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Funding: A.F.H. and L.L. were funded by Marie Sklodowska-Curie Intra-European Fellowships (PIEF-GA-2012-328377 and PIEF-GA-2013-623168, respectively). Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host’s immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. Publisher PDF

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively. GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component. Postprint

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high–medium–low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype., Author summary Complement plays a key role in the immunity against Neisseria meningitidis. The meningococcus uses the Factor H binding protein (fHbp), to bind a negative regulator of the alternative complement pathway, factor H, to its surface thus preventing complement deposition and lysis. The use of fHbp as an antigen in two licensed vaccines highlights its public health relevance. Therefore the levels of this antigen produced by the bacterium are pivotal on the one hand for the survival of N. meningitidis in blood and on the other hand for the susceptibility to vaccine-induced killing antibodies. Here, we identify the predominant nucleotide sequences that drive distinct levels of the fHbp antigen in circulating meningococcal strains. We cluster them into distinct groups with increasing levels and observe that strains expressing higher fHbp amounts are associated with invasive disease. Our findings show that the nucleotide sequence of the fHbp promoter can be used for the prediction of antigen levels of any given strain and consequently for both the assessment of its sensitivity to killing by fHbp antibodies and its likelihood to cause invasive disease.

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis., Author Summary Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide. Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen.

The Neisseria meningitidis regulator NadR was shown to repress expression of the NadA adhesin and play a major role in NadA phase-variable expression. In this study, we identified through microarray analysis over 30 genes coregulated with nadA in the NadR mutant and defined members of the NadR regulon through in vitro DNA-binding assays. Two distinct types of promoter architectures (I and II) were identified for NadR targets, differing in both the number and position of NadR-binding sites. All NadR-regulated genes investigated were found to respond to 4-hydroxyphenylacetic acid (4HPA), a small molecule secreted in human saliva, which was previously demonstrated to induce nadA expression by alleviating NadR-dependent repression. Interestingly, two types of NadR 4HPA responsive activities were found on different NadR targets corresponding to the two types of genes identified by different promoter architectures: while NadA and the majority of NadR targets (type I) are induced, only the MafA adhesins (type II) are corepressed in response to the same 4HPA signal. This alternate behavior of NadR was confirmed in a panel of strains in response to 4HPA and after incubation in saliva. The in vitro NadR binding activity at type I and type II promoter regions is differentially affected by 4HPA, suggesting that the nature of the NadR binding sites may define the regulation to which they will be subjected. We conclude that NadR coordinates a broad transcriptional response to signals present in human saliva, mimicked in vitro by 4HPA, enabling the meningococcus to adapt to the relevant host niche.

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis , we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.

The hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain and hspR and hrcA singly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.

Iron homeostasis is particularly important in pathogenic bacteria, which need to compete with the host for this essential cofactor. In Helicobacter pylori , a causative agent of several gastric pathologies, iron uptake and storage genes are regulated at the transcriptional level by the ferric uptake regulator Fur. The regulatory circuit of Fur has recently come under focus because of an intimate interlink with a broader regulatory network governing metal homeostasis, acidic response, and virulence. To dissect the Fur regulatory circuit and identify in vivo targets of regulation, we developed a genome-wide location analysis protocol which allowed the identification of 200 genomic loci bound by Fur as well as the investigation of the binding efficiency of the protein to these loci in response to iron. Comparative analysis with transcriptomes of wild-type and fur deletion mutant strains allowed the distinction between targets associated with Fur regulation and genes indirectly influenced by the fur mutation. The Fur regulon includes 59 genes, 25 of which appear to be positively regulated. A case study conducted by primer extension analysis of two oppositely regulated genes, hpn2 and flaB , suggests that negative regulation as well as positive regulation occurs at the transcriptional level. Furthermore, the results revealed the existence of 13 Fur targeted loci within polycistronic operons, which were associated with transcript deregulation in the fur mutant strain. This study provides a systematic insight of Fur regulation at the genome-wide level in H. pylori and points to regulatory functions extending beyond the classical Fur repression paradigm.

