bv. viciae 3841. Vector plasmid pBBR1MCS5 was intro-duced readily in these strains, but transconjugants with therepABCp42e-harboring derivative (pBBRrep1) were found atonly very low frequencies (Table 1). Plasmid profile analyses ofthese transconjugants revealed that in R. etli CIAT652, pA wascointegrated with pC (Fig. 3C) while in R. leguminosarum bv.

viciae 3841, pRL11 was cointegrated with pRL10 (Fig. 3D).These data indicate that plasmids p42e, pA, and pRL11 allbelong to the same incompatibility group and evade their elim-ination by cointegration with other replicons.

Introduction of pBBRrep1 into A. tumefaciens GMI9023(49) (a plasmid-free strain) also occurred at very low frequen-cies (Table 1), suggesting that repABCp42e is incompatible withthe only repABC replicon present in this strain, i.e., the A.tumefaciens linear chromosome. However, chromosome sizingof the transconjugants by PFGE failed to reveal any changes inthe size of Agrobacterium chromosomes (data not shown).

The inability to eliminate p42e or equivalent replicons byincompatibility, obtaining instead cointegrates with other rep-licons in the cell, is consistent with the view that loss of thesereplicons may be lethal even in rich medium, an attribute to beexpected in secondary chromosomes.

Participation of p42e in primary metabolic functions. Toascertain the participation of p42e-localized genes in primarymetabolic functions, we inactivated the corresponding genes oroperons by vector insertion mutagenesis (VIMS) (see Materi-als and Methods). We excluded the thiMED (30) and asnRPAB(43) operons and the glnT gene (10) from this analysis, whosefunctionality was demonstrated previously.

Inactivation of the nadABC operon provokes nicotinate aux-otrophy. The pathway for de novo biosynthesis of NAD inbacteria is the aspartate pathway, encoded by the nadABCDEgenes (47). In R. etli, the first three genes are localized in p42e,and the rest are localized on the chromosome. Inactivation ofthe nadABC operon in R. etli CE3 provoked a nicotinate aux-otrophy (see Fig. S1 in the supplemental material), consistentwith the participation of the NAD salvage pathway. This phe-notype was seen after a previous subculture in minimal me-dium, due to the need to deplete the endogenous NAD pool.

R. etli CE3 actP is hypersensitive to copper. Maintenance ofcopper homeostasis requires a P-type ATPase encoded byactP, which exports copper (46). A mutant on actP in p42edisplays hypersensitivity to copper (see Fig. S1 in the supple-mental material).

A minCDE mutant is affected in cell length but not ingrowth. The products of the minCDE genes participate in theaccurate placement of the division site, allowing septum for-mation in the middle of the cell (50). Hence, we evaluated thegrowth kinetics and cell length of the CE3 minC mutant byelectron microscopy. Despite the fact that growth was notaffected by inactivation of this operon (data not shown), sig-nificant differences (P �� 0.01) were found in distribution ofcell length between the mutant and the wild-type strain (seeFig. S2 in the supplemental material). The mutant displayed ashorter cell length than the wild type (median cell length, 1.53versus 2 �m, respectively); in fact, 13% of the mutant cellswere found in the size class of 0.5 to 1.0 �m, whereas this classwas absent in the wild type.

A cobalamin auxotrophic phenotype requires mutations inthe cobFGHIJKLM operon and in mmuM. In R. etli, cobalaminbiosynthesis proceeds by the aerobic pathway (59). Part of thepathway is encoded on p42e, while the rest is on the chromo-some. Cobalamin is a cofactor for essential enzymes, such asmethionine synthase and ribonucleotide reductase. Some bac-teria have two isozymes for each enzyme, one B12 dependentand another B12 independent (15, 18, 48). R. etli harbors two

