An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales.
The CrbS/R system is a two-component signal transduction system that regulates acetate utilization in Vibrio cholerae, P. aeruginosa, and P. entomophila. CrbS is a hybrid histidine kinase that belongs to a recently identified family, in which the signaling domain is fused to an SLC5 solute symporter domain through aSTAC domain. Upon activation by CrbS, CrbR activates transcription of the acs gene, which encodes an acetyl-CoA synthase (ACS), and the actP gene, which encodes an acetate/solute symporter. In this work, we characterized the CrbS/R system in Pseudomonas fluorescens SBW25. Through the quantitative proteome analysis of different mutants, we were able to identify a new set of genes under its control, which play an important role during growth on acetate. These results led us to the identification of a conserved DNA motif in the putative promoter region of acetate-utilization genes in the Gammaproteobacteria that is essential for the CrbR-mediated transcriptional activation of genes under acetate-utilizing conditions. Finally, we took advantage of the existence of a second SLC5-containing two-component signal transduction system in P. fluorescens, CbrA/B, to demonstrate that the activation of the response regulator by the histidine kinase is not dependent on substrate transport through the SLC5 domain.
Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.DOI: http://dx.doi.org/10.7554/eLife.16761.001
Bacteria of the order Rhizobiales are able to establish nitrogen-fixing symbioses with legumes. Commonly, genes for symbiosis are harbored on large symbiotic plasmids. Although the transfer of symbiotic plasmids is commonly detected in nature, there are few experimentally characterized examples. In Rhizobium etli, the product of rctA inhibits the conjugation of the symbiotic plasmid by reducing the transcription of the virB operon. rctA is transcribed divergently from this operon, and its product is predicted to have a DNA binding domain. In the present study, using DNase I footprinting and binding assays, we demonstrated the specific binding of RctA to the virB operon promoter. A 9-bp motif in the spacer region of this promoter (the rctA binding motif box) and the presence of a functional ؊10 region were critical elements for RctA binding. Transcriptional fusion analyses revealed that the elimination of either element provoked a relief of RctAmediated repression. These data support a model in which RctA inhibits the access of the RNA polymerase to the virB promoter. Interestingly, rctA expression levels were modulated by transcriptional interference from transcripts emanating from the virB promoter. This phenomenon adds another level of regulation for this system, thus revealing a novel mechanism of plasmid transfer regulation in the Rhizobiales.The ability to establish nitrogen-fixing symbioses is prevalent in bacteria of the order Rhizobiales. Commonly, most of the genes needed to establish symbiosis are either harbored on the so-called symbiotic plasmids (pSyms) or restricted to symbiosis islands (SI) located on the bacterial chromosome. As befits a trait that confers niche extension, there is evidence for the mobility of these genomic compartments. Indeed, sequence analyses of pSyms, including pRetCFN42d of Rhizobium etli (17), pNGR234a of Rhizobium sp. strain NGR234 (13), and pSymA of Sinorhizobium meliloti (1, 15), as well as of the SI of Bradyrhizobium japonicum (23,18) and Mesorhizobium loti (22,43), have lead to the identification of conjugation-related genes, mainly the virB1-to-virB11 and traA-traCDG systems, carried by these elements. Moreover, a common feature of these genetic compartments is that the GC contents of these elements differ significantly from those of the rest of the genomes. These data suggest that these gene clusters originated out of, and were transmitted to, other genetic systems. It is likely that these compartments may still be prone to lateral transfer.Evidence for the movement of pSyms among naturally occurring rhizobial populations has been inferred through phylogenetic and/or population genetics analyses of a variety of systems (45, 38). The transfer of SI, initially detected in field experiments investigating the SI of M. loti (41), was recently demonstrated for the SI of B. japonicum (16). Direct experimental evidence for lateral transfers has also been obtained, albeit such transfers have been found to occur at various rates (ranging from 10 Ϫ3 to 10 Ϫ9 transconjugants pe...
Streptomycetes, Gram-positive soil bacteria well known for the production of antibiotics feature a unique conjugative DNA transfer system. In contrast to classical conjugation which is characterized by the secretion of a pilot protein covalently linked to a single-stranded DNA molecule, in Streptomyces a double-stranded DNA molecule is translocated during conjugative transfer. This transfer involves a single plasmid encoded protein, TraB. A detailed biochemical and biophysical characterization of TraB, revealed a close relationship to FtsK, mediating chromosome segregation during bacterial cell division. TraB translocates plasmid DNA by recognizing 8-bp direct repeats located in a specific plasmid region clt. Similar sequences accidentally also occur on chromosomes and have been shown to be bound by TraB. We suggest that TraB mobilizes chromosomal genes by the interaction with these chromosomal clt-like sequences not relying on the integration of the conjugative plasmid into the chromosome.
Conjugation is a driving force in the evolution and shaping of bacterial genomes. In antibiotic producing streptomycetes even small plasmids replicating via the rolling-circle mechanism are conjugative. Although they encode only genes involved in replication and transfer, the molecular function of most plasmid encoded proteins is unknown. In this work we show that the conjugative plasmid pIJ101 encodes an overlooked protein, SpdA2. We show that SpdA2 is a DNA binding protein which specifically recognizes a palindromic DNA sequence (sps). sps is localized within the spdA2 coding region and highly conserved in many Streptomyces plasmids. Elimination of the palindrome or deletion of spdA2 in plasmid pIJ303 did not interfere with conjugative plasmid transfer or pock formation, but affected segregational stability.
Plant growth-promoting rhizobacteria are often utilized to improve crop health and productivity. Nevertheless, their positive effects can be hindered if they fail to withstand the environmental and ecological conditions of the regions where they are applied. An alternative approach to circumvent this problem is a tailored selection of bacteria for specific agricultural systems. In this work, we evaluated the plant growth promoting and pathogen inhibition activity of rhizobacteria obtained from the rhizosphere of Mariola (Solanum hindsianum), an endemic shrub from Baja California. Eight strains were capable of inhibiting Fusarium oxysporum in vitro, and thirteen strains were found to possess three or more plant-growth-promotion traits. Molecular identification of these strains, using 16 s rRNA partial sequences, identified them as belonging to the genera Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, and Streptomyces. Finally, the effect of selected plant growth-promoting rhizobacteria (PGPR) strains on the growth and suppression of Fusarium wilt in tomato was evaluated. Results showed that these strains improved tomato plants growth under greenhouse conditions and reduced Fusarium wilt effects, as reflected in several variables such as length and weight of roots and stem. This work highlights the potential of native plants related to regionally important crops as a valuable source of beneficial bacteria.
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