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The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi-chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division-replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.

Transposon mutagenesis in conjunction with identification of genomic transposon insertion sites is a powerful tool for gene function studies. We have implemented a protocol for parallel determination of transposon insertion sites by Illumina sequencing involving a hierarchical barcoding method that allowed for tracking back insertion sites to individual clones of an arrayed signature-tagged transposon mutant library. This protocol was applied to further characterize a signature-tagged mini‐Tn 5 mutant library comprising about 12,000 mutants of the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti ( Pobigaylo et al., 2006 ; Appl. Environ. Microbiol. 72, 4329–4337). Previously, insertion sites have been determined for 5000 mutants of this library. Combining an adapter-free, inverse PCR method for sequencing library preparation with next generation sequencing, we identified 4473 novel insertion sites, increasing the total number of transposon mutants with known insertion site to 9562. The number of protein-coding genes that were hit at least once by a transposon increased by 1231 to a total number of 3673 disrupted genes, which represents 59% of the predicted protein-coding genes in S. meliloti.

Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of sX13 impinged on Xcv virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of in vitro transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected Xcv virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the hfq mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding Xcv genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen Xanthomonas., Author Summary Since the discovery of the first regulatory RNA in 1981, hundreds of small RNAs (sRNAs) have been identified in bacteria. Although sRNA-mediated control of virulence was demonstrated for numerous animal- and human-pathogenic bacteria, sRNAs and their functions in plant-pathogenic bacteria have been enigmatic. We discovered that the sRNA sX13 is a novel virulence regulator of Xanthomonas campestris pv. vesicatoria (Xcv), which causes bacterial spot disease on pepper and tomato. sX13 contributes to the Xcv-plant interaction by promoting the synthesis of an essential pathogenicity factor of Xcv, i. e., the type III secretion system. Thus, in addition to transcriptional regulation, sRNA-mediated posttranscriptional regulation contributes to virulence of plant-pathogenic xanthomonads. To repress target mRNAs carrying G-rich motifs, sX13 employs C-rich loops. Hence, sX13 exhibits striking structural similarity to sRNAs in distantly related human pathogens, e. g., Staphylococcus aureus and Helicobacter pylori, suggesting that structure-driven target regulation via C-rich motifs represents a conserved feature of sRNA-mediated posttranscriptional regulation. Furthermore, sX13 is the first sRNA shown to control the mRNA level of hfq, which encodes a conserved RNA-binding protein required for sRNA activity and virulence in many enteric bacteria.

Depending on the phosphate concentration encountered in the environment Sinorhizobium meliloti 2011 synthesizes two different exopolysaccharides (EPS). Galactoglucan (EPS II) is produced under phosphate starvation but also in the presence of extra copies of the transcriptional regulator WggR (ExpG) or as a consequence of a mutation in mucR . The galactoglucan biosynthesis gene cluster contains the operons wga ( expA ), wge ( expE ), wgd ( expD ), and wggR ( expG ). Two promoters, differentially controlled by WggR, PhoB, and MucR, were identified upstream of each of these operons. The proximal promoters of the wga , wge , and wgd transcription units were constitutively active when separated from the upstream regulatory sequences. Promoter activity studies and the positions of predicted PhoB and WggR binding sites suggested that the proximal promoters are cooperatively induced by PhoB and WggR. MucR was shown to strongly inhibit the distal promoters and bound to the DNA in the vicinity of the distal transcription start sites. An additional inhibitory effect on the distal promoter of the structural galactoglucan biosynthesis genes was identified as a new feature of WggR in a mucR mutant. A regulatory model of the fine-tuning of galactoglucan production is proposed.

A 70 met oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1 h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis. (C) 2008 Elsevier B.V. All rights reserved.