FIG. 3. Rearrangements observed in Rhizobium strains harboringplasmid pBBRrepABCp42e. At the top of each panel, combinations ofletters (e.g., b-e) indicate which kind of cointegration has occurred.(A) Plasmid profile of R. etli recA (lane RE) and their recApBBRrep1derivatives. (B) Chromosomal profile of R. etli CE3 (lane RE) and arecApBBRrep1 derivative (lane e-chr); lane L, molecular size marker(Schizosaccharomyces pombe chromosomes [Bio-Rad]). Chromosomalsizes at the right of this panel are indicated in Mbp. (C) Plasmid profileof R. etli CIAT652 (lane RC) and a CIATpBBRrep1 derivative (laneA-C). (D) Plasmid profile of R. leguminosarum bv. viciae 3841 (laneRL) and a 3841pBBRrep1 derivative (lane 10-11). Letters or numberson the left of panels A, C, and D mark the locations of plasmids in R.etli CFN42 (panel A), R. etli CIAT652 (panel C), and R. leguminosarumbv. viciae 3841 (panel D). Asterisks indicate the locations of novelcointegrate plasmids.


chromosomally located ribonucleotide reductases, one depen-dent and another independent of cobalamin. In contrast, thereis a potential B12-dependent methionine synthase (metH) inthe chromosome, but the classical B12-independent methio-nine synthase (metE) is absent from this organism. Despitethis, a cobG R. etli mutant is still able to grow in minimalmedium (see Fig. S3 in the supplemental material). However,R. etli possess a homocysteine S-methyltransferase protein (en-coded in RHE_PC00163, localized in p42c) that is an orthologto the third methionine synthase (mmuM) of E. coli, which usesS-methylmethionine as a methyl donor and zinc as a cofactor(58). Simultaneous inactivation of cobG and mmuM in R. etlileads to an inability to grow in minimal medium, which iscorrected upon addition of either cobalamin or methionine tothe medium (see Fig. S3 in the supplemental material).

CE3 cyoA mutant is affected in growth in minimal mediumunder microaerobiosis. The cytochrome oxidases comprisecomplex IV in the electron transport chain. The cytochromesof R. etli expressed under free-living conditions are cytochromesaa3 and o; the former is expressed under aerobic conditions, whilethe latter is expressed under microaerobic conditions (56). Genesencoding the cytochrome c oxidase (also known as aa3) arelocated in the chromosome (ctaBCDE), while the cytochromeo oxidase is encoded in p42e (cyoABCDE). As expected, a CE3cyoA mutant grows well in both rich and minimal media underaerobic conditions but displays a severe growth reduction inminimal medium under microaerobiosis (see Fig. S3 in thesupplemental material).

A CE3 cls mutant fails to produce cardiolipin. Membranephospholipids of plant-associated bacteria are mainly phos-phatidylglycerol, cardiolipin, phosphatidylethanolamine (PE),and their methylated derivatives (monomethyl-PE and di-methyl-PE) and phosphatidylcholine. Cardiolipin is synthe-sized from phosphatidylglycerol through cardiolipin synthase(cls) (36). A CE3 cls mutant is unable to produce cardiolipin(see Fig. S3 in the supplemental material). This inability wasalso seen in strain CFNX185, which also lacks this gene (Fig. 1,�185 also, see Fig. S3 in the supplemental material). Theinability to produce cardiolipin, however, does not affectgrowth compared to that of the wild type (data not shown).

No distinctive phenotypes were found in mutants affected inselA, hutI, or pcaD. In the last two cases, this is due to theinability of the wild-type strain to grow on the correspondingmetabolites (histidine either as a carbon or nitrogen source;protocatechuic acid as a carbon source). The selA gene codesfor an L-seryl-tRNA(Ser) selenium transferase protein, whichincorporates selenium into proteins as selenocysteine (11). Al-though this gene is essential in bacteria that use selenocysteinefor recoding, R. etli and related bacteria lack other genesneeded for the selenium utilization trait, such as selB, selC, andselD (63, 64).

