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The nitrogen-fixing symbiosis fixation between legumes and Rhizobium helps the plant to survive and to compete effectively on nitrogen poor soils. The soil environment attached to the root system is characterized by intense microbial activity. This work aimed to study the diversity of indigenous rhizobia living in the rhizosphere of forage pea in eastern and central Algeria. Bacteria were trapped in vitro from collected soils. They were isolated from nodules by direct isolation or by bacterial trapping, and tested for nodulation. Further characterization was carried out to identify the isolates. Ninety-four isolates were obtained. Thirty-four were rhizobial strains that could, with two exceptions, establish root-nodule symbioses with the host plant. Different profiles of susceptibility to rhizobiophages indicated that the majority of isolates were likely to be members of the Rhizobium leguminosarum group of species. The strains revealed diverse plasmid profiles, and diverse restriction patterns of cpn60 gene amplicons and on Southern blot analysis of nodABC genes. Sequences of 16S rRNA gene showed that all isolates exhibited 99.85% identity with R. leguminosarum sv. viciae USDA 2370T, R. laguerreae FB206T and R. anhuiense CCBAU 23252T. Analysis of cpn60 sequences revealed two groups. One of them clustered with ≤99% identity with R. laguerreae FB403. The second was clustered with ≤99% identity with R. laguerreae FB206T. Our findings suggest that the isolates may belong to the species R. laguerreae.

Rhizobium leguminosarum strain VF39 contains a plasmid, pRleVF39b, which encodes a distinctive type of conjugation system (rhizobial type IVa) that is relatively widespread among rhizobial genomes. The cluster of genes encoding the transfer functions lacks orthologs to genes such as traCD, traF and traB, but contains 15 conserved genes of unknown function. We determined the importance of these genes in conjugation by constructing marked and unmarked mutations in each gene, and established that six genes, now designated trcA-F, played a significant role in plasmid transfer. Like the relaxase gene, traA, and the genes encoding the MPF system (trb genes), five of these genes, located in two divergently transcribed operons, are regulated by the Xre family repressor TrbR. The other gene, trcF encodes a protein with similarity to histidinol phosphatases, and its role in conjugation is unclear, but mutations in trcF are severely impaired for conjugation. TrcF does not play a role in regulation of other conjugation genes.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria., Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, the authors identify a thermosensitive enzyme that synthesizes c-di-GMP and modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a &lsquo, natural&rsquo, unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, &ldquo, R. indicum&rdquo, and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in Rhizobium leguminosarum. The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSymR7A is the prototypical member of a large family of “symbiosis ICEs” which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSymR7A enables recombination between the ICEMlSymR7A attachment site attP and the 3′ end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSymR7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSymR7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSymR7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSymR7A and related ICEs.

Plants are known to secrete chemical compounds that can change the behavior of rhizosphere-inhabiting bacteria. We investigated the effects of extracts from legume host plants on the swarming behavior of Rhizobium leguminosarum bv. viciae. We also investigated the effects on swarming when Rhizobium is exposed to extracts from an ancestor to vascular plants, the model bryophyte Physcomitrella patens. Lentil and faba bean seed exudates enhanced and inhibited swarming motility, respectively, whereas pea seed exudates had no observable effect on swarming. Swarming was also enhanced by the moss extracts. Exposure to lentil seed exudates and the moss extract increased flaA expression 2-fold, while faba bean seed exudates exposure decreased expression 3-fold, suggesting that the swarming effect could, in part, be due to regulation of flagellin gene expression. However, the exudates and extracts did not significantly affect flaA gene expression in planktonic motile cells, indicating that the response to flagellar regulation is specific to a physiology unique to the swarming cell. Transmission electron microscopy demonstrated that addition of the lentil seed exudate and the moss extract results in earlier differentiation into swarmer cells, which could contribute to the development of a larger swarming surface area. To gain further mechanistic insight into the effect of the moss extract on swarming, a moss strigolactone-deficient mutant (Ppccd8Δ) was tested. A reduction in the promotive effect was observed, suggesting that the plant hormone strigolactone may be a signalling molecule activating swarming motility in R. leguminosarum.

Summary Homoserine represents a substantial component of pea root exudate that may be important for plant–microbe interactions in the rhizosphere. We identified a gene cluster on plasmid pRL8JI that is required for homoserine utilization by Rhizobium leguminosarum bv. viciae. The genes are arranged as two divergently expressed predicted operons that were induced by L-homoserine, pea root exudate, and were expressed on pea roots. A mutation in gene pRL80083 that prevented utilization of homoserine as a sole carbon and energy source affected the mutant's ability to nodulate peas and lentils competitively. The homoserine gene cluster was present in approximately 47% of natural R. leguminosarum isolates (n = 59) and was strongly correlated with homoserine utilization. Conjugation of pRL8JI to R. leguminosarum 4292 or Agrobacterium tumefaciens UBAPF2 was sufficient for homoserine utilization. The presence of L-homoserine increased conjugation efficiency of pRL8JI from R. leguminosarum to a pRL8JI-cured derivative of R. leguminosarum 1062 and to A. tumefaciens UBAPF2, and induced expression of the plasmid transfer gene trbB; however, there was no difference in conjugation efficiency or trbB expression with A. tumefaciens UBAPF2pRL8-Gm as the donor suggesting that other genes in R. leguminosarum may contribute to regulating conjugation of pRL8 in the presence of homoserine.

Rhizobium leguminosarum strain VF39SM contains two plasmids that have previously been shown to be self-transmissible by conjugation. One of these plasmids, pRleVF39b, is shown in this study to carry a set of plasmid transfer genes that differs significantly from conjugation systems previously studied in the rhizobia but is similar to an uncharacterized set of genes found in R. leguminosarum bv. trifolii strain WSM2304. The entire sequence of the transfer region on pRleVF39b was determined as part of a genome sequencing project, and the roles of the various genes were examined by mutagenesis. The transfer region contains a complete set of mating pair formation (Mpf) genes, a traG gene, and a relaxase gene, traA , all of which appear to be necessary for plasmid transfer. Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster. A regulatory gene, trbR , was identified in the region between traA and traG and was mutated. TrbR was shown to function as a repressor of both trb gene expression and plasmid transfer.

In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes inRhizobium leguminosarumbiovarviciaestrains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involvinggusA–gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes ofR. leguminosarumcould be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression ofrem, while Rem positively regulates the expression offlaA,flaB,flaC,flaD,motA,motB,che1andmcpD. All of these genes exceptmcpDare located within the main motility and chemotaxis gene cluster ofR. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2operon,flaHfor VF39SM, andflaG) or are plasmid-borne (flaEandmcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, exceptflaEandflaH.flaEis not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM.flaHis under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility forR. leguminosarumunder free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem.

We have characterized swarming motility in Rhizobium leguminosarum strains 3841 and VF39SM. Swarming was dependent on growth on energy-rich media, and both agar concentration and incubation temperature were critical parameters for surface migration. A cell density-dependent lag period was observed before swarming motility was initiated. Surface migration began 3–5 days after inoculation and a full swarming phenotype was observed 3 weeks after inoculation. The swarming front was preceded by a clear extracellular matrix, from which we failed to detect surfactants. The edge of the swarming front formed by VF39SM was characterized by hyperflagellated cells arranged in rafts, whereas the cells at the point of inoculation were indistinguishable from vegetative cells. Swarmer cells formed by 3841, in contrast, showed a minor increase in flagellation, with each swarmer cell exhibiting an average of three flagellar filaments, compared with an average of two flagella per vegetative cell. Reflective of their hyperflagellation, the VF39SM swarmer cells demonstrated an increased expression of flagellar genes. VF39SM swarmed better than 3841 under all the conditions tested, and the additional flagellation in VF39SM swarm cells may contribute to this difference. Metabolism of the supplemented carbon source appeared to be necessary for surface migration as strains incapable of utilizing the carbon source failed to swarm. We also observed that swarmer cells have increased resistance to several antibiotics.

Rhizobium leguminosarum biovar viciae strain 3841 is a motile alpha-proteobacterium that can establish a nitrogen-fixing symbiosis within the roots of pea plants. In order to determine the contribution of chemotaxis to the lifestyle of R. leguminosarum, we have characterized the function of two chemotaxis gene clusters (che1 and che2) in controlling motility behaviour. We have found that both chemotaxis gene clusters modulate the motility swimming bias of R. leguminosarum cells and that the che1 cluster is the major pathway controlling swimming bias and chemotaxis. The che2 cluster also contributes to swimming bias, but has a minor effect on chemotaxis. Using competitive nodulation assays, we have demonstrated that a functional che1 cluster, but not the che2 cluster, promotes competitive nodulation of the peas. This finding implies that the environmental cue(s) triggering chemotaxis of R. leguminosarum bv. viciae cells towards the roots of pea and facilitating colonization are likely to be processed through the che1 cluster despite the contribution of both che clusters to swimming behaviour. A phylogenetic analysis of the distribution of che1 and che2 orthologues in the alpha-proteobacteria together with our results allow us to propose that che1 homologues are major controllers of chemotaxis and host association in the Rhizobiaceae.

A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. A Tn5-B22 mutant (19B-3) unable to grow on erythritol was isolated from a mutant library of R. leguminosarum strain VF39SM. The mutated gene eryF was cloned and partially sequenced, and determined to have a high homology to permease genes of ABC transporters. A cosmid complementing the mutation (pCos42) was identified and was shown to carry all the genes necessary to restore the ability to grow on erythritol to a VF39SM strain cured of pRleVF39f. In the genomic DNA sequence of strain 3841, the gene linked to the mutation in 19B-3 is flanked by a cluster of genes with high homology to the known erythritol catabolic genes from Brucella spp. Through mutagenesis studies, three distinct operons on pCos42 that are required for growth on erythritol were identified: an ABC-transporter operon (eryEFG), a catabolic operon (eryABCD) and an operon (deoR-tpiA2-rpiB) that encodes a gene with significant homology to triosephosphate isomerase (tpiA2). These genes all share high sequence identity to genes in the erythritol catabolism region of Brucella spp., and clustalw alignments suggest that horizontal transfer of the erythritol locus may have occurred between R. leguminosarum and Brucella. Transcription of the eryABCD operon is repressed by EryD and is induced by the presence of erythritol. Mutant 19B-3 was impaired in its ability to compete against wild-type for nodulation of pea plants but was still capable of forming nitrogen-fixing nodules.

