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In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guanosine monophosphate (cGMP). The prototypicEscherichia coliCAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr ofSinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr•cAMP and Clr•cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr•cNMP•CBS-DNA complexes to almost identical active conformations. Unlike the situation known for theE. coliCAP•cNMP complex, in the Clr•cNMP complex, the nucleobases of cGMP and cAMP are in thesyn-andanti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP16 μM). However, different affinities were determined in absence of this DNA (KDcGMP24 μM; KDcGMP6 μM). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr•cNMP•CBS-DNA crystal structures.IMPORTANCECyclic 3’,5’-adenosine monophosphate (cAMP) and cyclic 3’,5’-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins.Escherichia coliCAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP•cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr fromSinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

While the vast majority of bacterial genomic DNA molecules contain a single origin of replication, some natural isolates and engineered strains were reported to contain chromosomes derived from cointegration events of multiple replicons. We investigated effects of multiple DNA replication origins and terminus regions on spatial DNA organization and spatiotemporal replicon segregation in the alphaproteobacterium Sinorhizobium meliloti. Strains with a bi- and monopartite genome configuration were constructed by Cre/lox-mediated site-specific fusions of the secondary replicons pSymA and pSymB, and of the chromosome, pSymA and pSymB. The design of these strains maintained replichore ratios, GC skew, as well as distribution and orientation of KOPS and coding sequences. Growth of these strains was essentially unaffected, except for high salt conditions. Replication initiation at the three origins as well as key features of spatial organization and spatiotemporal segregation were maintained in the triple-replicon fusion strain. Cell growth was slowed down by deleting, either individually or together, the pSymA- and pSymB-derived replication initiator encoding repC genes with their intrinsic origin of replication from the dual or triple replicon cointegrates, respectively. Replication of the triple cointegrate, characterized by the chromosomal oriC as sole origin and a strongly disbalanced replichore ratio, terminated in the original chromosomal terC region, suggesting a replication trap. Progression of replication of the longer replichore was not blocked but impaired, possibly due to the retained secondary replicon’s terminus regions and reverse alignment of replichore-orienting sequence features following from the deletion of replication origins. Moreover, during the cell cycle of this strain, oriC aberrantly localized and served as replication initiation site in the mid cell area of the cell with the oldest cell pole. Growth deficiency of this strain was attenuated by a suppressor mutation causing amino acid substitution R436H in the cell cycle histidine kinase CckA.AUTHOR SUMMARYTo proliferate, any cell must reliably replicate its genomic DNA and allocate copies to the future cell compartments prior to cell division. While eukaryotic cells typically use multiple origins of replication per chromosome to initiate replication, bacteria usually rely on a single origin of replication per genomic DNA molecule. About 10 % of total-genome-sequenced bacterial species maintain multipartite genomes. Derivatives of such bacterial species with fused genomic DNA molecules - either natural bacterial isolates or engineered strains - demonstrate that bacteria can handle genomic DNA molecules with multiple origins of replication in their cell cycle. In our study we show that a cointegrate of three genomic replicons maintains key features of spatial organization and segregation dynamics of each replicon in the alphaproteobacterium Sinorhizobium meliloti. The spatial association of origin and terminus regions to specific cell areas, as well as preserving typical features of bacterial genome organization, such as replichore ratio and the distribution and DNA strand specificity of coding sequences and replichore-orienting sequence motifs, are likely key to maintaining genomic cointegrates with multiple replication origins in a bacterial cell without major loss of cell fitness.

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.

Rhizobia are α- and s-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.

Chromosome segregation typically occurs after replication has finished in eukaryotes but during replication in bacteria. Here, we show that the alphaproteobacterium Hyphomonas neptunium, which proliferates by bud formation at the tip of a stalk-like cellular extension, segregates its chromosomes in a unique two-step process. First, the two sister origin regions are targeted to opposite poles of the mother cell, driven by the ParABS partitioning system. Subsequently, once the bulk of chromosomal DNA has been replicated and the bud exceeds a certain threshold size, the cell initiates a second segregation step during which it transfers the stalk-proximal origin region through the stalk into the nascent bud compartment. Thus, while chromosome replication and segregation usually proceed concurrently in bacteria, the two processes are largely uncoupled in H. neptunium, reminiscent of eukaryotic mitosis. These results indicate that stalked budding bacteria have evolved specific mechanisms to adjust chromosome segregation to their unusual life cycle., In bacteria, DNA replication and segregation are commonly coupled. Here, by investigating the dynamics of these processes in the marine bacterium Hyphomonas neptunium, the authors unravel a two-step chromosomal segregation process reminiscent of eukaryotic mitosis, providing insights into the evolution of bacterial cell organization.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III–secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.

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.

Sinorhizobium meliloti Rm41 nodulates alfalfa plants, forming indeterminate type nodules. It is characterized by a strain-specific K-antigen able to replace exopolysaccharides in promotion of nodule invasion. We present the Rm41 genome, composed of one chromosome, the chromid pSymB, the megaplasmid pSymA, and the nonsymbiotic plasmid pRme41a.

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.