Sonia Paytubi

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.

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.

Integration host factor alleviates H-NS silencing of the Salmonella enterica serovar Typhimurium master regulator of SPI1, hilA.

Coordination of the expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 (SPI1) depends on a complex circuit involving several regulators that converge on expression of the hilA gene, which encodes a transcriptional activator (HilA) that modulates expression of the SPI1 virulence genes. Two of the global regulators that influence hilA expression are the nucleoid-associated proteins Hha and H-NS. They interact and form a complex that modulates gene expression. A chromosomal transcriptional fusion was constructed to assess the effects of these modulators on hilA transcription under several environmental conditions as well as at different stages of growth. The results obtained showed that these proteins play a role in silencing hilA expression at both low temperature and low osmolarity, irrespective of the growth phase. H-NS accounts for the main repressor activity. At high temperature and osmolarity, H-NS-mediated silencing completely ceases when cells enter the stationary phase, and hilA expression is induced. Mutants lacking IHF did not induce hilA in cells entering the stationary phase, and this lack of induction was dependent on the presence of H-NS. Band-shift assays and in vitro transcription data showed that for hilA induction under certain growth conditions, IHF is required to alleviate H-NS-mediated silencing.

A novel role for antibiotic resistance plasmids in facilitating Salmonella adaptation to non‐host environments

It is believed that the main role of plasmids that encode multiple antibiotic resistance is to confer their hosts the ability to survive in the presence of antimicrobial compounds. In the pathogenic bacterium Salmonella, plasmids of the incompatibility group HI1 account for a significant proportion of antibiotic resistance phenotypes. In this work, we show that plasmid R27 has a strong impact on the global transcriptome of Salmonella Typhimurium strain SL1344 when cells grow at low temperature and enter the stationary phase. Down-regulated genes include pathogenicity islands, anaerobic respiration and metabolism determinants. Up-regulated genes include factors involved in the response to nutrient starvation, antimicrobial resistance, iron metabolism and the heat shock response. Accordingly, cells harbouring R27 are more resistant to heat shock than plasmid-free cells. The use of a different IncHI1 plasmid, pHCM1, provided evidence that these plasmids facilitate adaptation of Salmonella to environmental conditions outside their host(s). This is consistent with the fact that conjugative transfer of IncHI1 plasmids only occurs at low temperature. A significant number of the R27-dependent alterations in gene expression could be correlated with expression of a plasmid-encoded orthologue of the global modulator H-NS, which is up-regulated when cells grow at low temperature.

The Hha protein facilitates incorporation of horizontally acquired DNA in enteric bacteria.

Hha-like proteins are an evolutive trait of members of the family Enterobacteriaceae. These proteins mimic the oligomerization domain of the nucleoid-associated protein H-NS and interact with this latter protein to modulate gene expression. In this report, we provide evidence that, as has been shown for H-NS, Hha-like proteins play an essential role facilitating acquisition of horizontally transferred DNA in both Escherichia coli and Salmonella. Incorporation of conjugative plasmids such as pHly152 or R27 results in a fitness cost in E. coli or Salmonella strains that lack Hha-like proteins. E. coli spontaneous derivatives from double hha ydgT mutants that showed an increased growth rate and a restored fitness overexpressed the H-NS protein. In addition to reinforcing the role of H-NS/Hha-modulating xenogeneic DNA, the results obtained demonstrate that the Enterobacteriaceae display regulatory features not found in other bacteria that facilitate incorporation of horizontally transferred DNA.

Essential residues in the H-NS binding site of Hha, a co-regulator of horizontally acquired genes in Enterobacteria

YdgT and H‐NS bind by nuclear magnetic resonance (View interaction) Hha and H‐NS bind by nuclear magnetic resonance (View Interaction 1, 2, 3) Hha physically interacts with H‐NS by pull down (View Interaction 1, 2).

Success in incorporating horizontally transferred genes: the H-NS protein

The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer.

Bacterial Hha-like proteins facilitate incorporation of horizontally transferred DNA

Horizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.

A High-Throughput Screening Platform of Microbial Natural Products for the Discovery of Molecules with Antibiofilm Properties against Salmonella

In this report, we describe a High-Throughput Screening (HTS) to identify compounds that inhibit biofilm formation or cause the disintegration of an already formed biofilm using the Salmonella Enteritidis 3934 strain. Initially, we developed a new methodology for growing Salmonella biofilms suitable for HTS platforms. The biomass associated with biofilm at the solid-liquid interface was quantified by staining both with resazurin and crystal violet, to detect living cells and total biofilm mass, respectively. For a pilot project, a subset of 1120 extracts from the Fundación MEDINAs collection was examined to identify molecules with antibiofilm activity. This is the first validated HTS assay of microbial natural product extracts which allows for the detection of four types of activities which are not mutually exclusive: inhibition of biofilm formation, detachment of the preformed biofilm and antimicrobial activity against planktonic cells or biofilm embedded cells. Currently, several extracts have been selected for further fractionation and purification of the active compounds. In one of the natural extracts patulin has been identified as a potent molecule with antimicrobial activity against both, planktonic cells and cells within the biofilm. These findings provide a proof of concept that the developed HTS can lead to the discovery of new natural compounds with antibiofilm activity against Salmonella and its possible use as an alternative to antimicrobial therapies and traditional disinfectants.

Role of plasmid- and chromosomally encoded Hha proteins in modulation of gene expression in E. coli O157:H7

H-NS and Hha belong to the nucleoid-associated family of proteins and modulate gene expression in response to environmental stimuli. Genes coding for these proteins can be either chromosomally or plasmid-encoded. In this work, we analyse the regulatory role of the Hha protein encoded in the virulence plasmid of the enterohemorrhagic Escherichia coli O157:H7 (Hha(pO157)). This plasmid is present in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both, Hha(pO157) and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant degree of similarity. The hha gene from plasmid pO157 is transcribed from its own putative promoter and is overexpressed in a chromosomal hha mutant. As its chromosomal counterpart, Hha(pO157) is able to interact with H-NS. Remarkably, Hha(pO157) targets only a subset of the genes modulated by Hhachr. This has been evidenced by both assaying the ability of Hha(pO157) to complement expression of a specific operon (i.e., the haemolysin operon) and by comparing the global transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives. Hha(pO157) and Hhachr share some common regulatory features, however they also display specific targeting of some genes and even a different modulatory role in some others.