Cognate Sigma Research Articles

Mechanistic Implications of the Unique Structural Features of the Anti‐Sigma Domain of the Pseudomonas Sigma Regulator, PupR

Gram‐negative bacteria tightly regulate intracellular iron, an essential nutrient. To ensure this strict control, some outer membrane TonB‐dependent transporters (TBDTs) responsible for iron import stimulate their own transcription in response to extracellular binding of ferric siderophore. This process is mediated by an inner membrane sigma regulator protein that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we employ the Pseudomonas putida ferric‐pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X‐ray crystal structure of the cytoplasmic anti‐sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small‐angle X‐ray scattering, and sedimentation velocity analytical ultracentrifugation (SV‐AUC) all indicate that, in contrast to other ASDs, the PupR‐ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by SV‐AUC and thermal denaturation circular dichroism spectroscopy. Additionally, we have demonstrated that the helical content of the PupR‐ASD increases in the presence of trifluoroethanol, and are investigating the interaction of the PupR‐ASD with its cognate sigma factor, PupI. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcriptional activation.Support or Funding InformationFunded by NSF ND EPSCoR DDA #FAR0025216 to JJ; NIH NINDS 1R03 NS090939 and NSF MCB‐1413525 to SS; NIH NIGMS 1R15 GM113227, NIH NCRR 2P20 RR015566, and NIH NIGMS P30 GM103332 to CC.

Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR.

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this strict control, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small-angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation all indicate that, in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.

Alternative zinc‐binding sites explain the redox sensitivity of zinc‐containing anti‐sigma factors

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.

Differential proteolysis of sigma regulators controls cell‐surface signalling in Pseudomonas aeruginosa

Cell-surface signalling systems are widespread in Gram-negative bacteria. In these systems gene expression occurs following binding of a ligand, commonly a siderophore, to a receptor protein in the outer membrane. The receptor interacts with a sigma regulator protein that extends from the periplasm into the cytoplasm to control the activity of a cognate sigma factor. The mechanisms of signal transduction in cell-surface signalling systems have not been determined. Here we investigate signal transduction in the pyoverdine, ferrichrome and desferrioxamine siderophore systems of Pseudomonas aeruginosa. When pyoverdine is present the sigma regulator FpvR undergoes complete proteolysis resulting in activation of two sigma factors PvdS and FpvI and expression of genes for pyoverdine synthesis and uptake. When pyoverdine is absent subfragments of FpvR inhibit PvdS and FpvI. Similarly, subfragments of the sigma regulators FoxR and FiuR are formed in the absence of desferrioxamine and ferrichrome. These are much less abundant when the siderophores are present and downstream gene expression takes place. In all three systems RseP (MucP/YaeL) is required for complete proteolysis of the sigma regulator and sigma factor activity. These findings indicate that regulated proteolysis is a general mechanism for signal transduction in cell-surface signalling.

Structural Characterization of the Multidomain Regulatory Protein Rv1364c from Mycobacterium tuberculosis

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain anda C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6Å) and a phosphatase/kinase dual domain construct (at 2.6Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding,affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).

Different roles for anti‐sigma factors in siderophore signalling pathways of Pseudomonas aeruginosa

Group IV (extracytoplasmic function) sigma factors direct the expression of a large number of regulons in bacteria. The activities of many Group IV sigma factors are inhibited by members of a family of anti-sigma factor proteins, with appropriate environmental signals causing the sigma factor to be released for interaction with core RNA polymerase and consequent transcription of target genes. One subgroup of Group IV sigmas directs expression of genes for uptake of siderophores (iron-chelating compounds) by Gram-negative bacteria. The activities of these sigma factors are controlled by anti-sigma factors that span the cytoplasmic membrane. Binding of siderophore by a receptor protein in the outer membrane results in signal transduction from the periplasmic portion to the cytoplasmic portion of the appropriate anti-sigma factor, with consequent activity of the cognate sigma factor and upregulation of the gene encoding the receptor protein. We have investigated receptor/anti-sigma/sigma factor signalling pathways for uptake of the siderophores ferrichrome and desferrioxamine by Pseudomonas aeruginosa. In these pathways the 'anti-sigma' proteins are normally required for sigma factor activity and the cytoplasmic parts of the 'anti-sigmas' have 'pro-sigma' activity. We suggest that the family of anti-sigma factor proteins may be better considered as 'sigma regulators'.

Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and the anti-anti sigma factor RsfA

M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73–Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX–RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73–Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.

Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c

The alternative sigma factors are regulated by a phosphorylation-mediated signal transduction cascade involving anti-sigma factors and anti-anti-sigma factors. The proteins regulating Mycobacterium tuberculosis sigma factor F (SigF), anti-SigF and anti-anti-SigF have been identified, but the factors catalyzing phosphorylation-dephosphorylation have not been well established. We identified a distinct pathogenic species-specific multidomain protein, Rv1364c, in which the components of the entire signal transduction cascade for SigF regulation appear to be encoded in a single polypeptide. Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of various domains, namely a phosphatase (RsbU) domain, an anti-SigF (RsbW) domain, and an anti-anti-SigF (RsbV) domain. We report that the RsbU domain of Rv1364c bears all the conserved features of the PP2C-type serine/threonine phosphatase family, whereas its RsbW domain has certain substitutions and deletions in regions important for ATP binding. Another anti-SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even more unfavorable substitutions in the kinase domain. Biochemical assay with the purified RsbW domain of Rv1364c and UsfX showed the loss of ability of autophosphorylation and phosphotransfer to cognate anti-anti-SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and UsfX protein display very weak binding with fluorescent ATP analogs, despite showing functional interactions characteristic of anti-SigF proteins. In view of conservation of specific interactions with cognate sigma and anti-anti-sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation-dependent interaction involved in SigF regulation in Mycobacterium.

Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary sigma factor

Bacterial anti-sigma factors typically regulate sigma factor function by restricting the access of their cognate sigma factors to the RNA polymerase (RNAP) core enzyme. The Escherichia coli Rsd protein forms a complex with the primary sigma factor, sigma(70), inhibits sigma(70)-dependent transcription in vitro, and has been proposed to function as a sigma(70)-specific anti-sigma factor, thereby facilitating the utilization of alternative sigma factors. In prior work, Rsd has been shown to interact with conserved region 4 of sigma(70), but it is not known whether this interaction suffices to account for the regulatory functions of Rsd. Here we show that Rsd and the Rsd orthologue AlgQ, a global regulator of gene expression in Pseudomonas aeruginosa, interact with conserved region 2 of sigma(70). We show further that Rsd and AlgQ can interact simultaneously with regions 2 and 4 of sigma(70). Our findings establish that the abilities of Rsd and AlgQ to interact with sigma(70) region 2 are important determinants of their in vitro and in vivo activities.

A Possible Extended Family of Regulators of Sigma Factor Activity inStreptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific sigma(F), suggesting that it is an antagonistic regulator of sigma(F). Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-sigma(F) during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.

Mycobacterium tuberculosis UsfX (Rv3287c) exhibits novel nucleotide binding and hydrolysis properties

The Mycobacterium tuberculosis UsfX protein is an anti-sigma factor which regulates its cognate sigma factor SigF. UsfX shares low sequence homology with other anti-sigma factors making it difficult to identify the nucleotide binding site and characterize its properties. We have identified that the NTP binding site occurs close to Trp106 and the area around the nucleotide binding site is predominantly negatively charged. UsfX binds to a variety of nucleotides unlike other reported anti-sigma factors and exhibits an unusual dual NTPase activity. In silico computational experiments have identified a XG SF S motif close to the nucleotide binding site for metal ion binding. This motif is analogous to the DX SX S motif reported earlier in the human integrin CR3 protein superfamily. Overall, the experiments suggest that the M. tuberculosis UsfX represents a distinct anti-sigma factor family with a novel nucleotide binding motif.

A Conserved Structural Module Regulates Transcriptional Responses to Diverse Stress Signals in Bacteria

A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV anti-sigmas. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate sigma factor.

Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors.

Extracytoplasmic function (ECF) sigma factors constitute a diverse family of proteins, within the class of the sigma 70 subunit of RNA polymerase. Most members of the family studied to date are known to regulate gene expression in response to stress conditions. The Bacillus subtilis genome encodes at least 17 distinct sigma factors, seven of which are members of the ECF subfamily. Among these, five sigma factors, namely SigV, SigW, SigX, SigY and SigM, are encoded by the first genes of the cognate sigma operons. Disruption or repressed expression of the downstream gene(s) resulted in transcriptional activation of the cognate sigma operon. Moreover, in vivo protein-protein interaction analyses by yeast two-hybrid experiments indicated that these immediate downstream gene products bind the cognate ECF sigma factor, suggesting that they function as anti-sigma factors by capturing sigma factor on the inner surface of the cytoplasmic membrane. Interaction with other sigma factors was not observed. The results presented here also show that these anti-sigma factors interact with ECF sigma factors through their N-terminal region, implying that the N-terminal domain resides inside the cytoplasmic membrane.

The extracytoplasmic function (ECF) sigma factors

Bacterial sigma (sigma) factors are an essential component of RNA polymerase and determine promoter selectivity. The substitution of one sigma factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent. As a class, alternative sigma factors play key roles in coordinating gene transcription during various stress responses and during morphological development. The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that are quite divergent in sequence relative to most other sigma factors. Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors and these regulators often outnumber all other types of sigma factor combined. Examples include Bacillus subtilis (7 ECF sigma factors), Mycobacterium tuberculosis (10), Caulobacter crescentus (13), Pseudomonas aeruginosa (approximately 19), and Streptomyces coelicolor (approximately 50). The roles and mechanisms of regulation for these various ECF sigma factors are largely unknown, but significant progress has been made in selected systems. As a general trend, most ECF sigma factors are cotranscribed with one or more negative regulators. Often, these include a transmembrane protein functioning as an anti-sigma factor that binds, and inhibits, the cognate sigma factor. Upon receiving a stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to stimulate transcription. In many ways, these anti-sigma:sigma pairs are analogous to the more familiar two-component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA-binding response regulator. Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane. Here, I review current knowledge of some of the better characterized ECF sigma factors, discuss the variety of experimental approaches that have proven productive in defining the roles of ECF sigma factors, and present some unifying themes that are beginning to emerge as more systems are studied.

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris.

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

An Escherichia coli chemoreceptor gene is temporally controlled in Caulobacter.

Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. The tsr gene was placed on the chromosome in single copy or on a low-copy-number plasmid. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Nuclease S1 mapping experiments showed that the tsr transcript was also controlled by the cell cycle, suggesting that the E. coli tsr gene is regulated by C. crescentus factors that mediate the timing of transcription initiation. The apparent transcription start site of the E. coli tsr gene was determined in both E. coli and C. crescentus, and we found that in both backgrounds the promoter used conforms to the consensus sequence for the promoters of the flagellar and chemosensory genes of Bacillus subtilis and E. coli. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter.