A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria.

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3' untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3' UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

Correction draft: RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli.

[This corrects the article DOI: 10.1371/journal.pone.0063781.].

Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer.

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

Stem-loops direct precise processing of 3' UTR-derived small RNA MicL.

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To better understand the sequence and structure determinants for the precise processing of a 3' UTR-derived sRNA, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and probably other sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

The Evasive Enemy: Insights into the Virulence and Epidemiology of the Emerging Attaching and Effacing Pathogen Escherichia albertii.

The diarrheic attaching and effacing (A/E) pathogen Escherichia albertii was first isolated from infants in Bangladesh in 1991, although the bacterium was initially classified as Hafnia alvei Subsequent genetic and biochemical interrogation of these isolates raised concerns about their initial taxonomic placement. It was not until 2003 that these isolates were reassigned to the novel taxon Escherichia albertii because they were genetically more closely related to E. coli, although they had diverged sufficiently to warrant a novel species name. Unfortunately, new isolates continue to be mistyped as enteropathogenic E. coli (EPEC) or enterohemorrhagic E. coli (EHEC) owing to shared traits, most notably the ability to form A/E lesions. Consequently, E. albertii remains an underappreciated A/E pathogen, despite multiple reports demonstrating that many provisional EPEC and EHEC isolates incriminated in disease outbreaks are actually E. albertii Metagenomic studies on dozens of E. albertii isolates reveal a genetic architecture that boasts an arsenal of candidate virulence factors to rival that of its better-characterized cousins, EPEC and EHEC. Beyond these computational comparisons, studies addressing the regulation, structure, function, and mechanism of action of its repertoire of virulence factors are lacking. Thus, the paucity of knowledge about the epidemiology, virulence, and antibiotic resistance of E. albertii, coupled with its misclassification and its ability to develop multidrug resistance in a single step, highlights the challenges in combating this emerging pathogen. This review seeks to synthesize our current but incomplete understanding of the biology of E. albertii.

Transcriptional and posttranscriptional regulation of Shigella shuT in response to host-associated iron availability and temperature.

Like most bacteria, Shigella must maintain a precise balance between the necessity and toxicity of iron; a balance that is achieved, at least in part, by regulating the production of bacterial iron acquisition systems in response to specific environmental signals. Using the Shigella heme utilization (Shu) system, S. dysenteriae is able to acquire iron from heme, a potentially rich source of nutritional iron within the otherwise iron-limited environment of the human host. Investigations presented within reveal two distinct molecular mechanisms underlying previously uncharacterized transcriptional and translational regulation of shuT, a gene encoding the periplasmic-binding component of the Shu system. While shuT transcription is regulated in response to iron availability via a process dependent upon the global regulator Fur and a Fur-binding site located immediately downstream of the promoter, shuT translation is regulated in response to environmental temperature via the activity of an RNA thermometer located within the 5' untranslated region of the gene. Such complex regulation likely increases the fitness of S. dysenteriae by ensuring maximal ShuT production when the pathogen is within the iron-limited and relatively warm environment of the infected host, the only environment in which heme will be encountered as a potential source of essential iron.

RNA-mediated thermoregulation of iron-acquisition genes in Shigella dysenteriae and pathogenic Escherichia coli.

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5' untranslated region. In addition, we have identified similar nucleotide sequences within the 5' untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5' untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source.