Construction and functional analyses of a comprehensive σ54 site-directed mutant library using alanine–cysteine mutagenesis

The σ54 factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. σ54 is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define σ54 residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of σ54 was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)σ54 were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of σ54 were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased σ54 isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after σ54 isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the σ54-core RNAP interface changes upon activation

Nitric oxide-responsive interdomain regulation targets the σ54-interaction surface in the enhancer binding protein NorR

Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain that represses the ATPase activity of the central AAA+ domain in the absence of nitric oxide. Here, we investigate the mechanism of interdomain repression in NorR by characterizing substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ54 during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ54. Biochemical studies suggest that the bypass mutations in the GAFTGA loop do not influence the DNA binding properties of NorR or the assembly of higher order oligomers in the presence of enhancer DNA, and as expected these variants retain the ability to activate open complex formation in vitro. We identify a crucial arginine residue in the GAF domain that is essential for interdomain repression and demonstrate that hydrophobic substitutions at this position suppress the bypass phenotype of the GAFTGA substitutions. These observations suggest a novel mechanism for negative regulation in bEBPs in which the GAF domain targets the σ54-interaction surface to prevent access of the AAA+ domain to the sigma factor

Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping

Comparative genomics of Pseudomonas fluorescens subclade III strains from human lungs

Abstract Background While the taxonomy and genomics of environmental strains from the P. fluorescens species-complex has been reported, little is known about P. fluorescens strains from clinical samples. In this report, we provide the first genomic analysis of P. fluorescens strains in which human vs. environmental isolates are compared. Results Seven P. fluorescens strains were isolated from respiratory samples from cystic fibrosis (CF) patients. The clinical strains could grow at a higher temperature (>34 °C) than has been reported for environmental strains. Draft genomes were generated for all of the clinical strains, and multi-locus sequence analysis placed them within subclade III of the P. fluorescens species-complex. All strains encoded type- II, −III, −IV, and -VI secretion systems, as well as the widespread colonization island (WCI). This is the first description of a WCI in P. fluorescens strains. All strains also encoded a complete I2/PfiT locus and showed evidence of horizontal gene transfer. The clinical strains were found to differ from the environmental strains in the number of genes involved in metal resistance, which may be a possible adaptation to chronic antibiotic exposure in the CF lung. Conclusions This is the largest comparative genomics analysis of P. fluorescens subclade III strains to date and includes the first clinical isolates. At a global level, the clinical P. fluorescens subclade III strains were largely indistinguishable from environmental P. fluorescens subclade III strains, supporting the idea that identifying strains as ‘environmental’ vs ‘clinical’ is not a phenotypic trait. Rather, strains within P. fluorescens subclade III will colonize and persist in any niche that provides the requirements necessary for growth.http://deepblue.lib.umich.edu/bitstream/2027.42/116129/1/12864_2015_Article_2261.pd

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm

Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator