RNase E is required for induction of the glutamate-dependent acid resistance system in Escherichia coli

The Escherichia coli RNase E is an essential endoribonuclease involved in processing and/or degradation of rRNAs, tRNAs, and non-coding small RNAs as well as many mRNAs. It is known that RNase E activity is somehow regulated by an RNA-binding protein Hfq, at least in some cases. We searched for proteins that showed changes in expression in both hfq::cat and rne-1 mutant cells as compared with the wild type, and found that a protein band of 49-kDa decreased in these mutant cells at 42 degrees C, the restrictive temperature for rne-1. N-terminal amino acid sequencing identified it as a mixture of GadA and GadB, two isozymes of glutamate decarboxylase involved in glutamate-dependent acid resistance. The rne-1 mutant as well as the hfq mutant showed decreased survival under acidic conditions (pH 2.5). Hfq is known to regulate the expression of GadA/B in RpoS- and GadY small RNA-dependent ways. We examined the expression of these two regulators in rne-1 mutant cells. In the mutant cells, the induction of GadY was defective at 42 degrees C, but the expression of RpoS was normal. These results indicate that RNase E is required for induction of the glutamate-dependent acid resistance system in a RpoS-independent manner.     

Negative regulatory role of the Escherichia coli hfq gene in cell division

We found that the hfq::cat mutant strain produced minicells at high frequency. Minicell production by the mutant strain was more prominent in poor media and in the stationary phase than in rich media and in the exponentially growing phase. The amount of the cell division protein FtsZ increased up to two- to threefold of the wild-type cells in the hfq::cat mutant in the stationary phase, while such differences were not observed in the exponentially growing phase. Increased ftsZ mRNA levels were also observed in the hfq::cat mutant in the stationary phase. These results suggest a negative regulatory role of the DNA-, RNA-binding protein Hfq in cell division in the stationary phase.     

The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli.

The hfq-encoded RNA-binding protein HF-I has long been known as a host factor for phage Qbeta RNA replication and has recently been shown to be essential for translation of rpoS, which encodes the sigmaS subunit of RNA polymerase. Here we demonstrate that an hfq null mutant does not synthesize glycogen, is starvation and multiple stress sensitive, and exhibits strongly reduced expression of representative sigmaS-regulated genes. These phenotypes are consistent with strongly reduced sigmaS levels in the hfq mutant. However, the analysis of global protein synthesis patterns on two-dimensional O'Farrell gels indicates that approximately 40% of the more than 30 proteins whose syntheses are altered in the hfq null mutant are not affected by an rpoS mutation. We conclude that HF-I is a global regulator involved in the regulation of expression of sigmaS and sigmaS-independent genes.     

Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth

Catecholamines may stimulate enteric bacteria including the foodborne pathogen Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by two mechanisms in vivo: as a quorum sensing signal and a supplier of iron. To identify genes of Salmonella Typhimurium that respond to norepinephrine, transposon mutagenesis and DNA microarray analysis were performed. Insertional mutations in the following genes decreased norepinephrine-enhanced growth: degS, entE, entF, fes, gpmA, hfq, STM3846. DNA microarray and real-time RT-PCR analyses revealed a decrease in the expression of several genes involved in iron acquisition and utilization during norepinephrine exposure, signifying the iron-limiting conditions of serum-SAPI minimal medium and the siderophore-like activity of norepinephrine. Unlike the wild-type parent strain, growth of neither a fepA iroN cirA mutant nor a fepC mutant, harboring deletional mutations in the outer and inner membrane transporters of enterochelin, respectively, was enhanced by norepinephrine. However, growth of the fepC and the fepA iroN cirA mutants could be rescued by an alternative siderophore, ferrioxamine E, further validating the role of norepinephrine in supplying the organism with iron via the catecholate-specific iron transport system. Contrary to previous reports using small animal models, the fepA iroN cirA mutant of Salmonella Typhimurium colonized the swine gastrointestinal tract, as did the fepC mutant.     

A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq

The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.     

Negative effect of glucose on ompA mRNA stability: a potential role of cyclic AMP in the repression of hfq in Escherichia coli

Glucose is a carbon source that is capable of modulating the level of cyclic AMP (cAMP)-regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose (40 mM) was present in Luria-Bertani (LB) medium. This effect was associated with a low level of cAMP induced by the glucose. The results were confirmed with an adenylyl cyclase mutant with low levels of cAMP that are not modulated by glucose. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were downregulated in the presence of cAMP. Furthermore, we showed that a complex of cAMP receptor protein (CRP) and cAMP binds to a specific P3(hfq) promoter region of hfq and regulates hfq expression. The regulation of the hfq gene was confirmed in vivo using an hfq-deficient mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that expression of hfq was repressed by the CRP-cAMP complex. The presence of glucose resulted in increased Hfq protein levels, which decreased ompA mRNA stability. An additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to the stability of other mRNA in E. coli.     

RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli

Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.     

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.