Tn5-induced and spontaneous switching of Sinorhizobium meliloti to faster-swarming behavior.
Tn5 mutants of Sinorhizobium meliloti RMB7201 which swarmed 1.5 to 2. 5 times faster than the parental strain in semisolid agar, moist sand, and viscous liquid were identified. These faster-swarming (FS) mutants outgrew the wild type 30- to 40-fold within 2 days in mixed swarm colonies. The FS mutants survived and grew as well as or better than the wild type under all of the circumstances tested, except in a soil matrix subjected to air drying. Exopolysaccharide (EPS) synthesis was reduced in each of the FS mutants when they were grown on defined succinate-nitrate medium, but the extent of reduction was different for each. It appears that FS behavior likely results from a modest, general derepression of motility involving an increased proportion of motile and flagellated cells and an increased average number of flagella per cell and increased average flagellar length. Spontaneous FS variants of RMB7201 were obtained at a frequency of about 1 per 10,000 to 20,000 cells by either enrichment from the periphery of swarm colonies or screening of colonies for reduced EPS synthesis on succinate-nitrate plates. The spontaneous FS variants and Tn5 FS mutants were symbiotically effective and competitive in alfalfa nodulation. Reversion of FS variants to wild-type behavior was sporadic, indicating that reversion is affected by unidentified environmental factors. Based on phenotypic and molecular differences between individual FS variants and mutants, it appears that there may be multiple genetic configurations that result in FS behavior in RMB7201. The facile isolation of spontaneous FS variants of Escherichia coli and Pseudomonas aeruginosa indicates that switching to FS behavior may be fairly common among bacterial species. The substantial growth advantage of FS mutants and variants wherever nutrient gradients exist suggests that switching to FS forms may be an important behavioral adaptation in natural environments. (+info)
Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB.
Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation. Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI). Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of orfA. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in S. meliloti and to the consensus PHO box in Escherichia coli. Introduction of a phoB mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the ackA::lacZ fusion. Mutations in either ackA alone or both pta and ackA did not affect the nodulation or nitrogen fixation phenotype of S. meliloti. (+info)
Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti.
Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in S. meliloti and R. leguminosarum also acted as very effective competitors of [14C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for S. meliloti and R. leguminosarum did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S. meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed. (+info)
Differential regulation of two divergent Sinorhizobium meliloti genes for HPII-like catalases during free-living growth and protective role of both catalases during symbiosis.
Two catalases, KatA and KatB, have been detected in Sinorhizobium meliloti growing on rich medium. Here we characterize a new catalase gene encoding a third catalase (KatC). KatC activity was detectable only at the end of the stationary phase in S. meliloti growing in minimum medium, whereas KatA activity was found during the exponential phase. Analysis with a katC-lacZ fusion demonstrated that katC expression is mainly regulated at the transcription level. An increase of catalase activity correlating with KatA induction was detected in bacteroids. A dramatic decrease of nitrogen fixation capacity in a katA katC double mutant was observed, suggesting that these catalases are very important for the protection of the nitrogen fixation process. (+info)
Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR.
The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the alternative exopolysaccharide galactoglucan (EPS II) in addition to succinoglycan (EPS I). In the wild-type strain EPS II production is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was identified in the exp gene cluster. Using exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG gene on the transcription of all exp complementation groups was determined. Phosphate limitation also resulted in increased expression of the exp-lacZ fusions. This increase was reduced in strains characterized by a deletion of expG. The previously reported high level of exp gene transcription in a mucR mutant was further elevated under phosphate-limiting conditions. The expA, expD, expG and expE promoters contain sequences with similarities to the PHO box known as the PhoB-binding site in phosphate-regulated promoters in Escherichia coli. The S. meliloti phoB gene was required for the activation of exp gene expression under phosphate limitation, but not for induction of exp expression by MucR or ExpG. (+info)
Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine.
Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine. The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine. Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases. Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival. This is believed to be the first report of a hmgA-homologous gene in bacteria. (+info)
Co-transcription of Rhizobium meliloti lysyl-tRNA synthetase and glutamyl-tRNA synthetase genes.
An open reading frame encoding a putative polypeptide very similar to several lysyl-tRNA synthetases was found 10 nucleotides downstream of Rhizobium meliloti gltX encoding glutamyl-tRNA synthetase. Expression of this gene complemented a mutation in lysS of Escherichia coli and led to the overexpression of a polypeptide of the expected mass (62 kDa), thus confirming that it encodes R. meliloti lysyl-tRNA synthetase. Reverse transcription/polymerase chain reaction was used to demonstrate that this lysS gene is co-transcribed with gltX in R. meliloti. This is the first reported case of two immediately adjacent and co-transcribed genes encoding aminoacyl-tRNA synthetases. (+info)
Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria.
The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested. (+info)