José María Nieto

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

Abstract The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin.

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

The hha gene modulates haemolysin expression in Escherichia coli

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202‐312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.

Microbial community structure of a heavy fuel oil-degrading marine consortium: linking microbial dynamics with polycyclic aromatic hydrocarbon utilization.

A marine microbial consortium obtained from a beach contaminated by the Prestige oil spill proved highly efficient in removing the different hydrocarbon families present in this heavy fuel oil. Seawater cultures showed a complete removal of all the linear and branched alkanes, an extensive attack on three to five-ring polycyclic aromatic hydrocarbons [PAHs; including anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, and benzo(a)pyrene] (30-100%), and a considerable depletion of their alkyl derivatives. Community dynamics analysis revealed that Alcanivorax species, known alkane degraders, predominated in the initial stages. This was followed by an increase in Alphaproteobacteria (i.e. Maricaulis, Roseovarius), which coincided with the depletion of low molecular PAHs. Finally, these were succeeded by Gammaproteobacteria (mainly Marinobacter and Methylophaga), which were involved in the degradation of the high molecular-weight PAHs. The role of these populations in the removal of the specific components was confirmed by the analysis of subcultures established using the aliphatic or the aromatic fraction of the fuel oil, or single PAHs, as carbon sources. The genus Marinobacter seemed to play a major role in the degradation of a variety of hydrocarbons, as several members of this group were isolated from the different enrichment cultures and grew on plates with hexadecane or single PAHs as sole carbon sources.

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.

Role of the Hha/YmoA family of proteins in the thermoregulation of the expression of virulence factors.

Virulent bacteria are able to sense temperature changes and respond by modifying the expression of--among others--genes that code for virulence factors. The chromatin-associated protein H-NS has been shown to play a role in the thermomodulation of virulence factor expression. In addition to H-NS, proteins of the Hha/YmoA family have also been identified in different enterobacteria as participating in the thermoregulation of some virulence factors. For one of these proteins, the Hha protein, it has been shown that it interacts with H-NS, and both proteins form a nucleoid-protein complex responsible for the thermoregulation of, at least, E. coli hemolysin. The presence of genes coding for homologues of both proteins on some conjugative plasmids and their relation to thermoregulation suggests that this complex could also play a role in the regulation of plasmid transfer.

Interaction between the bacterial nucleoid associated proteins Hha and H-NS involves a conformational change of Hha

The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 degrees C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS.

Phenol removal from hypersaline wastewaters in a Membrane Biological Reactor (MBR): Operation and microbiological characterisation

In this study, two Membrane Biological Reactors (MBR) with submerged flat membranes, one at lab-scale conditions and the other at pilot-plant conditions, were operated at environmental temperature to treat an industrial wastewater characterised by low phenol concentrations (8-16 mg L(-1)) and high salinity (∼ 150-160 mS cm(-1)). During the operation of both reactors, the phenol loading rate was progressively increased and less than 1mg phenol L(-1) was detected even at very low HRTs (0.5-0.7 days). Membrane fouling was minimized by the cross flow aeration rate inside the MBRs and by intermittent permeation. Microbial community analysis of both reactors revealed that members of the genera Halomonas and Marinobacter (gammaproteobacteria) were major components. Growth-linked phenol degradation by pure cultures of Marinobacter isolates demonstrated that this bacterium played a major role in the removal of phenol from the bioreactors.

Functional replacement of the oligomerization domain of H-NS by the hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.

Interaction of the Nucleoid-Associated Proteins HAA and H-NS to Modulated Expression of the Hemolysin Operon in Escherichia coli

Both the in vitro and in vivo data reported here strongly support the hypothesis that Hha and H-NS interact with DNA to generate a nucleoprotein complex that influences gene expression. The Hha-H-NS complex seems to be mainly responsible for the thermoosmotic modulation of the expression of the hly operon of plasmid pHly152. Binding sites for Hha and H-NS along the regulatory region of the hly operon, as well as the topological changes mediated by the interaction of both proteins with DNA, are currently being investigated.