Regulation, sensory domains and roles of two Desulfovibrio desulfuricans ATCC27774 Crp family transcription factors, HcpR1 and HcpR2, in response to nitrosative stress.

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2 . We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator.

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

Mechanism of [4Fe-4S](Cys)4 cluster nitrosylation is conserved among NO-responsive regulators.

The Fumarate nitrate reduction (FNR) regulator from Escherichia coli controls expression of >300 genes in response to O2 through reaction with its [4Fe-4S] cluster cofactor. FNR is the master switch for the transition between anaerobic and aerobic respiration. In response to physiological concentrations of nitric oxide (NO), FNR also regulates genes, including the nitrate reductase (nar) operon, a major source of endogenous cellular NO, and hmp, which encodes an NO-detoxifying enzyme. Here we show that the [4Fe-4S] cluster of FNR reacts rapidly in a multiphasic reaction with eight NO molecules. Oxidation of cluster sulfide ions (S(2-)) to sulfane (S(0)) occurs, some of which remains associated with the protein as Cys persulfide. The nitrosylation products are similar to a pair of dinuclear dinitrosyl iron complexes, [Fe(I)2(NO)4(Cys)2](0), known as Roussin's red ester. A similar reactivity with NO was reported for the Wbl family of [4Fe-4S]-containing proteins found only in actinomycetes, such as Streptomyces and Mycobacteria. These results show that NO reacts via a common mechanism with [4Fe-4S] clusters in phylogenetically unrelated regulatory proteins that, although coordinated by four Cys residues, have different cluster environments. The reactivity of E. coli FNR toward NO, in addition to its sensitivity toward O2, is part of a hierarchal network that monitors, and responds to, NO, both endogenously generated and exogenously derived.

Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis.

The ESX-1 secretion system of Mycobacterium tuberculosis has to be precisely regulated since the secreted proteins, although required for a successful virulent infection, are highly antigenic and their continued secretion would alert the immune system to the infection. The transcription of a five-gene operon containing espACD-Rv3613c-Rv3612c, which is required for ESX-1 secretion and is essential for virulence, was shown to be positively regulated by the EspR transcription factor. Thus, transcription from the start site, found to be located 67 bp upstream of espA, was dependent upon EspR enhancer-like sequences far upstream (between 884 and 1,004 bp), which we term the espA activating region (EAR). The EAR contains one of the known binding sites for EspR, providing the first in vivo evidence that transcriptional activation at the espA promoter occurs by EspR binding to the EAR and looping out DNA between this site and the promoter. Regulation of transcription of this operon thus takes place over long regions of the chromosome. This regulation may differ in some members of the M. tuberculosis complex, including Mycobacterium bovis, since deletions of the intergenic region have removed the upstream sequence containing the EAR, resulting in lowered espA expression. Consequent differences in expression of ESX-1 in these bacteria may contribute to their various pathologies and host ranges. The virulence-critical nature of this operon means that transcription factors controlling its expression are possible drug targets.

Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins.

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide-sensitive iron-sulfur cluster.

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections.

DNA recognition by the Salmonella enterica serovar Typhimurium transcription factor SlyA.

The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA.

Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches.

Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested. Here the properties of the E. coli, Mycobacterium tuberculosis and Pseudomonas putida CRPs are considered in the context of the ecological niches occupied by these bacteria. It appears that the cyclic-AMP-CRP regulatory system has been adapted to respond to distinct external and internal inputs across a broad sensitivity range that is, at least in part, determined by bacterial lifestyles.

Mycobacterium tuberculosis WhiB1 represses transcription of the essential chaperonin GroEL2.

A central feature of TB pathogenesis is the formation of Mycobacterium tuberculosis latent infections that can persist for decades. Nitric oxide produced by infected lung macrophages promotes expression of genes associated with dormancy, and impaired nitric oxide production can lead to reactivation of latent disease. Recently, WhiB1 was identified as a nitric oxide-responsive transcription factor. Here it is shown that apo-WhiB1 binds to groEL2 (Rv0440) promoter DNA. Apo-WhiB1 inhibited transcription from the groEL2 promoter in vitro and the transcript start was located ∼181 bases upstream of the groEL2 start codon. Electrophoretic mobility shift assays with sub-fragments of the groEL2 promoter indicated that the complete Rv0439c-Rv0440 intergenic region was required for WhiB1 binding, suggesting that this region possessed more than one WhiB1-binding site. DNase I footprinting identified a WhiB1-binding region that overlapped the -35 element of the groEL2 promoter. The CRP-family transcription factor Cmr (Rv1675c) was shown to bind the groEL2 promoter and activate transcription in vitro in the presence or absence of cAMP. Therefore, it is suggested that WhiB1 acts to oppose Cmr-mediated cAMP-independent activation of groEL2 expression in the presence of nitric oxide by promoter occlusion.

Structure-function relationships of the Mycobacterium tuberculosis transcription factor WhiB1.

BACKGROUND: Members of the WhiB-like (Wbl) protein family possess iron-sulfur clusters and are implicated in the regulation of developmental processes in Actinomycetes. Mycobacterium tuberculosis possesses seven Wbl proteins. The [4Fe-4S] cluster of M. tuberculosis WhiB1 is relatively insensitive to O(2) but very sensitive to nitric oxide (NO). Nitric oxide nitrosylates the WhiB1 iron-sulfur cluster and promotes DNA-binding; the apo-forms of WhiB1 also bind DNA. However, the molecular requirements for iron-sulfur cluster acquisition and for DNA-binding by WhiB1 are poorly characterized. METHODS AND FINDINGS: WhiB1 variants were created by site-directed mutagenesis and the abilities of the corresponding proteins to acquire an iron-sulfur cluster and/or bind to whiB1 promoter DNA were assessed. All four Cys residues (Cys9, 37, 40, and 46) in the N-terminal region of WhiB1 were required for incorporation of a [4Fe-4S] cluster, whereas a possible alternative cluster ligand Asp13 (by analogy with M. smegmatis WhiB2) was not. The C-terminal region of WhiB1 is predicted to house the DNA-binding domain of the protein consisting of a predicted ß-turn ((58)GVWGG(62)) followed by two amino acid motifs ((72)KRRN(75) and (78)TKAR(81)) that are conserved in WhiB1 proteins. Gly residues (Gly58, 61 and 62) in the ß-turn and positively-charged residues (Lys72, Arg73, Arg74, Lys79 and Arg81) in the downstream conserved regions were required for binding of WhiB1 DNA. CONCLUSIONS: Site-directed mutagenesis of M. tuberculosis whiB1 and characterization of the corresponding proteins has been used to explore structure-function relationships of the NO-responsive transcription factor WhiB1. This showed that all four conserved Cys residues in the N-terminal region are required for incorporation of iron-sulfur clusters but not for DNA-binding. Analysis of variants with amino acid substitutions in the C-terminal region revealed the crucial roles played by a predicted ß-turn and two conserved positively-charged motifs in facilitating DNA-binding, but not iron-sulfur cluster acquisition, by WhiB1.

Systems analysis of transcription factor activities in environments with stable and dynamic oxygen concentrations.

Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

Identification of conserved antigens from staphylococcal and streptococcal pathogens.

The design of vaccines containing epitopes shared between different human pathogens may lead to cross-species protection. In order to identify potentially conserved bacterial antigens, bacteriophage expression libraries of genomic DNA from Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes were probed with human sera from Staphylococcus aureus-infected and healthy individuals. By comparison with previous screening data from Staphylococcus epidermidis and Staph. aureus, putative antigenic, conserved domains across the genera were identified. In particular, three potentially antigenic conserved regions were identified based on the N-terminal domain of SACOL0609 (SdrD), the C-terminal domain of SACOL0723 (ScaB) and the C-terminus of SACOL1140 (IsdA) from Staph. aureus. The three domains were overexpressed, recombinant proteins were purified and polyclonal antisera raised against them recognized cell surface-located proteins from both staphylococcal and streptococcal species. The antisera were also able to opsonize both Staph. aureus and Strep. agalactiae thereby increasing their phagocytic uptake by human neutrophils. The conserved antigenic domains therefore represent potential cross-protective vaccine candidates.

Characterization of IsaA and SceD, two putative lytic transglycosylases of Staphylococcus aureus.

Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

Identification of in vivo-expressed antigens of Staphylococcus aureus and their use in vaccinations for protection against nasal carriage.

A spectrum of in vivo-expressed Staphylococcus aureus antigens was identified by probing bacteriophage expression libraries of S. aureus with serum samples from infected and uninfected individuals. Eleven recombinant antigenic proteins were produced, and specific antibody titers in a large collection of human serum samples were determined. Significantly increased concentrations of reactive immunoglobulin G (IgG) to 7 antigens were found in serum samples from ill individuals, compared with those in healthy individuals. Significantly higher concentrations of reactive IgG to 4 antigens, including iron-responsive surface determinant (Isd) A and IsdH, were found in serum samples from healthy individuals who were not nasal carriers of S. aureus, compared with those in healthy carriers. Vaccination of cotton rats with IsdA or IsdH protected against nasal carriage. Also, IsdA is involved in adherence of S. aureus to human desquamated nasal epithelial cells and is required for nasal colonization in the cotton rat model. Thus, vaccination with these antigens may prevent S. aureus carriage and reduce the prevalence of human disease.

PhoP-responsive expression of the Salmonella enterica serovar typhimurium slyA gene.

The SlyA protein of Salmonella enterica serovar Typhimurium is a member of the MarR family of transcription regulators and is required for virulence and survival in professional macrophages. Isolated SlyA protein was able to bind a specific DNA target without posttranslational modification. This suggested that SlyA might not be activated by directly sensing an external signal but rather that the intracellular concentration of SlyA is enhanced in appropriate environments through the action of other transcription factors. Analysis of slyA transcription reveals the presence of a promoter region located upstream of the previously recognized SlyA repressed promoter. The newly identified upstream promoter region did not respond to SlyA but was activated by Mg(II) starvation in a PhoP-dependent manner. We present here evidence for a direct link between two transcription factors (PhoP and SlyA) crucial for Salmonella virulence.

Interaction of the Salmonella typhimurium transcription and virulence factor SlyA with target DNA and identification of members of the SlyA regulon.

The SlyA protein from Salmonella typhimurium is a transcription factor that contributes to virulence. It is shown that a slyA mutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyA mutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyA mutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyA gene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyA promoter prevented open-complex formation, consistent with the pattern of slyA::lacZ expression parental and slyA mutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonella unfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZ fusion was reduced in a slyA mutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimurium genome annotation is complete.

Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA.

The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the -35 element and one downstream of the -10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3' end of the extended upstream footprint, and H-NS II, located at the 5' end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.

DNA recognition by the Salmonella enterica serovar Typhimurium transcription factor SlyA

The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA (AU)

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