Acquired β-lactamase genes in Pseudomonas aeruginosa are often associated with transposons and integrons and carried on R plasmids, which can be classified into 13 incompatibility groups (P1 to 13) with a wide range of sizes (8 to 483 kb). The IncP2 plasmids are described as particularly common in P. aeruginosa (50% of the transmissible plasmids), are very large, and are distributed worldwide (13). Carriage of metallo-β-lactamase (MBL) genes of the blaIMP or blaVIM type has been reported infrequently on plasmids in P. aeruginosa. A limited number of blaIMP/VIM genes, such as blaIMP-1, -3, -10, and -12 and blaVIM-2, have been reported on plasmids with a size range of 31 to 56 kb in P. aeruginosa or P. putida (5, 10, 11, 15, 20, 24). Some studies have failed to find plasmids in carbapenem-resistant P. aeruginosa even though the resistance marker was transferred by conjugation (16). The carbapenem resistance rates in P. aeruginosa isolates in the city of Guangzhou, China, have been reported to be 16 to 18% during the period 1998 to 2001, according to a multicenter surveillance of antibiotic resistance in nosocomial isolates from that area (25). In this report, we describe the identification of a new variant of the IMP-type plasmid-mediated MBL gene in carbapenem-resistant isolates of P. aeruginosa from that area. In the year 2000, a total of 301 clinically significant and nonduplicated P. aeruginosa isolates were collected from the 12 participating centers, and 54 isolates from 11 centers were found to be resistant to imipenem by disk screening (zone diameter, ≤17 mm) (Clinical and Laboratory Standards Institute [formerly National Committee on Clinical and Laboratory Standards]); 29 of them were randomly selected as representative isolates from each of the 11 centers. The standard strains used in this study as quality controls or for conjugation and plasmid incompatibility testing are listed in Table ​Table1.1. The PCR primers used are listed in Table ​Table22. TABLE 1. Bacterial strains used in this study TABLE 2. Oligonucleotide primers used for PCR amplification and sequencing in this study By the Etest MBL (AB BIODISK, Solna, Sweden), 19 of the 29 P. aeruginosa isolates were positive for production of MBL. Seven of the 29 isolates screened with PCR primers IMP-1 and -2 were positive. No blaVIM genes were detected in the 29 isolates. The discrepancy between the results of Etest MBL (19/29 positive) and PCR (7/19) could be explained by the presence of carbapenemases other than IMP- or VIM-type enzymes. The blaIMP-positive isolates were found to carry a new blaIMP variant, named blaIMP-9, as identified by direct sequencing of both strands of the PCR amplicon with primers IMP-13 and IMP-15 (3). It was 82 to 91% homologous to other blaIMP alleles, and its product was 78 to 91% identical to the other IMP-type enzymes (http://www.ncbi.nlm.nih.gov/). In isolate 96, the blaIMP-9 gene was found to be carried on a gene cassette inserted between a 5′- and a 3′-conserved segment typical of sul-1-associated integrons, as determined by PCR mapping and sequencing with primers listed in Table ​Table22 (EMBL/GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY033653","term_id":"496684371"}}AY033653). The integron cassette array included five gene cassettes: aacA4→blaIMP-9→aacA4→catB8→blaOXA-10. The blaIMP-9 gene was found in an identical genetic context in the other six blaIMP-positive isolates. Interestingly, 59be of the blaIMP-9 gene cassette is most closely related (only one nucleotide difference, position 776 G→C in {"type":"entrez-nucleotide","attrs":{"text":"AB074437","term_id":"17026027"}}AB074437) to that of blaIMP-11 (89% homologous to blaIMP-9) found in P. aeruginosa from Japan (S. Iyobe et al., unpublished data), while it was more divergent (83% homology) from that of blaIMP-5, despite a higher similarity (91%) in their β-lactamase genes. Nucleotide sequence analysis of the amplicon obtained from isolate 96 showed a hybrid Pant promoter identical to that of In1 in R46 (17) and also to that carried by the blaIMP-4 gene-containing integron described in a Citrobacter youngae from the same area (7). The β-lactam MICs determined by the agar dilution method (19) and clinical data of the seven blaIMP-carrying P. aeruginosa isolates are shown in Table ​Table3.3. Interestingly, upon MIC testing some isolates appeared to be carbapenem intermediate or even susceptible and meropenem appeared to be consistently more active than imipenem against those isolates. The isolates were sensitive or borderline resistant to ciprofloxacin, amikacin, or gentamicin. All seven blaIMP-positive isolates were also resistant to potassium tellurite, with MICs of 10−3 M by a quantitative method (23). TABLE 3. Susceptibility profiles and MBL production of blaIMP-9-carrying P. aeruginosa isolates and their transconjugants Conjugal transfer of resistance (1) to carbapenems and other β-lactams was demonstrated with four of the blaIMP-9-carrying P. aeruginosa isolates with P. aeruginosa NCTC 50814 as the recipient, but not with Escherichia coli UB1637/R (4). In all cases, the transfer of blaIMP-9 was confirmed by Southern hybridization with a specific probe and PCR amplification with primers IMP-1 and -2 (Fig. 1A and B and data not shown). Resistance to tellurite was also transferred. Tellurite MICs for transconjugants were higher than that for recipient strain NCTC 50814 (10−4 M versus ≤10−5 M), although they were lower than those for donors (see above). FIG. 1. Plasmids (A), hybridization profiles (B), and sizing of plasmids (C) of IMP-9-producing P. aeruginosa isolates and their transconjugants. The plasmids were prepared by a modification of the Eckhardt method. (A) Plasmid patterns of the isolates of 96 and ... The plasmid content of P. aeruginosa 96 and its transconjugant was investigated by a modification of the Eckhardt method for isolation of large plasmids (8, 9). The size of the plasmids carried by P. aeruginosa 96 and by its transconjugant 96T was almost the same as that of the larger plasmid of Agrobacterium tumefaciens C58 (pAtC58 [543 kb]), the D plasmid (pRL10JI) of Rhizobium leguminosarum 3841 (488 kb), and the D plasmid (ca. 500 kb) of R. leguminosarum VF39 by visual estimation (Fig. ​(Fig.1C).1C). From the plotted standard curve, the size of the plasmid was determined to be about 450 to 500 kb and the plasmid was designated pOZ176. IncP group incompatibility tests (22) showed that pOZ176 and an IncP9-carrying plasmid (R2) could be transferred reciprocally and could replicate in the same cell. Their coreplication was confirmed by the phenotypic changes in resistance markers, plasmid profile, and PCR detection of the blaIMP gene. In contrast, introduction of pOZ176 eliminated the IncP2 plasmid from P. aeruginosa PAO1(pBS31), P. putida ML4262(pBS228), and P. putida ML4262(R2) (Table ​(Table1),1), as confirmed by changes in the properties of the transconjugants. Furthermore, to test for the possibility of plasmid hybridization and incompatibility between IncP2 plasmids, P. aeruginosa 96 was crossed with PU21 (12) and PU21(pOZ176) was then crossed with PAO2003(CAM) (containing an IncP2 plasmid encoding functions for camphor degradation) (2), and PAO2003 was used as a control. Loss of the Cam+ phenotype (14) was observed in 90% of the resulting transconjugants, indicating the incompatibility of the IncP2 plasmids from P. aeruginosa PAO2003 and 96. However, the phenotypes of both CAM and pOZ176 were also found in some of the transconjugants, which suggests the possibility of generation of CAM-pOZ176 recombinant plasmids; such IncP2 hybrid plasmid formation was first reported in P. aeruginosa in 1974 (12). The evidence for pOZ176 being an IncP2 plasmid is as follows: (i) pOZ176 mediates transferable tellurite resistance (13); (ii) pOZ176 was not stable with an IncP2 plasmid and was able to form a recombinant plasmid with a Cam+ plasmid (IncP2), possibly via homologous DNA recombination or transposon-mediated cointegration (14); and (iii) the plasmid is large with a limited host range (13). Randomly amplified polymorphic DNA typing (18) showed that the seven blaIMP-positive isolates from four hospitals were nonclonal, except isolates 96 and 121, which were from the same hospital and ward. This finding, together with identification of the blaIMP-9 gene on a transferable plasmid, suggests that spreading of carbapenem resistance mediated by IMP-9 MBL in P. aeruginosa in our area is largely due to horizontal transfer of the gene, most likely on a large R plasmid similar to pOZ176.

Regulation of methyl-accepting chemotaxis protein (MCP) genes of Rhizobium leguminosarum was studied under symbiotic conditions. Transcriptional fusions using both beta-galactosidase and beta-glucuronidase genes within two different mcp genes demonstrated that mcp expression decreased significantly during nodulation. Immunoblots using an anti-MCP antibody detected MCPs in free-living cells but not in bacteroids. Down-regulation during nodulation was not dependent upon known regulatory proteins involved in induction of expression of genes involved in nitrogen fixation. Environmental conditions found in the bacteroid that may trigger down-regulation were investigated by growing free-living cultures under a variety of growth conditions. Growth under low oxygen concentration or using succinate as a sole carbon source did not lower expression of the mcp gene fusions.

A new method for preparing alkyl esters of indole-3-acetic acid (IAA) in aqueous solution is adapted from the chloroformate method originally described by Husek for the analysis of amino acids. This method has the significant advantage of avoiding the generation and use of diazomethane, and is done in aqueous solution without the need to dry the sample with concomitant non-specific losses of IAA. The effectiveness of this method is demonstrated by its use in an isotope dilution gas chromatography-mass spectrometry (GC-MS) assay of IAA and L-tryptophan (Trp) in the culture supernatant of a series of Sinorhizobium meliloti and Rhizobium leguminosarum bv. trifolii strains that can interact with rice to either enhance or inhibit rice plant growth. We were testing the hypothesis that the rice growth inhibition was related to the biosynthesis of IAA. It was found that S. meliloti and Rhizobium strains produced high amounts of IAA in Trp supplemented BIII minimal medium compared to BIII media. All the strains produced more than the minimum amount of IAA required to inhibit rice growth and thus IAA is not the major inhibitory factor of rice seedling growth from S. meliloti and Rhizobium strains.

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

The six plasmids of Rhizobium leguminosarum VF39SM comprise nearly 35% of the bacterium's genome and are all repABC replicons. The repABC operons of the three largest plasmids of VF39SM were found to have strong incompatibility determinants in the non-protein coding regions. However, in all three repABC operons, the intergenic region between repB and repC was the strongest incompatibility factor; this intergenic region has been shown, for most repABC plasmids, to encode a counter-transcribed RNA (ctRNA) that regulates RepC abundance and therefore also rate of initiation of replication. To understand the way in which the ctRNA regulates replication and incompatibility, we carried out mutagenesis on this region from all three plasmids, using error-prone PCR. Mutants with altered incompatibility were detected by screening for their ability to co-exist in the same cell as the parent plasmid. Mutations that abolished the strong incompatibility phenotype were nearly all localized to the predicted ctRNA promoter regions. RT-PCR analysis confirmed that ctRNA was still produced in these promoter mutants, but transcriptional fusions of these mutated promoters to a gusA reporter gene showed a 10- to 50-fold decrease in activity when compared with the wild type promoter. For the repABC operons in this study, the intergenic region is critical in establishing incompatibility, and this appears to require a high level of transcription of the ctRNA.

Cross-testing of a number of strains of Rhizobium leguminosarum for bacteriocin production revealed that strain 306 produced at least two distinct bacteriocins. Further analysis involving plasmid transfer to Agrobacterium and other hosts demonstrated that there were bacteriocin determinants on plasmids pRle306b and pRle306c, as well as a third bacteriocin. The bacteriocin encoded by pRle306b was indistinguishable from the bacteriocin encoded by strain 248, whereas the bacteriocin encoded by plasmid pRle306c had a distinctive spectrum of activity against susceptible strains, as well as different physical properties from other bacteriocins that we have studied in our lab. Two mutants altered in production of the pRle306c bacteriocin were generated by transposon Tn5 mutagenesis, and the DNA flanking the transposon inserts in these mutants was cloned and characterized. DNA sequence analysis suggested that the pRle306c bacteriocin was a large protein belonging to the RTX family, and that a type I secretion system involving an ABC type transporter was required for export of the bacteriocin. A mutant unable to produce this bacteriocin was unaltered in its competitive properties, both in broth and in nodulation assays, suggesting that the bacteriocin may not play a major role in determining the ecological success of this strain.Key words: Rhizobium, bacteriocins, RTX proteins, plasmids.

The rpoN gene, which codes for the alternative transcription factor sigma54, was cloned and sequenced from Rhizobium leguminosarum strain VF39SM. Construction of a rpoN mutant allowed analysis of the role of RpoN as a transcriptional regulator of genes carrying lacZ reporter fusions. Analysis of a rpoN::lacZ transcriptional fusion in the rpoN background revealed that this gene was negatively autoregulated. Site-directed mutagenesis was used to demonstrate that this autoregulation was dependent on a reverse complement RpoN binding site located upstream of the rpoN gene. rpoN was shown to be required for full microaerobic expression of both copies of fixGHIS, as well as of fixNOQP, despite the absence of apparent rpoN binding sites upstream of fixG. Moreover, rpoN was found to be required for full microaerobic expression of fnrN, which in turn is absolutely required for microaerobic induction of fixGHIS. This suggests that the reduced fixG::lacZ expression seen in the rpoN background is due to the dependence of fnrN expression on RpoN.

A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn 5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn 5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both R. leguminosarum and Sinorhizobium meliloti . A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.

Cosmids carrying genes involved in utilization of rhamnose, sorbitol, and adonitol were isolated from a genomic library of Rhizobium leguminosarum by complementation of plasmid-cured derivatives of strain Rlt100 that were unable to grow on these carbon sources. Transposon mutagenesis was used to identify regions of each cosmid necessary for catabolism of the respective carbon source; partial DNA sequencing, as well as analysis of gene fusions created with transposon Tn5-B20, helped to determine the orientation and possible function of genes required for growth on the three substrates. Representative Tn5 insertions in the cosmids were recombined into the wild-type strain Rlt100 by gene replacement to generate isogenic strains unable to use either rhamnose, sorbitol, or adonitol. These strains were tested for their nodulation competitiveness compared with Rlt100 in co-inoculation experiments on clover plants. While sorbitol and adonitol catabolic mutants were unaltered in their competitive behavior, the nodulation competitiveness of three different rhamnose utilization mutants was significantly impaired. This result, coupled with the fact that the rhamnose catabolic genes were inducible by clover root extracts, suggests an important role for rhamnose catabolism in the early stages of the interaction of R. leguminosarum with clover plants. Hybridization studies with probes derived from the rhamnose, sorbitol, and adonitol catabolic loci demonstrated that these genes are plasmid encoded in virtually all R. leguminosarum strains, including representatives from all three biovars from a variety of different geographic locations.

Methyl-accepting chemotaxis proteins (MCPs) play important roles in the chemotactic response of many bacteria. Oligonucleotide primers designed to amplify the conserved signalling domain of MCPs by PCR were used to identify potential MCP-encoding genes in Rhizobium leguminosarum. Using a PCR-derived probe created from these primers a genomic library of R. leguminosarum VF39SM was screened; at least five putative MCP-encoding genes (termed mcpB to mcpF) were identified and isolated from the library. One of these putative genes (mcpC) is located on one of the indigenous plasmids of VF39SM. Fifteen different cosmids showing homology to an mcpD probe were also isolated from a genomic library. The complete DNA sequences of mcpB, mcpC and mcpD were obtained. All three genes code for proteins with characteristics typical of MCPs. However, the protein encoded by mcpB has a relatively large periplasmic domain compared to that in other MCPs. Partial DNA sequences of mcpE and mcpF had strong similarity to sequences from the methylation domains of known MCPs. Mutants defective in mcpB, mcpC, mcpD or mcpE were created using insertional mutagenesis strategies. Mutation of mcpB resulted in impairment of chemotaxis to a wide range of carbon sources on swarm plates; phenotypes for the other three mutants have yet to be elucidated. The mcpB, mcpC and mcpD mutants were tested for loss of nodulation competitiveness. When co-inoculated with the wild-type, the mcpB and mcpC mutants formed fewer nodules than the wild-type, whereas the mcpD mutant was just as competitive as the wild-type. The results overall suggest that R. leguminosarum possesses mcp-like genes, and that at least some of these play a role in early steps in the plant-microbe interaction.

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncP alpha classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.

The δ -endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a δ -endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and δ -endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that

Plasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium leguminosarum. We isolated the locus responsible for glycerol utilization from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase, respectively. All the genes except the regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp operon was strongly induced by both glycerol and glycerol 3-phosphate, as well as by pea seed exudate. GlpR repressed the operon in the absence of inducer. Mutation of genes encoding the ABC transporter abolished all transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other organisms such as Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R. leguminosarum and Rhizobium etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol were deficient in competitiveness for nodulation of peas compared with the wild-type, suggesting that glycerol catabolism confers an advantage upon the bacterium in the rhizosphere or in the infection thread.

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 x 10(-7) per donor and 1.11 x 10(-5) per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 x 10(-6) per donor and 4.70 x 10(-8) per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.

An amylase gene was identified in a Streptococcus bovis 033 lambda gtWES lambda B genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, lambda SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.

This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures. The pairs of plasmids p42f-pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of p42c with pRL10 are shown to be similar, whereas the symbiotic plasmids (p42d and pRL10) are structurally unrelated and seem to follow distinct evolutionary paths. Even though purifying selection is acting on the whole genome, the accessory component is evolving more rapidly. This component is constituted largely for proteins for transport of diverse metabolites and elements of external origin. The present analysis allows us to conclude that a heterogeneous and quickly diversifying group of plasmids co-exists in a common genomic framework.

Analysis of plasmid profiles was used to type Rhizobium leguminosarum biovar viciae strains isolated from nodules of peas, lentils and faba beans grown in two different soils. One soil was from a native pasture with no previous history of cultivation, the other was from a plot in a rotation study which included lentils every 2 years. The results indicated a strong preference of both peas and faba beans for strains having certain specific plasmid profiles. Strains belonging to one plasmid profile group (group 2) formed over half the nodules on peas grown in soil from the rotation plot but were never found on faba beans grown in the same soil, while strains from another group (group 5) formed nearly all of the nodules on faba beans grown in soil from the rotation plot, but no nodules on peas. Competitiveness for pea nodulation was correlated with an ability to catabolize homoserine, an amino acid found in large quantities in pea root exudate. Strains having plasmid profiles corresponding to those of strains that have been used in commercial inoculants over the last few years were isolated only rarely, regardless of the soil and host plant studied. Key words: Rhizobium, competition, plasmids, legumes, nodulation.

Summary A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRIeVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRIeVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix− nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRIeVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.

Rhizobium leguminosarum bv. trifolii is a gram-negative aerobic soil bacterium that can exist as a free-living heterotropic saprophyte or can form nitrogen-fixing nodules on various species of clovers (Trifolium spp.). The interaction of rhizobia with their hosts is a sequence of events that begins with an exchange of signals in the rhizosphere, followed by the invasion of the plant through infection threads and ultimately by release of the bacteria into plant cells, where they differentiate into nitrogen-fixing bacteroids. Through this symbiotic association, the plant provides the bacteria with energy for growth, and in return the Rhizobium provides the plant with fixed nitrogen. Although a great deal has been elucidated regarding the actual steps during nodulation (27, 56) and the metabolic pathways utilized while in the bacteroid state (39), comparatively little is known about what influences the growth of the bacteria prior to or during their interaction with plant roots. The rhizosphere has been described as the region directly under the influence of secreted compounds from the plant root (12). Competition for nodule occupancy exists between strains of Rhizobium within the rhizosphere and has been well documented in the literature (16, 54). A large number of biotic and abiotic factors influence competition between strains for nodule occupancy. Some of the best-characterized strain-dependent factors include production of antibiotics and bacteriocins (36, 42, 57) and the ability to catabolize specific compounds present in the root environment (11, 13, 18, 22, 23, 35, 43, 49, 55), as well as the synthesis or utilization of specific vitamins (38, 50). Rhamnose is a methyl-pentose sugar and is found as a constituent of pectin in the form of rhamnogalacturonan within the cell walls of dicotyledonous plants (28). It has also been found in the mucilage of a number of legume plants (26). R. leguminosarum mutants unable to utilize rhamnose as a sole carbon source were found to be significantly impaired in their nodulation competitiveness (35). The reason for the uncompetitive nature of these mutants is not understood. It was hypothesized that if the genetic region responsible for the catabolism of rhamnose were characterized, a better understanding of why these mutants were uncompetitive could be achieved. The objective of the present work, therefore, was to identify and characterize the genes in this region.

We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms. The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene. This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions. In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed. This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids. A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives. As an example of the use of these tools, three Rhizobium leguminosarum bv. viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2. None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.

In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia.Key words: Rhizobium, malate synthase, glyoxylate cycle, arabinose metabolism.

R. leguminosarum plasmids encode a large number of genes responsible for catabolism of a wide variety of sugars, amino acids and other carbon sources. Analysis of the role some of the catabolic genes play in influencing competition for nodulation, using isogenic mutant strains, has demonstrated that catabolism of rhamnose plays a significant role in the clover symbiosis, whereas catabolism of ribitol and sorbitol does not. Since chemotaxis towards carbon sources may also be important in competition, we have analysed a number of genes potentially encoding chemotaxis chemoreceptors, and shown that two of these do affect competitive success of a R. leguminosarum bv. viciae strain on pea plants. These chemoreceptor genes appear to be down regulated during symbiosis.

Antirhizobial compounds such as bacteriocins, inhibitory agents which limit the growth of closely related strains, may play a role in interstrain competition in the field. Some strains produce larger molecular weight molecules referred to here as rhizobiocins (medium bacteriocins) which have remained uncharacterized. The rhizobiocin determinants from R. leguminosarum strains by. viciae 248 and by. trifolii 162Y10 were isolated and, through Southern analysis, shown to be plasmid encoded. Characterization was carried out through transposon mutagenesis and sequence analyses. In both cases, the production region demonstrated substantial homology to the calcium-binding regions of RTX toxins, a family of pore-forming cytolysins produced by Gram-negative bacterial pathogens. Support for a calcium-binding function for the rhizobiocin from strain 248 was obtained by a bioassay of bacteriocin production against other sensitive strains of R. leguminosarum which demonstrated calcium-dependence. Even though both 248 and 162Y10 bacteriocins show homology to RTX toxins, cross-hybridization was not observed by Southern analysis. In the case of 162Y10, a region encoding a putative ABC (ATP-binding transporter)-type transporter is necessary for rhizobiocin production and is located adjacent to the RTX locus. This is in contrast to 248 for which a specific transporter has not yet been identified.

Most R. leguminosarum strains produce bacteriocins which have been traditionally classified as small and medium. based on apparent size (Hirsch, 1979). The small bacteriocin has been identified as a N-acetylated homoserine lactone (Schripsema et al., 1996) but the structure and function of the medium bacteriocins remain uncharacterized. We isolated and began characterization of the medium encoding regions from two different R. leguminosarum strains, bv. viciae 248 and bv. trifola 162Y10. In both cases, sequencing data for the production region demonstrated substantial homology to the calcium-binding regions of RTX toxins such as haemolysin (hly A) and leukotoxin (lkt A). These pore-forming cytolysins are produced by Gram-negative bacterial pathogens such as E. coli and Pasteurella haemolytica (Welch, 1991). Indeed support for a calcium-binding function for the bacteriocin from strain 248 was obtained when the bacteriocin bioassay for this strain demonstrated exogenous calcium-dependence. Even though both 248 and 162Y10 bacteriocins show homology to RTX toxins, cross-hybridization was not observed by Southern analysis under low stringency conditions. In fact, probes from both the 162Y10 and 248 RTX-like regions did not detect homologous determinants in any other medium producing Rhizobium strains tested indicating that these bacteriocins are unique. The 162Y10 bacteriocin also requires an ABC-type transporter for expression which is a novel feature of this strain since it is not found in 248.

