Laurent Sauviac

AP2-ERF Transcription Factors Mediate Nod Factor–Dependent Mt ENOD11 Activation in Root Hairs via a Novel cis-Regulatory Motif

Rhizobium Nod factors (NFs) are specific lipochitooligosaccharides that activate host legume signaling pathways essential for initiating the nitrogen-fixing symbiotic association. This study describes the characterization of cis-regulatory elements and trans-interacting factors that regulate NF-dependent and epidermis-specific gene transcription in Medicago truncatula. Detailed analysis of the Mt ENOD11 promoter using deletion, mutation, and gain-of-function constructs has led to the identification of an NF-responsive regulatory unit (the NF box) sufficient to direct NF-elicited expression in root hairs. NF box–mediated expression requires a major GCC-like motif, which is also essential for the binding of root hair–specific nuclear factors. Yeast one-hybrid screening has identified three closely related AP2/ERF transcription factors (ERN1 to ERN3) that are able to bind specifically to the NF box. ERN1 is identical to an ERF-like factor identified recently. Expression analysis has revealed that ERN1 and ERN2 genes are upregulated in root hairs following NF treatment and that this activation requires a functional NFP gene. Transient expression assays in Nicotiana benthamiana have further shown that nucleus-targeted ERN1 and ERN2 factors activate NF box–containing reporters, whereas ERN3 represses ERN1/ERN2-dependent transcription activation. A model is proposed for the fine-tuning of NF-elicited gene transcription in root hairs involving the interplay between repressor and activator ERN factors.

An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in Sinorhizobium meliloti

Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as well as upon entry into stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be up-regulated under both conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon or nitrogen starvation. We also present evidence that the product of the gene cotranscribed with rpoE2 negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes encoding proteins with known functions in stress responses, namely, katC and rpoH2, as well as a second ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a major global regulator of the general stress response in S. meliloti. Despite these observations, and although this sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from the wild-type strain could be detected for rpoE2 mutants. This therefore suggests that other important actors in the general stress response have still to be identified in S. meliloti.

Dual control of Sinorhizobium meliloti RpoE2 sigma factor activity by two PhyR-type two-component response regulators.

RpoE2 is an extracytoplasmic function (ECF) sigma factor involved in the general stress response of Sinorhizobium meliloti, the nitrogen-fixing symbiont of the legume plant alfalfa. RpoE2 orthologues are widely found among alphaproteobacteria, where they play various roles in stress resistance and/or host colonization. In this paper, we report a genetic and biochemical investigation of the mechanisms of signal transduction leading to S. meliloti RpoE2 activation in response to stress. We showed that RpoE2 activity is negatively controlled by two paralogous anti-sigma factors, RsiA1 (SMc01505) and RsiA2 (SMc04884), and that RpoE2 activation by stress requires two redundant paralogous PhyR-type response regulators, RsiB1 (SMc01504) and RsiB2 (SMc00794). RsiB1 and RsiB2 do not act at the level of rpoE2 transcription but instead interact with the anti-sigma factors, and we therefore propose that they act as anti-anti-sigma factors to relieve RpoE2 inhibition in response to stress. This model closely resembles a recently proposed model of activation of RpoE2-like sigma factors in Methylobacterium extorquens and Bradyrhizobium japonicum, but the existence of two pairs of anti- and anti-anti-sigma factors in S. meliloti adds an unexpected level of complexity, which may allow the regulatory system to integrate multiple stimuli.

