Genetic Dissection of an Exogenously Induced Biofilm in Laboratory and Clinical Isolates of E. coli

Microbial biofilms are a dominant feature of many human infections. However, developing effective strategies for controlling biofilms requires an understanding of the underlying biology well beyond what currently exists. Using a novel strategy, we have induced formation of a robust biofilm in Escherichia coli by utilizing an exogenous source of poly-N-acetylglucosamine (PNAG) polymer, a major virulence factor of many pathogens. Through microarray profiling of competitive selections, carried out in both transposon insertion and over-expression libraries, we have revealed the genetic basis of PNAG-based biofilm formation. Our observations reveal the dominance of electrostatic interactions between PNAG and surface structures such as lipopolysaccharides. We show that regulatory modulation of these surface structures has significant impact on biofilm formation behavior of the cell. Furthermore, the majority of clinical isolates which produced PNAG also showed the capacity to respond to the exogenously produced version of the polymer

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation

Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>In <it>Escherichia coli</it>, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of <it>E. coli </it>with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.</p> <p>Results</p> <p>We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three <it>cis</it>-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.</p> <p>Conclusions</p> <p>This comprehensive screen for <it>E. coli </it>sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the <it>E. coli </it>genome. We constructed the <it>Escherichia coli </it>Small RNA Browser (ECSBrowser; <url>http://rna.iab.keio.ac.jp/</url>), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study.</p

Geochemical consequences of increased atmospheric carbon dioxide on coral reefs

A coral reef represents the net accumulation of calcium carbonate (CaCO3) produced by corals and other calcifying organisms. If calcification declines, then reef-building capacity also declines. Coral reef calcification depends on the saturation state of the carbonate mineral aragonite of surface waters. By the middle of the next century, an increased concentration of carbon dioxide will decrease the aragonite saturation state in the tropics by 30 percent and biogenic aragonite precipitation by 14 to 30 percent. Coral reefs are particularly threatened, because reef-building organisms secrete metastable forms of CaCO3, but the biogeochemical consequences on other calcifying marine ecosystems may be equally severe

The excludon: a new concept in bacterial antisense RNA-mediated gene regulation

International audienceIn recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria