The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA. ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P exsA ) located immediately upstream of exsA. P exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P exsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCEVfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa. Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA. Pseudomonas aeruginosa is an environmental bacterium typically found in soil and water. The organism is also an important opportunistic pathogen of humans, especially in those with neutropenia, severe burns, and cystic fibrosis (1, 2). Both the physical and host environments expose P. aeruginosa to unique stresses that challenge survival. Reprogramming gene expression is critical for adaptation. The host signals to which the bacteria respond are not entirely clear but likely include contact with host cell surfaces or host-derived macromolecules, temperature, osmolarity, pH, iron limitation, and oxidative stress (3-5). Bacterial genes induced within mammalian hosts include those important for iron acquisition, carbon utilization, and ...

R NA-binding proteins play an integral role in the posttranscriptional regulation of protein synthesis by altering translation initiation, mRNA stability, and/or RNA processing. The CsrA family of RNA-binding proteins regulates carbon metabolism, virulence factor production, and motility in a number of Gramnegative bacteria (1-5). CsrA proteins usually bind sites on target mRNAs that overlap the Shine-Dalgarno sequence to prevent translation initiation (6-8). Although considerable sequence variability exists between natural CsrA-binding sites, a common feature is a core GGA sequence that is usually presented in the loop portion of a stem-loop structure (9, 10). High-affinity interactions between CsrA and RNA targets have been analyzed by two powerful techniques. First, nuclear magnetic resonance (NMR) spectroscopy was used to show that RsmE, a CsrA homolog in Pseudomonas fluorescens, makes optimal contact with the sequence 5=-(A/U)CANGGANG(U/A), where N is any nucleotide (10). RsmE functions as a molecular clamp and gathers the ANGGAN core into a hexaloop, with the flanking nucleotides forming a 3-bp stem (10). The second approach, a systematic evolution of ligands by exponential enrichment (SELEX), was used to identify high-affinity RNA ligands of Escherichia coli CsrA (9). SELEX is a method used to select for RNA ligands that bind proteins of interest (11). Evolution of the ligands is based on repeated cycles of in vitro selection (12). The selection is driven toward optimized RNA targets that bind to the protein of interest with Citation Schulmeyer KH, Diaz MR, Bair TB, Sanders W, Gode CJ, Laederach A, Wolfgang MC, Yahr TL. 2016. Primary and secondary sequence structure requirements for recognition and discrimination of target RNAs by Pseudomonas aeruginosa RsmA and RsmF.

Type III secretion systems (T3SS) serve as a primary anti-host defense mechanism for many Gram-negative plant and animal pathogens. T3SS production is tightly controlled and activated by host-associated signals. Although transcriptional responses represent a significant component of the activation cascade, recent studies have uncovered diverse post-transcriptional mechanisms that also contribute to T3SS production. Targets for post-transcriptional control are often AraC/XylS transcription factors that promote T3SS gene expression. Commons mechanisms of post-transcriptional regulation include direct control of either the activity of AraC/XylS transcription factors by protein ligands, small molecules, or post-translational modification, or transcription factor synthesis. In the latter case, RNA-binding proteins such as Hfq, CsrA/RsmA, and components of the RNA degradosome alter mRNA stability and/or the rate of translation initiation to control transcription factor synthesis. Here we summarize post-transcriptional mechanisms that contribute to the exquisite regulation of T3SS gene expression.

Although populations of planctomycete bacteria are nearly ubiquitous in aquatic habitats, we lack a detailed understanding of their diversity and structure. This is due to difficulty in obtaining cultured representatives, but also to low recovery rates for planctomycete 16S rRNA genes when universal PCR primers are employed for sequencing studies. In an attempt to expand recoverable planctomycete diversity, we investigated the use of primers targeting the Planctomycetes phylum. Planctomycete populations present during an algal bloom in a eutrophic lake were characterized by clone library sequencing and pyrosequencing of 16S rRNA genes, using planctomycetetargeted primers. We analyzed samples recovered from the sediment, water column, and algal mats within the lake's littoral zone. Sequences related to 9 planctomycete genera were identified within the 6287 planctomycete sequences and 1730 operational taxonomic units (OTUs) recovered. We observed variation in the specificity of the planctomycete primers, with more non-planctomycete sequences recovered through pyrosequencing than through cloning-based sequencing. Nevertheless, the results of our study suggest that phylum-targeted pyrosequencing is a useful tool for better describing the diversity of bacterial sub-populations. This methodology could be employed for future testing of hypotheses regarding spatial and temporal differences in planctomycete diversity and abundance. Candidate hypotheses arising from this preliminary study include (1) members of certain planctomycete genera (particularly Rhodopirellula) are enriched in algal mats relative to other planctomycete genera; (2) sediments harbor the most diverse planctomycete populations; and (3) most planctomycete OTUs are not shared between lake habitats. KEY WORDS: Planctomycetes · Freshwater lake · Algal bloomResale or republication not permitted without written consent of the publisher

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