Next-Generation Annotation of Prokaryotic Genomes with EuGene-P: Application to Sinorhizobium meliloti 2011

The availability of next-generation sequences of transcripts from prokaryotic organisms offers the opportunity to design a new generation of automated genome annotation tools not yet available for prokaryotes. In this work, we designed EuGene-P, the first integrative prokaryotic gene finder tool which combines a variety of high-throughput data, including oriented RNA-Seq data, directly into the prediction process. This enables the automated prediction of coding sequences (CDSs), untranslated regions, transcription start sites (TSSs) and non-coding RNA (ncRNA, sense and antisense) genes. EuGene-P was used to comprehensively and accurately annotate the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti strain 2011, leading to the prediction of 6308 CDSs as well as 1876 ncRNAs. Among them, 1280 appeared as antisense to a CDS, which supports recent findings that antisense transcription activity is widespread in bacteria. Moreover, 4077 TSSs upstream of protein-coding or non-coding genes were precisely mapped providing valuable data for the study of promoter regions. By looking for RpoE2-binding sites upstream of annotated TSSs, we were able to extend the S. meliloti RpoE2 regulon by ∼3-fold. Altogether, these observations demonstrate the power of EuGene-P to produce a reliable and high-resolution automatic annotation of prokaryotic genomes.

CYP76C2, an Arabidopsis thaliana cytochrome P450 gene expressed during hypersensitive and developmental cell death

The characterisation of an Arabidopsis thaliana cytochrome P450‐encoding cDNA clone, B72, preferentially expressed during the hypersensitive response (HR) provoked by the bacterial pathogen Pseudomonas syringae pathovar maculicola, is reported. The B72 cDNA clone corresponded to the CYP76C2 gene, which belongs to a small multigene family comprising four genes. HR‐triggering bacteria harbouring different avirulence genes induced the accumulation of transcripts of this P450 gene. CYP76C2 gene expression was moreover associated with various processes leading to cell death such as leaf senescence, ageing of cell cultures, wounding as well as with treatment with the necrotising heavy metal salt, lead nitrate.

Response of Sinorhizobium meliloti to Elevated Concentrations of Cadmium and Zinc

ABSTRACT Whole-genome transcriptional profiling was used to identify genes in Sinorhizobium meliloti 1021 that are differentially expressed during exposure to elevated concentrations of cadmium and zinc. Mutant strains with insertions in metal-regulated genes and in genes encoding putative metal efflux pumps were analyzed for their metal sensitivities, revealing a crucial role for the SMc04128-encoded P-type ATPase in the defense of S. meliloti against cadmium and zinc stress.

A Putative Bifunctional Histidine Kinase/Phosphatase of the HWE Family Exerts Positive and Negative Control on the Sinorhizobium meliloti General Stress Response

The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.

Sinorhizobium meliloti Sigma Factors RpoE1 and RpoE4 Are Activated in Stationary Phase in Response to Sulfite

Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis with legume plants. Both in soil and in planta, rhizobia spend non-growing periods resembling the stationary phase of in vitro-cultured bacteria. The primary objective of this work was to better characterize gene regulation in this biologically relevant growth stage in Sinorhizobium meliloti. By a tap-tag/mass spectrometry approach, we identified five sigma factors co-purifying with the RNA polymerase in stationary phase: the general stress response regulator RpoE2, the heat shock sigma factor RpoH2, and three extra-cytoplasmic function sigma factors (RpoE1, RpoE3 and RpoE4) belonging to the poorly characterized ECF26 subgroup. We then showed that RpoE1 and RpoE4 i) are activated upon metabolism of sulfite-generating compounds (thiosulfate and taurine), ii) display overlapping regulatory activities, iii) govern a dedicated sulfite response by controlling expression of the sulfite dehydrogenase SorT, iv) are activated in stationary phase, likely as a result of endogenous sulfite generation during bacterial growth. We showed that SorT is required for optimal growth of S. meliloti in the presence of sulfite, suggesting that the response governed by RpoE1 and RpoE4 may be advantageous for bacteria in stationary phase either by providing a sulfite detoxification function or by contributing to energy production through sulfite respiration. This paper therefore reports the first characterization of ECF26 sigma factors, the first description of sigma factors involved in control of sulphur metabolism, and the first indication that endogenous sulfite may act as a signal for regulation of gene expression upon entry of bacteria in stationary phase.