Characterization of two plasmid-borne lps beta loci of Rhizobium etli required for lipopolysaccharide synthesis and for optimal interaction with plants.

In Rhizobium etli CFN42, both the symbiotic plasmid (pd) and plasmid b (pb) are required for effective bean nodulation. This is due to the presence on pb of a region (lps beta) involved in lipopolysaccharide (LPS) biosynthesis. We report here the genetic array and functional features of this plasmid-borne region. The sequence analysis of a 3,595-bp fragment revealed the presence of a transcriptional unit integrated by two open reading frames (lps beta 1 and lps beta 2) essential for LPS biosynthesis and symblosis. The lps beta 1 encodes a putative 193 amino acid polypeptide that shows strong homology with glucosyl-1P and galactosyl-1P transferases. The deduced amino acid sequence of the protein encoded by lps beta 2 was very similar to that of proteins involved in surface polysaccharide biosynthesis, such as Pseudomonas aeruginosa WpbM, Bordetella pertussis BpIL, and Yersinia enterocolitica TrsG. DNA sequences homologous to lps beta 1 and lps beta 2 of R. etli CFN42 were consistently found in functionally equivalent plasmids of R. etli, R. leguminosarum bv. viciae, and R. leguminosarum hv. trifolii strains, but not in R. meliloti, R. loti, R. tropici, R. fredii, Bradyrhizobium, Azorhizobium, and Agrobacterium tumefaciens. Even though Rhizobium and Agrobacterium do not share lps beta sequences, their presence is required for crown-gall tumor induction by R. etli transconjugants carrying the Ti plasmid.

Rhizobium plasmids in bacteria-legume interactions.

The functional analysis of plasmids in Rhizobium strains has concentrated mainly on the symbiotic plasmid (pSym). However, genetic information relevant to both symbiotic and saprophytic Rhizobium life cycles, localized on other 'cryptic' replicons, has also been reported. Information is reviewed which concerns functional features encoded in plasmids other than the pSym: biosynthesis of cell surface polysaccharides, metabolic processes, the utilization of plant exudates, aromatic compounds and diverse sugars, and features involved symbiotic performance. In addition, factors which affect plasmid evolution through their influence on structural features of the plasmids, such as conjugative transfer and genomic rearrangements, is discussed. Based on the overall data, we propose that together the plasmids and the chromosome constitute a fully integrated genomic complex, entailing structural features as well as saprophytic and cellular functions.

In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons.

Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles.

Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis.

Fifty rhizobial isolates from root nodules of Mimosa affinis, a small leguminous plant native to Mexico, were identified as Rhizobium etli on the basis of the results of PCR-RFLP and RFLP analyses of small-subunit rRNA genes, multilocus enzyme electrophoresis and DNA-DNA homology. They are, however, a restricted group of lineages with low genetic diversity within the species. The isolates from M. affinis differed-from the R. etli strains that orginated from bean plants (Phaseolus vulgaris) in the size and replicator region of the symbiotic plasmid and in symbiotic-plasmid-borne traits such as nifH gene sequence and organization, melanin production and host specificity. A new biovar, bv. mimosae, is proposed within R. etli to encompass Rhizobium isolates obtained from M. affinis. The strains from common bean plants have been designated previously as R. etli bv. phaseoli. Strains of both R. etli biovars could nodulate P. vulgaris, but only those of bv. mimosae could form nitrogen-fixing nodules on Leucaena leucocephala.

Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance.

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.

High-frequency rearrangements in Rhizobium leguminosarum bv. phaseoli plasmids.

High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv. phaseoli CFN42 were analyzed. This strain contains six large plasmids ranging in size from 200 to 600 kb. In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids. These data support the concept that the R. leguminosarum bv. phaseoli genome is a dynamic structure and imply that strains are mixtures of similar but not identical cells.