The results shown in this section clearly demonstrate theparticipation of several genes located on p42e in differentaspects of primary metabolism. Important as these genes are, itis appropriate to stress that none of the mutations analyzedcompromise growth on rich medium. Therefore, putative es-sential genes on p42e must be looked for in other sectors ofthis replicon.

Deletion analysis of p42e reveals regions essential forgrowth in R. etli. In order to find regions on p42e essential for

growth on rich medium, we undertook a programmed deletionanalysis of this replicon, using a modification of the vector-mediated excision system (2). In this system, the region to bedeleted is flanked by loxP sites (introduced by cointegration) indirect orientation; excision of the region of interest is achievedby expression of the Cre recombinase, at frequencies ap-proaching 90% (see Materials and Methods).

Ten deletions affecting predefined sectors of p42e wereobtained using this system (Fig. 1, outer arcs). The sizes ofthe deleted regions range from 27 kb to 447 kb; these de-letions, together with the spontaneous deletion harbored bystrain CFNX185 (5), provide a nearly complete deletioncoverage of p42e. The only regions not covered by deletionsinclude the cobFGHIJKLM operon, the repABC operon, andgene RHE_PE00001 (see below). Nine of these deletions (de-letions 1, 2, 3, 4, 5, 9, 10, 11, and 185) do not noticeably affectthe growth of the corresponding strains on rich medium. Dis-tinctive phenotypes for strains bearing these deletions wereseen only on minimal medium, in a manner consistent with theloss of recognized genes (for instance, the �1 strain showednicotinate auxotrophy and a min phenotype, the �185 straindid not produce cardiolipin and was unable to degrade melibi-ose). The largest deletion strain analyzed was the �5 strain(447-kb deletion); thus, it is possible to eliminate nearly 90% ofp42e without severely impairing growth in rich medium.

A marked reduction in growth on rich medium was seen forstrains harboring either deletion 6 or 7. Upon growth of thesestrains on rich medium plates, only pinpoint colonies wereseen, which took twice as long to acquire a detectable size ascolonies formed by the wild-type strain. A precise evaluation ofthe growth retardation in these strains was hindered both bytheir slow growth and by the frequent appearance of revertantsof unknown origin. Both �6 and �7 strains have lost a sectorencompassing genes RHE_PE00003 to RHE_PE00035 (Fig.1); since a strain harboring �9 (lacking the region comprisingRHE_PE00003 to RHE_PE00023) grows normally, we inferthat the region responsible for the slow-growth phenotypeon rich medium lies in the sector spanning from genesRHE_PE00024 to RHE_PE00035. In fact, deletion of thisinterval turned out to be impossible to obtain, despite theflanking of this interval with appropriate loxP sites.

To find out which gene on this sector is needed for growthin rich medium, systematic inactivation of the transcriptionalunits in this interval was done with the VIMS approach (seeMaterials and Methods). Inactivation of transcriptional units en-compassing RHE_PE00025, RHE_PE00026 to RHE_PE00030(the cyoABCDE operon), RHE_PE00031, RHE_PE00032 toRHE_PE00033, and RHE_PE00034 were readily obtainedthrough this approach. Inactivation of RHE_PE00024 was im-possible to obtain by VIMS; interestingly, inactivation of thisgene was readily obtained only upon introduction of supernu-merary copies of RHE_PE00024. These data indicate thatRHE_PE00024 (encoding a probable two-component sensorhistidine kinase/response regulator hybrid protein) is an im-portant determinant for growth in rich medium.

Attempts to generate deletions lacking RHE_PE00001 werefrustrated by the inability to get insertions on this gene, even byemploying the VIMS approach. This inability can be circum-vented by introduction of additional copies of RHE_PE00001.As expected, corresponding insertions occurred either on


RHE_PE00001 in p42e or in the supernumerary copy. Thesedata are consistent with the proposal that RHE_PE00001 is agene that is essential for growth of R. etli on rich medium.

