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Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

ABSTRACT: Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragment was cloned and used as a hybridization probe for cloning of the R. etli sigA gene. Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors. The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides. Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase. On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors.

SUBMITTER: Luka S

PROVIDER: S-EPMC178625 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

Luka S S Patriarca E J EJ Riccio A A Iaccarino M M Defez R R

Journal of bacteriology 19961201 24

Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragme ...[more]

PMID: 8955394

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Project description:Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Project description:BackgroundA traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer.ResultsAnalysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.ConclusionsThis study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The unusual presence of panCB genes in plasmids of Rhizobiales may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.

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Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

Publications

Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

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