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Title: Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS}
Article:
Abstract:
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.
Introduction:
Acquisition of DNA by horizontal gene transfer( HGT) is a crucial mechanism by which bacteria increase genetic variability. Among others, functions that enable bacterial cells to cause disease( virulence factors) as well as to overcome the effect of antimicrobial drugs are often encoded in HGT DNA( i. e., bacterial plasmids or genomic islands). While HGT DNA may provide a potential advantage in host colonization, the incorporation of foreign DNA may constitute a potential perturbation for the regulation of the core genome, resulting in a significant fitness cost. An efficient mechanism that enables the bacterial cell to control the expression of foreign DNA is exemplified by the H-NS protein[for a review see 1]. H-NS belongs to the superfamily of bacterial nucleoid-associated proteins and is involved in the adaptative response of bacterial cells to changes in environmental factors such as temperature or osmolarity. The regulatory region of H-NS-modulated genes usually contains two separated target sequences, which have often been characterized by being AT-rich curved DNA stretches[2]. Interaction of H-NS molecules with their target sequences results in protein oligomerization and the generation of a DNA loop. When this nucleoprotein complex is formed, transcription is switched off[2]–[4]. Silencing is relieved when changes in physicochemical parameters( i. e., temperature) affect either DNA properties or the capacity of H-NS to oligomerize[2],[5],[6]. In some instances, H-NS-mediated silencing requires the participation of proteins of the Hha/YmoA family[for a review see 7]. Hha-like proteins have been identified on the basis of their role in modulating several virulence determinants[8]–[11]. Their molecular mass is about half of that of H-NS-like proteins. They show structural mimicry to the H-NS oligomerization domain, bind to H-NS and appear to comodulate the expression of several genes with this latter protein[7]. H-NS targets both core genome and HGT genes[12],[13] and provides an efficient mechanism that enables bacterial cells to control the expression of foreign DNA. Mapping of H-NS binding sites on the Salmonella enterica serovar Typhimurium chromosome by a ChlP on chip approach showed that H-NS binds preferentially to AT-rich HGT DNA[13],[14]. This finding has been interpreted as H-NS playing a relevant role in the silencing of unwanted expression of these sequences and has led to the proposal of a predominant role of the H-NS protein as a genome sentinel[15]. Several conjugative plasmids, such as those of the IncH1 group, also encode plasmidic forms of H-NS and Hha. IncH1 plasmids are common in the causal agent of typhoid fever, Salmonella enterica subsp. enterica serovar Typhimurium, and are associated with the multi-drug resistance( MDR) phenotype that some isolates exhibit[16]. A well-characterized representative of this incompatibility group is plasmid R27[17]. This plasmid was isolated from Salmonella enterica serovar Typhimurium in the 1960s and since then has been detected in several S. Typhi outbreaks. R27 is 180 kbp in length, confers tetracycline resistance and shows a temperature-dependent conjugative phenotype. R27 encodes single copies of hns and hha genes( ORFs 164 and 182 respectively). Both chromosomal- and plasmid-encoded H-NS and Hha proteins interact to modulate R27 temperature-dependent conjugative transfer[18]: either plasmid- or chromosomally-encoded H-NS and Hha proteins can repress conjugative transfer at high temperature. The role of Sfh, an H-NS-like protein encoded by plasmid pSf-R27, 99. 7% identical to R27, has been also addressed[19]–[21]. In an elegant series of experiments, it was shown that Sfh provides a stealth function that allows the plasmid to be transmitted to new bacterial cells without reducing fitness[22]. Plasmid-encoded H-NS would prevent the depletion of resident H-NS by AT-rich HGT sequences, for which H-NS shows a strong preference. To date, plasmid- and chromosome-encoded forms of H-NS proteins have been assumed to be functionally equivalent[18],[22]. Here we provide evidence that plasmid-encoded H-NS-like proteins have evolved to selectively target HGT and not core genome DNA. We also show that chromosomally-encoded H-NS targets both HGT and core genomic DNA, but differentially modulates them by using Hha-like proteins to specifically silence HGT genes. Altogether, our results suggest that in enteric bacteria the H-NS modulator may have evolved to discriminate between vertically and horizontally inherited DNA sequences, efficiently silencing the latter. This feature could provide a fitness advantage by allowing the presence of a large number of silent virulence genes to be available, without interfering with the regulation of the bacterial core genome.
