Miguel A. Ramírez-Romero

The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments

BackgroundSymbiotic bacteria known as rhizobia interact with the roots of legumes and induce the formation of nitrogen-fixing nodules. In rhizobia, essential genes for symbiosis are compartmentalized either in symbiotic plasmids or in chromosomal symbiotic islands. To understand the structure and evolution of the symbiotic genome compartments (SGCs), it is necessary to analyze their common genetic content and organization as well as to study their differences. To date, five SGCs belonging to distinct species of rhizobia have been entirely sequenced. We report the complete sequence of the symbiotic plasmid of Rhizobium etli CFN42, a microsymbiont of beans, and a comparison with other SGC sequences available.ResultsThe symbiotic plasmid is a circular molecule of 371,255 base-pairs containing 359 coding sequences. Nodulation and nitrogen-fixation genes common to other rhizobia are clustered in a region of 125 kilobases. Numerous sequences related to mobile elements are scattered throughout. In some cases the mobile elements flank blocks of functionally related sequences, thereby suggesting a role in transposition. The plasmid contains 12 reiterated DNA families that are likely to participate in genomic rearrangements. Comparisons between this plasmid and complete rhizobial genomes and symbiotic compartments already sequenced show a general lack of synteny and colinearity, with the exception of some transcriptional units. There are only 20 symbiotic genes that are shared by all SGCs.ConclusionsOur data support the notion that the symbiotic compartments of rhizobia genomes are mosaic structures that have been frequently tailored by recombination, horizontal transfer and transposition.

Structural Elements Required for Replication and Incompatibility of the Rhizobium etli Symbiotic Plasmid

The symbiotic plasmid of Rhizobium etli CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes, repA, repB, and repC, encoded by the same DNA strand. A long intergenic sequence (igs) between repB and repC is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the repABC genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two cis-acting incompatibility regions, one located in the igs (incalpha) and the other downstream of repC (incbeta) (the former is essential for replication); and (v) RepA is a trans-acting incompatibility factor. We suggest that incalpha is a cis-acting site required for plasmid partitioning and that the origin of replication lies within incbeta.

Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem-loop structure.

The repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB). The latter two proteins negatively regulate the transcription of the operon. In this article we have identified two novel regulatory elements, located within the conserved repB–repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli. One of them is a small antisense RNA and the other is a stem–loop structure in the repABC mRNA that occludes the Shine‐Dalgarno sequence of repC. According to in vivo and in vitro analyses, the small antisense RNA (57–59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho‐independent terminator, (iv) the antisense RNA is a strong trans‐incompatibility factor and (v) its presence reduces the level of repC expression. Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.

RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon

The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans‐incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site‐directed mutagenesis of the putative −35 and −10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.

The Rhizobium etli RpoH1 and RpoH2 sigma factors are involved in different stress responses.

The physiological role and transcriptional expression of Rhizobium etli sigma factors rpoH1 and rpoH2 are reported in this work. Both rpoH1 and rpoH2 were able to complement the temperature-sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite and hydrogen peroxide, whereas the rpoH2 mutant was sensitive to NaCl and sucrose. The rpoH2 rpoH1 double mutant had increased sensitivity to heat shock and oxidative stress when compared with the rpoH1 single mutant. This suggests that in R. etli, RpoH1 is the main heat-shock sigma factor, but a more complete protective response could be achieved with the participation of RpoH2. Conversely, RpoH2 is involved in osmotic tolerance. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2 rpoH1 mutants still elicited nodule formation, but exhibited reduced nitrogenase activity and bacterial viability in early and late symbiosis compared with nodules produced by rpoH2 mutants and wild-type strains. In addition, nodules formed by R. etli rpoH1 and rpoH2 rpoH1 mutants showed premature senescence. It was also determined that fixNf and fixKf expression was affected in rpoH1 mutants. Both rpoH genes were induced under microaerobic conditions and in the stationary growth phase, but not in response to heat shock. Analysis of the upstream region of rpoH1 revealed a sigma70 and a probable sigmaE promoter, whereas in rpoH2, one probable sigmaE-dependent promoter was detected. In conclusion, the two RpoH proteins operate under different stress conditions, RpoH1 in heat-shock and oxidative responses, and RpoH2 in osmotic tolerance.

Role of the Extracytoplasmic Function Sigma Factor RpoE4 in Oxidative and Osmotic Stress Responses in Rhizobium etli

The aims of this study were to functionally characterize and analyze the transcriptional regulation and transcriptome of the Rhizobium etli rpoE4 gene. An R. etli rpoE4 mutant was sensitive to oxidative, saline, and osmotic stresses. Using transcriptional fusions, we determined that RpoE4 controls its own transcription and that it is negatively regulated by rseF (regulator of sigma rpoE4; CH03274), which is cotranscribed with rpoE4. rpoE4 expression was induced not only after oxidative, saline, and osmotic shocks, but also under microaerobic and stationary-phase growth conditions. The transcriptome analyses of an rpoE4 mutant and an rpoE4-overexpressing strain revealed that the RpoE4 extracytoplasmic function sigma factor regulates about 98 genes; 50 of them have the rpoE4 promoter motifs in the upstream regulatory regions. Interestingly, 16 of 38 genes upregulated in the rpoE4-overexpressing strain encode unknown putative cell envelope proteins. Other genes controlled by RpoE4 include rpoH2, CH00462, CH02434, CH03474, and xthA1, which encode proteins involved in the stress response (a heat shock sigma factor, a putative Mn-catalase, an alkylation DNA repair protein, pyridoxine phosphate oxidase, and exonuclease III, respectively), as well as several genes, such as CH01253, CH03555, and PF00247, encoding putative proteins involved in cell envelope biogenesis (a putative peptidoglycan binding protein, a cell wall degradation protein, and phospholipase D, respectively). These results suggest that rpoE4 has a relevant function in cell envelope biogenesis and that it plays a role as a general regulator in the responses to several kinds of stress.

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors—abundant at infection sites—and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

The Rhizobium etli σ70 (SigA) factor recognizes a lax consensus promoter

A collection of Rhizobium etli promoters was isolated from a genomic DNA library constructed in the promoter-trap vector pBBMCS53, by their ability to drive the expression of a gusA reporter gene. Thirty-seven clones were selected, and their transcriptional start-sites were determined. The upstream sequence of these 37 start-sites, and the sequences of seven previously identified promoters were compared. On the basis of sequence conservation and mutational analysis, a consensus sequence CTTGACN16–23TATNNT was obtained. In this consensus sequence, nine on of twelve bases are identical to the canonical Escherichia coli σ70 promoter, however the R.etli promoters only contain 6.4 conserved bases on average. We show that the R.etli sigma factor SigA recognizes all R.etli promoters studied in this work, and that E.coli RpoD is incapable of recognizing them. The comparison of the predicted structure of SigA with the known structure of RpoD indicated that regions 2.4 and 4.2, responsible for promoter recognition, are different only by a single amino acid, whereas the region 1 of SigA contains 72 extra residues, suggesting that the differences contained in this region could be related to the lax promoter recognition of SigA.

RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock.

BackgroundRegulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci.ResultsAnalysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions.ConclusionsOur data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation.

Transcriptional interference and repression modulate the conjugative ability of the symbiotic plasmid of Rhizobium etli.

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 RctA-mediated 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.