Gene RHE_PE00001 encodes a hypothetical protein, con-taining DUF1612 (a domain of unknown function, conservedin Rhizobiales), as well as a probable winged helix-turn-helixmotif (HTH-13). Searching for more-distant homologs usingPSI-BLAST revealed similarity with members of the Fic(filamentation induced by cyclic AMP [cAMP]) protein fam-ily (data not shown). Interestingly, an analysis of the pre-dicted RHE_PE00001 protein in the PSIPRED server, usingpGenTHREADER (data not shown), revealed a structuralfolding similar to that of the Fic family protein of Shewanellaoneidensis (12). The significance of these similarities is ex-plored in Discussion.

DISCUSSION

In this work, we present four lines of evidence in support ofour proposal that p42e and related replicons should be con-sidered secondary chromosomes in Rhizobiales: (i) the pres-ence of a significant fraction of genes functionally involved inprimary metabolism; (ii) the finding of replicons equivalent top42e, in content and gene order, in other Rhizobium species;(iii) the inability to eliminate p42e or equivalent replicons byincompatibility; and (iv) the identification of two genes whosepresence is needed for normal growth in rich medium.

Regarding the first point, our data show that nearly 10% ofthe genes in p42e participate in different aspects of primarymetabolism, including amino acid biosynthesis and degra-dation, vitamin and cofactor biosynthesis, membrane lipidformation, sugar utilization, electron transport, and respi-ratory ability, as well as septum location. Interestingly, sev-eral of these aspects reveal a certain degree of functionalsolidarity among the different replicons, in the sense that partof the pathway is located in p42e and the rest in other repli-cons, such as the main chromosome. This was clearly evincedfor cobalamin, thiamine, and NAD biosynthesis, as well as forseptum location.

It is important to note that the number of primary metabolicfunctions encoded in p42e may be as yet underestimated. Inour analysis, we concentrated on genes with a functional an-notation which had no additional copies elsewhere in the ge-nome. This leaves out genes for which functional reiteration iscommonplace. Among genes on p42e that may fall in thiscategory are those encoding a type 1C penicillin binding pro-tein (pbpC), a peptide methionine sulfoxide reductase (msrAe),four different adenylate cyclases (cyaCe, cyaA, RHE_PE00043,and RHE_PE00045), an extracytoplasmic sigma factor and itscorresponding anti-sigma (RHE_PE00003 and RHE_PE00004),a DNA topoisomerase type IB (RHE_PE00209), glutathioneS-transferase (RHE_PE00210), and a set of genes that mayparticipate in double-strand break repair via the Ku pathway(RHE_PE00249 to RHE_PE00252). Although the functional-ity of most of these genes remains to be evaluated, there isevidence that at least the cyaA copy is active in R. etli (57).Analyses of genes that participate in double-strand break re-pair via the Ku pathway in S. meliloti revealed functional reit-eration; genes on the so-called pSymB (orthologs to the onespresent in p42e) are required for a full repair activity (32).

The finding of replicons equivalent to p42e in other Rhizo-bium species is a further argument for its role as a secondarychromosome. Other than the respective chromosome, p42eand their relatives are the largest genomic elements sharedbetween different Rhizobium spp., including R. etli and R. le-guminosarum biovars viciae and trifolii. The finding that p42e,pA, and pRL11 belong to the same incompatibility group alsoargues in favor of a common origin for these replicons.

Our inability to achieve elimination of p42e, pA, or pRL11by incompatibility curing can be explained by the finding of twoloci on p42e (and corresponding replicons) that are essentialfor growth in rich medium. Based on this view, cells that losep42e are unable to grow in rich medium. The strong pressureimposed by another replicon of the same incompatibility groupin the cell makes it more likely to select low-frequency recA-independent cointegration events of p42e (and pA or pRL11)with other replicons in the cell. These cointegrates should bestable, since p42e and their relatives now employ the replica-tion system from the replicon in which it was cointegrated,while the plasmid used for incompatibility curing harbors aseparate replication system (pBBRMCS).

Although the exceptional stability of p42e and its relativesshould be explained by their possession of two essential genes,recent data suggest that there are additional factors. At-tempts to eliminate p42e providing supernumerary copies ofRHE_PE00001 and RHE_PE00024 proved to be unsuccessful(data not shown), suggesting that other factors, such as thepresence of an addiction system, may have a role in the highstability of this replicon.

Two regions relevant for growth in rich medium were iden-tified in this work. One of them (RHE_PE00001) encodes ahypothetical protein that contains DUF1612 (a domain of un-known function, conserved in Rhizobiales), as well as a proba-ble winged helix-turn-helix motif (HTH-13). This protein isconserved among all sequenced rhizobial species. As men-tioned in Results, this protein shows detectable similarity inboth primary structure and folding with members of the Ficprotein family. Members of the Fic family are thought to par-ticipate together with cAMP in regulatory processes for celldivision, probably involving folate metabolism. Some membersof this family are death-on-curing (Doc) proteins, involved intoxin-antitoxin systems (12). Intriguing as these similarities are,we believe that RHE_PE00001 does not possess these func-tions, because it lacks both the well-conserved HPFXXGNGmotif (12) and the Fido motif (31), characteristic of this pro-tein family. Thus, it is possible that a Fic folding was recruitedon RHE_PE00001 for a different process, perhaps involvingDNA interactions.

The second region comprises gene RHE_PE00024, whichwas annotated as a probable two-component sensor histidinekinase/response regulator hybrid protein, also conserved in theRhizobiales. Even though its precise function remains un-known, it is plausible to invoke a role as a global regulator,controlling an essential aspect of cell metabolism. In this re-gard, it is germane to mention that although many two-com-ponent systems are dispensable (60, 62), at least the WalK/WalR two-component system in low-G�C Gram-positivebacteria (16) and the two-component sensor histidine kinase/response regulator hybrid protein CckA in Caulobacter cres-centus (28) are essential proteins. Although the exact roles of


RHE_PE00001 and RHE_PE00024 will be explored in futurestudies, this work reveals the complexities inherent to infergene essentiality based solely on sequence data. None of thesegenes was recognized as essential, based on sequence annota-tion or through screenings in the Database of Essential Genes(http://tubic.tju.edu.cn/deg/).

Data presented here are consistent with a scenario in whichthe ancestor of p42e and their relatives was a typical repABCplasmid. Several genes located on the main chromosome un-derwent migration to this plasmid replicon without gene du-plication, thence generating a second essential replicon. Themigration process invoked here is probably a consequence ofthe activity of the recombinational systems of this group ofbacteria. The case of p42e and their relatives is notable in thesense that accretion of chromosomal genes was particularlyactive, entailing the acquisition of some genes essential forgrowth in rich medium. The exploration of the processes forgeneration and possible selective advantages of this particulargenome architecture constitute an interesting field for furtherresearch.

ACKNOWLEDGMENTS

We gratefully acknowledge Rafael Díaz for valuable help with thePFGE, Patricia Bustos and Victor Gonzalez for help with bioinfor-matic analysis, Mario Sandoval and Christian Sohlenkamp for lipidanalysis, Laura Cervantes for skillful technical assistance, Paul Gaytanand Eugenio Lopez (Unidad de Síntesis de Oligonucleotidos, Institutode Biotecnología, UNAM) for help with oligonucleotide synthesis,Rodolfo Paredes (Unidad de Microscopía, Instituto de Fisiología Ce-lular, UNAM) for help with the microscopy analysis, and VeronicaRohen for advice on the statistical tests.

C.L. was supported during the Ph.D. program (Programa de Doc-torado en Ciencias Biomedicas, Universidad Nacional Autonoma deMexico) by a scholarship from Consejo Nacional de Ciencia y Tec-nología (Mexico).

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Plasmids with a chromosome-like role in rhizobia

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