The ferric uptake regulator Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. In this study we used comparative transcriptome analysis to define the role of the Fur protein in the global control of gene transcription and iron regulation in Neisseria meningitidis . By using the Fur-null mutant and its complemented derivative, we identified 83 genes whose transcription is controlled by Fur. We report that Fur may control differential expression of these genes by binding directly to their promoters or through indirect mechanisms. In addition, mutation of the fur gene resulted in the induction of the heat shock response, and transcription of these genes does not respond to iron limitation. Furthermore, analysis of the iron starvation stimulon in the Fur-null mutant provided evidences of iron-responsive regulation that is independent of Fur. We began to dissect the regulatory networks of Fur and the heat shock (stress) response in N. meningitidis , and the observed interlink between the two circuits is discussed.

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

We have previously shown that in the human pathogen Neisseria meningitidis group B (MenB) more than 200 genes are regulated in response to growth with iron. Among the Fur-dependent, upregulated genes identified by microarray analysis was a putative operon constituted by three genes, annotated as NMB1436, NMB1437 and NMB1438 and encoding proteins with so far unknown function. The operon was remarkably upregulated in the presence of iron and, on the basis of gel retardation analysis, its regulation was Fur dependent. In this study, we have further characterized the role of iron and Fur in the regulation of the NMB1436-38 operon and we have mapped the promoter and the Fur binding site. We also demonstrate by mutant analysis that the NMB1436-38 operon is required for protection of MenB to hydrogen peroxide-mediated killing. By using both microarray analysis and S1 mapping, we demonstrate that the operon is not regulated by oxidative stress signals. We also show that the deletion of the NMB1436-38 operon results in an impaired capacity of MenB to survive in the blood of mice using an adult mouse model of MenB infection. Finally, we show that the NMB1436-38 deletion mutant exhibits increased susceptibility to the killing activity of polymorphonuclears (PMNs), suggesting that the 'attenuated' phenotype is mediated in part by the increased sensitivity to reactive oxygen species-producing cells. This study represents one of the first examples of the use of DNA microarray to assign a biological role to hypothetical genes in bacteria.

The HspR repressor regulates transcription of the groESL , hrcA-grpE-dnaK , and cbpA-hspR-orf operons of Helicobacter pylori . Here we show that two of the HspR-regulated operons, namely, the groESL and dnaK operons, encoding the major cellular chaperone machineries are also regulated by the H. pylori homologue of the HrcA repressor. Similarly to the hspR mutation, deletion of the hrcA gene also leads to complete derepression of the P gro and P hrc promoters. The presence of both HspR and HrcA is therefore necessary for regulated transcription from these promoters. HrcA binds directly to P gro and P hrc , likely contacting two inverted repeats with similarity to the CIRCE motif, which are present on both promoters. HrcA regulation is, however, shown to depend on binding of the HspR protein, since deletion of the HspR-binding site of the P gro promoter leads to loss of heat inducibility of this promoter. In contrast, transcription from the P cbp promoter is regulated solely by HspR. HspR is also shown to form oligomers in vivo through a stretch of hydrophobic repeats between amino acid positions 66 and 97. The implications of these findings for the elucidation of the networks regulating heat shock gene expression in H. pylori are discussed.

Summary Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. Four promoters of Neisseria meningitidis predicted to have Fur-binding boxes were selected to study the molecular interactions between Fur and the promoter regions of genes expected to play a central role in survival and pathogenesis. We demonstrate that Fur acts not only as a repressor, but also as an activator of gene expression both in vivo and in vitro. We report that Fur binds to operators located upstream of three promoters that are positively regulated in vivo by Fur and iron, whereas Fur binds to an operator overlapping the classically iron-repressed tbp promoter. Deletion of the upstream operator in the norB promoter abolished activation of transcription in vivo in response to iron and in vitro in response to Fur. The role of such a dual mechanism of Fur regulation during infection is discussed.

