Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization. Acquisition of DNA by horizontal gene transfer (HGT) significantly increases bacterial genetic variability. Relevant issues are the mechanisms that bacterial cells have evolved to efficiently integrate the newly acquired DNA into the host cell regulatory machinery. In Gram negative cells, the nucleoid associated protein H-NS has been shown to bind AT-rich sequences of HGT DNA and silence unwanted expression of these genes. This has led to consider H-NS as a “genome sentinel.” Nevertheless, this proposed role must be compatible with its role modulating core genome genes. Weak expression of recently transferred genes must be coordinated with proper expression levels of housekeeping genes. In this paper, we describe a strategy that enteric bacteria have developed to differentially modulate HGT and core genome genes. Two independent lines of experimental evidence suggest that the H-NS system of enteric bacteria may have evolved to discriminate between core genome and HGT DNA. The plasmid R27-encoded H-NS protein selectively modulates HGT genes. This avoids plasmid-encoded H-NS interfering with modulation of core functions. We also show that, for efficient silencing of HGT genes, resident chromosomal H-NS recruits the Hha protein and forms heteromeric complexes with DNA. In contrast, housekeeping genes are modulated by H-NS alone.

IncHI plasmids encode multiple-antibiotic resistance inSalmonella entericaserovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHI1 plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30°C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression inEscherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33°C), transcription of several ORFs in both transfer region 1 (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomalhhaandhnsmutations are compensated in cells harboring R27.

Salmonella is one of the most frequently reported causes of food-borne illness worldwide. Non-typhoideal serovars cause gastroenteritis in humans. Salmonella can grow on surfaces forming biofilms, contributing to its persistence since biofilms are difficult to eradicate due to the high resistance to antimicrobials and disinfectants. It has been described that there are two crucial biofilm promoting factors in Salmonella: curli and cellulose. The expression of both factors is coordinately regulated by the transcriptional regulator CsgD. Most biofilm studies of Salmonella have been performed by growing bacteria in low osmolarity rich medium and low temperature (25ºC). In such conditions, the biofilm is formed at the air-liquid interface (pellicle biofilm). Remarkably, when Salmonella grow in minimal medium, biofilm formation switches from the air-liquid interface to the solid-liquid interface (bottom biofilm). In this report the switching between pellicle and bottom biofilm has been characterized. Our data indicate that curli, but not cellulose, is crucial for the formation of both kinds of biofilms. In minimal medium, conditions promoting formation of bottom biofilm, a high transcriptional expression of csgD and consequently of the genes involved in the synthesis of curli and cellulose was detected. The nutritional status of the cells seems to be pivotal for the spatial distribution of the biofilms formed. The data reported let us conclude that when bacteria is growing in minimal medium the presence of amino acids downregulates the expression of csgB and causes the switch between bottom and pellicle biofilm. The crosstalk between general metabolism and biofilm formation is also highlighted by the fact that the metabolic sensor cAMP modulates the type of biofilm generated by Salmonella. Moreover, cAMP regulates transcriptional expression of csgD and stimulates pellicle biofilm formation, suggesting that the physiological conditions define the type of biofilm formed by Salmonella. The consequences of the switching between pellicle and bottom biofilm during either infection or survival in natural environments remain undercover.

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOS^SIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOS^SIAL and the ability to invade eukaryotic cells.

Summary: 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 S almonella , plasmids of the incompatibility group HI 1 account for a significant proportion of antibiotic resistance phenotypes. In this work, we show that plasmid R 27 has a strong impact on the global transcriptome of S almonella T yphimurium strain SL 1344 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 R 27 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 S almonella 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 R 27‐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.

Summary: Plasmid conjugation is a major horizontal gene transfer mechanism. The acquisition of a plasmid may cause a perturbation of the cell functions in addition to provide advantageous properties for the recipient cell, such as the gaining of antibiotic resistances. The interplay between plasmid and chromosomal functions has been studied using the IncHI1 plasmid R27. Plasmids of the incompatibility group HI1, isolated from several Gram‐negative pathogens, are associated with the spread of multidrug resistance. Their conjugation is tightly regulated by temperature, being repressed at temperatures within the host (37°C). In this report, we described that at permissive temperature, when conjugation of plasmid R27 is prompted, a reduction in the motility of the cells is observed. This reduction is mediated by the plasmid‐encoded regulators TrhR/TrhY, which together with HtdA form a plasmid‐borne regulatory circuit controlling R27 conjugation. TrhR/TrhY, required to induce R27 conjugation, is responsible for the downregulation of the flagella synthesis and the consequent decrease in motility. TrhR/TrhY repress, direct or indirectly, the expression of the specific flagellar sigma subunit FliA and, consequently, the expression of all genes located bellow in the flagellar expression cascade.