Differential spectrum of mutations that activate the Escherichia coli bgl operon in an rpoS genetic background

The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a beta-glucoside-negative (Bgl-) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures. The rpoS-encoded sigmaS, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds. We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells. Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells. The physiological significance of these differences is discussed in the context of survival of natural populations of E. coli.     

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.     

The StpA Protein Functions as a Molecular Adapter To Mediate Repression of the bgl Operon by Truncated H-NS in Escherichia coli

The mechanism of repression of the β-glucoside utilization (bgl) operon of Escherichia coli by a carboxy-terminally truncated derivative of the nucleoid-associated protein H-NS which is defective in DNA binding was investigated. The DNA-binding function of the H-NS-like protein StpA was found to be necessary for repression, which is consistent with a role for StpA as a DNA-binding adapter for mutant derivatives of H-NS.     

Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli

The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.     

LeuO antagonizes H-NS and StpA-dependent repression in Salmonella enterica ompS1

The ompS1 gene encodes a quiescent porin in Salmonella enterica. We analysed the effects of H-NS and StpA, a paralogue of H-NS, on ompS1 expression. In an hns single mutant expression was derepressed but did not reach the maximum level. Expression in an stpA single mutant showed the same low repressed level as the wild type. In contrast, in an hns stpA background, OmpS1 became abundant in the outer membrane. The expression of ompS1 was positively regulated by LeuO, a LysR-type quiescent regulator that has been involved in pathogenesis. Upon induction of the cloned leuO gene into the wild type, ompS1 was completely derepressed and the OmpS1 porin was detected in the outer membrane. LeuO activated the P1 promoter in an OmpR-dependent manner and P2 in the absence of OmpR. LeuO bound upstream of the regulatory region of ompS1 overlapping with one nucleation site of H-NS and StpA. Our results are thus consistent with a model where H-NS binds at a nucleation site and LeuO displaces H-NS and StpA.     

Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli

The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.     

H-NS regulation of IraD and IraM antiadaptors for control of RpoS degradation

RpoS, the master sigma factor during stationary phase and under a variety of stress conditions, is regulated at multiple levels, including regulated degradation. Degradation is dependent upon ClpXP and the RssB adaptor protein. H-NS, a nucleoid-associated protein, affects the regulated degradation of RpoS; in the absence of H-NS, RpoS is stable. The mechanisms involved in this regulation were not known. We have found that H-NS inhibits the expression of iraD and iraM, the genes coding for two antiadaptor proteins that stabilize RpoS when overexpressed. The regulation by H-NS of iraM is independent from the previously demonstrated regulation by the PhoP/PhoQ two-component system. Moreover, differences in the behavior of several hns alleles are explained by a role for StpA, an H-NS-like protein, in the regulation of RpoS stability. This finding parallels recent observations for a role of StpA in regulation of RpoS stability in Salmonella.     

The LysR-like regulator LeuO in Escherichia coli is involved in the translational regulation of rpoS by affecting the expression of the small regulatory DsrA-RNA

The translation of rpoS , which encodes the general stress sigma factor, σ^S, in Escherichia coli , is stimulated by various stress conditions. Regulatory factors involved in this control are the RNA-binding Hfq (HF-I) protein, the histone-like protein H-NS and the small regulatory DsrA-RNA (with the last being specifically required for increased rpoS translation at low temperature). Here, we report the characterization of a transposon insertion mutant (Tn 10 -8) with reduced σ^S levels that led to the identification of an additional factor involved in the regulation of rpoS translation, the LysR-like regulator LeuO. Tn 10 -8 decreases rpoS translation predominantly at low growth temperature. The mutation results in similarly strongly reduced DsrA-RNA expression and does not affect rpoS expression in a dsrA null mutant background, indicating that it affects rpoS translation via DsrA-RNA. Tn 10 -8 is inserted 26 bp upstream of the leuO open reading frame, which encodes a putative LysR-like regulator of unknown function. Instead of being a leuO null mutation, Tn 10 -8 activates leuO expression as a result of the p_out promoter on IS 10 _L reading into leuO , indicating that LeuO represses dsrA and thereby reduces rpoS translation at low temperature. LeuO does not contribute to temperature regulation of dsrA since its own expression is rather low and not temperature dependent. In a mutant deficient for H-NS, however, leuO is strongly derepressed. We conclude that rpoS translation is controlled by a regulatory network that includes Hfq, H-NS, LeuO and DsrA-RNA. In this network, H-NS plays a dual role by interfering with rpoS translation in general and, via LeuO, influencing the synthesis of its own low-temperature antagonist, DsrA-RNA.     

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.     

YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes. We also demonstrate that YdgT interacts both with H-NS and with StpA. Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell. The StpA protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.