The Fur protein acts as a regulator of iron-dependent gene transcription in bacteria. In Helicobacter pylori, Fur regulates iron-activated and iron-repressed promoters. It also acts as an autoregulatory rheostat of transcription to fine-tune its own expression in response to iron by binding to three operators at its own promoter Pfur. Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at -110) is essential for iron regulation of Pfur and functions as an anti-repression site that is bound by the iron-free form of Fur to induce transcription. Furthermore, operator I (centred at -50) may have a dual role both as a high-affinity binding site for Fur and as an UP element. We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level. Our data supports a novel promoter architecture and mechanism of regulation by Fur.

The antifungal metabolite 2,4-diacetylphloroglucinol plays a major role in the biocontrol capabilities of Pseudomonas fluorescens. The phloroglucinol biosynthetic locus of P. fluorescens F113 has been isolated previously. From nucleotide sequence data, a putative regulator gene (phlF) was identified upstream and divergently transcribed from the phlACBD phloroglucinol biosynthetic genes. PhlF shows similarity to various transcriptional repressors in the EMBL database and exhibits a helix–turn–helix motif in its amino acid sequence. phlF was cloned into an expression vector and the PhlF protein product was purified. Gel retardation experiments demonstrated PhlF to be a DNA-binding protein and showed that it binds to the phlA–phlF intergenic region. Introduction of phlF into P. fluorescens F113 in multiple copies resulted in repression of phloroglucinol production in this strain. This effect was mediated at the transcription level since the expression of a phloroglucinol biosynthetic gene fusion in this background was equally repressed. Furthermore, the inactivation of phlF results in derepression of phloroglucinol production in this strain.

Neisseria meningitidis is a major cause of septicemia and meningitis. The hypervirulent clonal complex 41/44 (cc41/44) has emerged as the predominant cause of serogroup B meningococcal disease, having been responsible for recent outbreaks and epidemics worldwide. However, the meningococcal factors that enable transition from asymptomatic carriage to rapidly progressing disease are poorly understood. Here we describe a novel phase-variable DNA methyltransferase, ModD, which was identified in the genome sequence of a New Zealand epidemic isolate. Investigation of the distribution of modD in the wider meningococcal population, by PCR and sequence analysis of genetically diverse N. meningitidis strains, revealed the presence of modD in 20/27 strains in cc41/44, but in only 2/47 strains from other clonal complexes, indicating a significant association of modD with cc41/44 (Fisher's exact P value=3×10(-10)). The modD gene contains 5'-ACCGA-3' repeats that mediate phase variation, leading to reversible on/off switching of modD expression. Microarray analysis of modD-on/off variants revealed that ModD regulates expression of multiple genes involved in colonization, infection, and protection against host defenses, with increased catalase expression in the modD-on variant conferring increased resistance to oxidative stress. The modulation of gene expression via the ModD phase-variable regulon (phasevarion), and its significant association with the cc41/44, suggest a role in the fitness and/or pathogenesis of strains belonging to the cc41/44.

In this study we characterized a genetic locus that is predicted to encode one of the three AraC-like regulators of Neisseria meningitidis, a homologue of MpeR of Neisseria gonorrhoeae which is specific to the pathogenic Neisseria species. Previous microarray studies have suggested that this gene is a member of the Fur regulon. In strain MC58, it is a pseudogene (annotated as two ORFs, NMB1879 and NMB1878) containing a frameshift mutation which we show is common to all strains tested belonging to the ST-32 hypervirulent clonal complex. Using primer extension and S1 nuclease protection assays, we mapped two promoters in the upstream intergenic region: the mpeR promoter and the NMB1880 promoter. The latter promoter drives transcription of the divergent upstream locus, which is predicted to encode a high-affinity iron uptake system. We demonstrated that both promoters are induced during iron limitation and that this regulation is also mediated by the Fur regulator. DNA-binding studies with the purified MpeR protein revealed that it binds to a region directly upstream of the NMB1880 divergent promoter, suggesting a role in its regulation. Mutants of N. meningitidis strains lacking MpeR or overexpressing MpeR showed no significant differences in expression of the P NMB1880 promoter, nor did global transcriptional profiling of an MpeR knockout identify any deregulated genes, suggesting that the MpeR protein is inactive under the conditions used in these experiments. The presence of MpeR in a regulatory cascade downstream of the Fur master iron regulator implicates it as being expressed in the iron-limiting environment of the host, where it may in turn regulate a group of genes, including the divergent iron transport locus, in response to signals important for infection.

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus.

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals., Author Summary Diversification strategies, through genetic switches that randomly turn genes on and off, occur in many pathogenic bacterial populations and confer adaptive advantages to new environments and evasion of host immune responses. This is often mediated by spontaneous changes in the length of short DNA sequence repeats located in protein-coding regions or upstream regulatory regions, leading to deactivation or alteration of the associated genes. In this study we describe how a repeat sequence, distally upstream of the promoter region, alters the expression of an important adhesin of N. meningitidis. We identify the major mediator of this control, a negative regulator NadR, which binds to sequences flanking the variable repeat. Changes in the spacing between these sequences affect the ability of NadR to shut down expression from the promoter. We also identify a relevant metabolite that can block NadR activity and therefore act as a signal to induce adhesin expression. This finding sheds new light on the role of DNA-repeats identified in intergenic regions for which no role could be hypothesised, and may be a model mechanism used by bacterial pathogens for fine-tuning diversity within the host. Elucidating these mechanisms can aid in our understanding and prevention of disease.

Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. The fur gene in Neisseria meningitidis has been described as an essential gene that may regulate a broad array of genes. We succeeded in obtaining an N. meningitidis mutant with the fur gene knocked out and used it to undertake studies of fur -mediated iron regulation. We show that expression of both Fur and the transferrin binding protein Tbp2 is iron regulated and demonstrate that this regulation is Fur mediated for the Tbp2 protein. Footprinting analysis revealed that Fur binds to two distinct sites upstream of its coding region with different affinities and that these binding sites overlap two promoters that differentially control transcription of the fur gene in response to iron. The presence of two independently regulated fur promoters may allow meningococcus to fine-tune expression of this regulator controlling iron homeostasis, possibly during infection.

The major chaperone genes of Helicobacter pylori are negatively regulated by HspR, a homologue of the repressor of the dnaK operon of Streptomyces coelicolor. Using an in vitro selection and amplification approach we identified two new chromosomal binding sites of the HspR protein. Both binding sites were characterized by footprinting analysis with purified HspR protein. Intriguingly, these HspR binding sites are located at the 3′ ends of two genes coding for predicted proteins with functions unrelated to those of chaperones. This suggests that H. pylori HspR may regulate the expression of genes encoding proteins with diverse functions. Nucleotide sequence alignment of HspR-binding sites highlights conserved nucleotides extending outside the previously proposed consensus binding sequence with structural features predicting geometry of HspR binding as an oligomer. © 2002 Elsevier Science B.V. All rights reserved.

We have overexpressed and purified the Helicobacter pylori Fur protein and analyzed its interaction with the intergenic regions of divergent genes involved in iron uptake ( frpB and ceuE ) and oxygen radical detoxification ( katA and tsaA ). DNase I footprint analysis showed that Fur binds specifically to a high-affinity site overlapping the P frpB promoter and to low-affinity sites located upstream from promoters within both the frpB-katA and ceuE-tsaA intergenic regions. Construction of an isogenic fur mutant indicated that Fur regulates transcription from the P frpB promoter in response to iron. In contrast, no effect by either Fur or iron was observed for the other promoters.

A common strategy used by both Gram-negative and Gram-positive bacterial pathogens is based on the synchronisation of virulence gene expression using a variety of regulatory systems and networks to overcome host defence. During the last decade an exponentially growing number of studies on Helicobacter pylori, a human pathogen associated with diverse stomach diseases, have mainly focussed on the elucidation of mechanisms and functions of virulence factors. A subset of these studies were focussed on the molecular mechanisms regulating gene transcription in H. pylori with the aim of understanding the profound physiological changes that this pathogen, as well as other bacteria, undergoes during infection. Despite the limited number of putative regulatory proteins, as deduced from genome sequence analyses, evidence is accumulating for the existence of new and complex circuits regulating gene transcription and virulence of this bacterium. Here we will focus on the molecular mechanisms used by H. pylori to control gene transcription.