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 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.     

Investigation of the self-association and hetero-association interactions of H-NS and StpA from Enterobacteria

The nucleoid-associated protein H-NS and its paralogue StpA are global regulators of gene expression and form an integral part of the protein scaffold responsible for DNA condensation in Escherichia coli and Salmonella typhimurium . Although protein oligomerization is a requirement for this function, it is not entirely understood how this is accomplished. We address this by reporting on the self-association of H-NS and its hetero-association with StpA. We identify residues 1–77 of H-NS as being necessary and sufficient for high-order association. A multi-technique-based approach was used to measure the effects of salt concentration on the size distribution of H-NS and the thermal stability of H-NS and StpA dimers. The thermal stability of the StpA homodimer is significantly greater than that of H-NS₁₋₇₄. Investigation of the hetero-association of H-NS and StpA proteins suggested that the association of H-NS with StpA is more stable than the self-association of either H-NS or StpA with themselves. This provides a clear understanding of the method of oligomerization of these important proteins in effecting DNA condensation and reveals that the different associative properties of H-NS and StpA allow them to perform distinct, yet complementary roles in the bacterial nucleoid.     

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.     

Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli

The nucleoid-associated proteins H-NS and StpA in Escherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS-StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpA(F21C), forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.     

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.     

Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium highly adapted to a wide variety of environments and widely used in food and feed fermentations. Proteomes of two strains of L. plantarum, one isolated from spontaneously fermented cereal-based feed (strain REB1), and the other from white cabbage (strain MLBPL1), were studied to elucidate the strain-specific variation and the physiological changes occurring between the growth (lag, early-exponential, late-exponential and early-stationary) phases of this bacterium when cultivated in a standard rich medium. A total of 231 protein spots were identified by LC-MS/MS. These proteins showed that strain MLBPL1 had more proteins with growth phase-dependent expression than REB1, which possesses a more constant expression profile. The proteins with growth phase-dependent expression in REB1 and MLBPL1 were mainly associated with energy metabolism (glycolysis, phosphoketolase pathway and ribose metabolism), all having preferential expression in the early-exponential phase, confirming the use of different carbohydrates simultaneously. Indication of energy production was also seen in lag and early-stationary phases.     

Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.     

Gene silencing H-NS paralogue StpA forms a rigid protein filament along DNA that blocks DNA accessibility

Nucleoid-associated proteins are bacterial proteins that are responsible for chromosomal DNA compaction and global gene regulation. One such protein is Escherichia coli Histone-like nucleoid structuring protein (H-NS) which functions as a global gene silencer. Whereas the DNA-binding mechanism of H-NS is well-characterized, its paralogue, StpA which is also able to silence genes is less understood. Here we show that StpA is similar to H-NS in that it is able to form a rigid filament along DNA. In contrast to H-NS, the StpA filament interacts with a naked DNA segment to cause DNA bridging which results in simultaneous stiffening and bridging of DNA. DNA accessibility is effectively blocked after the formation of StpA filament on DNA, suggesting rigid filament formation is the important step in promoting gene silencing. We also show that >1 mM magnesium promotes higher order DNA condensation, suggesting StpA may also play a role in chromosomal DNA packaging.