This chapter is about the “general genetic knowledge” of the family Rhizobiaceae, Since this “family” is not a true family in any phylogenetic sense, it is very difficult to generalise about the genetics of the group (see Chapter 1). The phylogeny of its members will be discussed elsewhere, but suffice it to say here that the “family” has traditionally been defined in terms of the ability of the bacteria belonging to it to induce the formation of certain types of growths (e.g. nodules, tumours) on plants (Jordan, 1982). Since it has usually been because of this ability to interact with plants that individual members of the “family” have been studied, much information available about the genetics of these organisms is biased towards study of genes involved in symbiotic or pathogenic interactions. It is our intent in this chapter to review the basic genetics of some of the better-studied members of the Rhizobiaceae with an emphasis on tools available for genetic study. Where useful information regarding more “exotic” bacteria is available it will be introduced. The discussion will centre on Rhizobium leguminosarum, Rhizobium (syn. Sinorhizobium) meliloti, Agrobacterium spp. and Bradyrhizobium japonicum.

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.

Early seedling root growth of the nonlegumes canola (Brassica campestris cv. Tobin, Brassica napus cv. Westar) and lettuce (Lactuca saliva cv. Grand Rapids) was significantly promoted by inoculation of seeds with certain strains of Rhizobium leguminosarum, including nitrogen- and nonnitrogen-fixing derivatives under gnotobiotic conditions. The growfh-promotive effect appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. Auxotrophic Rhizobium mutants requiring tryptophan or adenosine (precursors for indole-3-acetic acid and cytokinin synthesis, respectively) did not promote growth to the extent of the parent strain. The findings of this study demonstrate a new facet of the Rhizobium–plant relationship and that Rhizobium leguminosarum can be considered a plant growth-promoting rhizobacterium (PGPR).Key words: Rhizobium, plant growth-promoting rhizobacteria, PGPR, indole-3-acetic acid, cytokinin, roots, auxotrophic mutants., La croissance précoce des racines de plantules de non-légumineuses, le colza (Brassica campestris cv. Tobin, Brassica napus cv. Westar) et la laitue (Lactuca sauva cv. Grand Rapids), a été accrue de façon significative par suite de l'inoculation des graines avec certaines souches de Rhizobium leguminosarum incluant des dérivés fixateurs et non fixateurs d'azote, ceci dans des conditions gnotobiotiques. L'effet promoteur sur la croissance semble avoir été direct, avec une implication possible de l'acide indole-3-acétique et de la cytokinine, régulateurs de la croissance végétale. Des mutants auxotrophes de Rhizobium qui ont besoin de tryptophane ou d'adénosine, lesquels sont respectivement les précurseurs de la synthèse de l'acide indole-3-acétique et de la cytokinine, n'ont pas autant favorisé la croissance que les souches non auxotrophes. Les résultats de cette étude montrent une nouvelle facette de la relation plante–Rhizobium et que le Rhizobium leguminosarum peut être considéré comme une rhizobactérie...

Populations of Rhizobium leguminosarum bv. phaesoli and bv. viceae in southern Alberta soils were measured over a period of 4 years using a most probable number method. Five fields cropped to bean (Phaseolus vulgaris L.), five fields cropped to pea (Pisum sativum L.), and two fields cropped to wheat were used as test sites. Legume crops had received appropriate legume inoculants. Fields were sampled in the fall of the crop year and in the spring of the following 3 years during which fields were cropped to nonlegumes or left fallow. Numbers of R. leguminosarum bv. phaseoli were 100 to 1000 times higher in fields that had been planted to bean than in fields that had been planted to pea or wheat. Fields that had been planted to pea maintained populations of R. leguminosarum bv. viceae 10 to 100 times higher than fields that had been planted to bean or wheat. Wheat fields, which had never had legumes grown in them, contained between 1 and 100 rhizobia per gram of soil of both biovars of R. leguminosarum, indicating that both biovars are native to southern Alberta soils. The numbers of rhizobia did not decrease in proportion to the population of other bacteria in the soil over the duration of the experiment. Plasmid profiles of soil Rhizobium isolates obtained in the last year of the experiment showed that none of the isolates had plasmid profiles similar to those of strains added as inoculants in the 1st year of the experiment. These results show that fields cropped to legumes and receiving rhizobial inoculants in this study maintained high populations of rhizobia for several years after harvest of the legume crop.Key words: Rhizobium leguminosarum bv. phaseoli, Rhizobium leguminosarum bv. viceae, nodule, plasmid profiles, inoculum potential, rhizobium competition.

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.