Regulation and mode of action of the second small RNA activator of RpoS translation,RprA

Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB-regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA-dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.     

Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of CysE enhances biofilm formation in Escherichia coli

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.     

Regulation of RpoS by a novel small RNA: the characterization of RprA

Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double-stranded RNA stem-loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double-strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem-loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non-essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.     

Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA'::lux fusions.

A set of genetically engineered Escherichia coli strains was constructed, in which the promoter of the fabA gene is fused to Vibrio fischeri luxCDABE either in a multi-copy plasmid or as a single copy chromosomal integration. The fabA gene codes for beta-hydroxydecanoyl-ACP dehydrase, a key enzyme in the synthesis of unsaturated fatty acids, and is induced when fatty acid biosynthesis pathways are interrupted. A dose-dependent and highly sensitive bioluminescent response to a variety of chemicals was controlled by the fadR gene. A tolC mutant E. coli host displayed generally lower detection threshold for toxicants. A chromosomal integration of a single copy of the fabA'::lux fusion led to a markedly lower background luminescence, but did not yield an improvement in overall performance. It is proposed that these or similarly constructed reporters of fatty acid biosynthesis inhibition may serve as novel microbial toxicity biosensors.     

RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.