Effects of multicopy LeuO on the expression of the acid-inducible lysine decarboxylase gene in Escherichia coli.

We previously reported that mutations in hns, the structural gene for the histone-like protein H-NS, cause derepressed expression of cadA, which encodes the acid-inducible lysine decarboxylase at noninducing pH (pH 8.0). This study reports the characterization of a plasmid isolated from an Escherichia coli library that suppresses the effect of an hns mutation on cadA expression. A previously sequenced open reading frame, leuO, proves to be the gene that causes the hns-complementing phenotype. The mechanism for this phenotype appears to be overexpression of leuO from a multicopy plasmid, which drastically reduces production of CadC, the essential activator for cadA induction. These results show an in vivo regulatory phenotype for leuO, consistent with its proposed protein sequence.     

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.     

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.     

rpoS Function Is Essential for bgl Silencing Caused by C-Terminally Truncated H-NS in Escherichia coli

From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein. It was found that sigma(S) is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.     

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.