Discussion:
It is well-documented and accepted that a key role of the global modulator H-NS is to silence large stretches of AT-rich HGT DNA[13]–[15],[27]. The capacity of this protein to preferentially bind to AT-rich motifs that display planar curvature probably underlies HGT DNA silencing. Efficient silencing of unwanted expression of foreign DNA appears, in turn, as a critical issue to facilitate the integration of newly acquired DNA into the host regulatory network. Nevertheless, several reports have also shown that H-NS modulates the expression of housekeeping genes, such as proV or leuO. Hence, a role for H-NS as a genome sentinel must be compatible with its function as a general gene regulator[15]. Our results open up a new perspective about how these two roles are accomplished by H-NS in enteric bacteria. To date, plasmid-encoded H-NS-like proteins have been considered to be functionally equivalent to the corresponding chromosomally-encoded paralogues. Indeed, functional replacements and equivalent sets of interactions have been shown for plasmid- and chromosomal- H-NS-like proteins[18],[20],[22]. On the basis of this equivalence, the presence of H-NS-like proteins in plasmids has been interpreted as providing an additional source of H-NS that can compensate the depletion of resident H-NS caused by binding of the protein to AT-rich stretches of newly acquired plasmids[22]. The results reported here evidence that plasmidic and chromosomal H-NS proteins show considerable functional differences. H-NSR27 may contribute to reducing the fitness cost of housing HGT DNA by targeting plasmid sequences while leaving chromosomal H-NS available for its corresponding chromosomal targets, as suggested[22]. In addition, H-NSR27 does not influence the intrinsic regulation of H-NS- sensitive housekeeping genes, such as rcsA, psiF, proV or stpA. The identification of a HGT-specific plasmid-encoded H-NS protein suggests that H-NS sensitive genes form two distinct genetic compartments. A relevant question is how resident chromosomal H-NS recognizes and differentially modulates genes from both compartments. We show that H-NS interacts with members of the Hha/YmoA family to specifically silence HGT but not core genome genes. Coregulation of gene expression by H-NS/Hha proteins was first evidenced for the E. coli hly operon[8]. Further data demonstrated that other genes in several enteric bacteria are modulated by an H-NS/Hha complex[11]–[13], rather than by H-NS alone. A recent global transcriptomic analysis has shown that, when S. Typhimurium SV5015 is grown in LB medium at 37°C, the set of genes sensitive to modulation by Hha and/or its paralogue YdgT is coincident with the set of H-NS-modulated genes that map in HGT DNA[26]. Remarkably, Hha/YdgT proteins appear to silence mainly HGT genes. The effect of hha/ydgT mutations on the transcriptome of E. coli strain BSN26 is very limited. Only a small number of genes is affected( our unpublished results). We interpreted these data as strain BSN26 containing a very limited amount of HGT DNA. Genes sensitive to Hha/YdgT modulation are silenced under several in vitro growth conditions( i. e., low and high temperature, low and high osmolarity), and a significant number are silenced by H-NS under the same conditions. H-NS-controlled weak expression of HGT DNA[28] must be compatible with the expression of several housekeeping genes, which are readily switched on in response to specific stimuli. A good example of the latter is osmolarity-dependent modulation of proV, which is silenced by H-NS alone and is insensitive to Hha silencing. We found a significant coincidence between the set of genes modulated by H-NS/Hha and those sensitive to H-NSR27. Plasmidic genes and chromosomal genes incorporated by HGT are prominent members of the set that are both silenced by H-NSR27 or require Hha to be silenced by the chromosomal form of H-NS. This observation strengthens the notion that H-NS-modulated genes can be assigned to two genetic compartments. The first includes genes encoding housekeeping functions that are modulated by chromosomal H-NS alone and are insensitive or only weakly modulated by plasmidic H-NSR27. In contrast, the genes belonging to the second compartment, which includes mostly horizontally acquired genes, require a helper protein of the Hha family for their complete silencing by chromosomal H-NS, and can also be modulated by plasmidic H-NS. For many years, it was considered that H-NS did not recognize a consensus DNA sequence, but bound to AT-rich curved stretches of DNA[1]. In recent years, significant efforts have been devoted to defining high-affinity binding sites for H-NS[29],[30]. Our results suggest that H-NS-sensitive promoters fall into at least two categories. H-NS sensitive promoters may share some basic common characteristics but display differences that can be picked up by the plasmidic H-NS form or by H-NS/Hha complexes. Differential affinity of H-NS-like proteins for DNA regions targeted by H-NS has also recently been proposed[31]. The StpA paralogue binds to DNA regions similar to those bound by H-NS in E. coli wt cells. In contrast, only one-third of these sequences are bound by StpA in the absence of H-NS. This partial binding results in only partial StpA-mediated modulation of H-NS-sensitive genes in hns mutants. While the basis for such differential affinity may be the generation of either StpA-H-NS hetero- or StpA-StpA homodimers, in the example reported here structural differences between H-NS and H-NSR27 might account for the differential affinity of these two proteins for some promoter regions. Sequence conservation between H-NSR27 and H-NS in the N- and C-terminal domains was very high; however, significant differences between H-NS and H-NSR27 were located in the linker domain( 53% of the positions were different)( data not shown). The linker domain is predicted to be partially unstructured in the isolated protein and is associated with protein oligomerization although it has also been implicated in the modulation of DNA binding[32]. While H-NS-like proteins are widely distributed within γ-proteobacteria, both Hha-like proteins and the presence of H-NS-like proteins in conjugative plasmids appear to be an evolutionary trait of members of the Enterobacteriaceae[33]. Members of this family display the ability to use the H-NS protein to silence HGT regions with the help of co-repressors of the Hha family of proteins. Specialized plasmidic H-NS-like molecules also have the capacity to modulate HGT DNA, but have evolved not to interfere with core genes. These features should facilitate the incorporation of HGT DNA, leading to more complex genomes with increased capability to adapt to new environments. This adaptive capacity may also explain why several enterobacterial representatives, such as virulent E. coli strains, have become such successful pathogens[34],[35].
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 .