Anti-sigma Domain Research Articles

Initial Characterization of the Extra Cytoplasmic Function Sigma Factor (FpvI) and Sigma Regulator (FpvR) Anti Sigma Domain Interaction in Pseudomonas aeruginosa

ObjectiveThe objective of this study is to structurally characterize the interaction between the Pseudomonas aeruginosa PA01extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, and the sigma regulator, FpvR.HypothesisThe extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, interacts with high affinity with the N‐terminal anti‐sigma domain of FpvR. It can be purified to homogeneity and used for structural studies by X‐ray crystallography.SummaryTranscription of the pyoverdine transport system of Pseudomonas aeruginosa PA01 is mediated by Cell Surface Signaling (CSS). This process involves binding of the siderophore ferric‐pyoverdine to the outer membrane transporter FpvA. This event triggers the regulated intramembrane proteolysis of the sigma regulator, FpvR. Release of the FpvR anti‐sigma domain (ASD) in complex with the extra‐cytoplasmic‐function (ECF) sigma factor FpvI, directs RNA polymerase to the promoter of FpvA to initiate the transcription. The molecular details of the interaction between FpvR ASD and FpvI are not known. We are using a dual expression plasmid to produce the FpvR ASD and a his‐tagged construct of FpvI. Immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography is used to purify the complex. We have characterized the complex by Circular dichroism (CD) to show it is well folded. This protein is being further characterized by sparse matrix crystal screening and Small‐angle X‐ray scattering (SAXS).

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

Here, we show that a partner-switching system of the aquatic Proteobacterium Shewanella oneidensis regulates post-translationally σS (also called RpoS), the general stress response sigma factor. Genes SO2118 and SO2119 encode CrsA and CrsR, respectively. CrsR is a three-domain protein comprising a receiver, a phosphatase, and a kinase/anti-sigma domains, and CrsA is an anti-sigma antagonist. In vitro, CrsR sequesters σS and possesses kinase and phosphatase activities toward CrsA. In turn, dephosphorylated CrsA binds the anti-sigma domain of CrsR to allow the release of σS. This study reveals a novel pathway that post-translationally regulates the general stress response sigma factor differently than what was described for other proteobacteria like Escherichia coli. We argue that this pathway allows probably a rapid bacterial adaptation.

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.

The Prc and RseP proteases control bacterial cell‐surface signalling activity

Extracytoplasmic function (ECF) sigma factors play a key role in the regulation of vital functions in the bacterial response to the environment. In Gram-negative bacteria, activity of these sigma factors is often controlled by cell-surface signalling (CSS), a regulatory system that also involves an outer membrane receptor and a transmembrane anti-sigma factor. To get more insight into the molecular mechanism behind CSS regulation, we have focused on the unique Iut system of Pseudomonas putida. This system contains a hybrid protein containing both a cytoplasmic ECF sigma domain and a periplasmic anti-sigma domain, apparently leading to a permanent interaction between the sigma and anti-sigma factor. We show that the Iut ECF sigma factor regulates the response to aerobactin under iron deficiency conditions and is activated by a proteolytic pathway that involves the sequential action of two proteases: Prc, which removes the periplasmic anti-sigma domain, and RseP, which subsequently removes the transmembrane domain and thereby generates the ECF active transcriptional form. We furthermore demonstrate the role of these proteases in the regulation of classical CSS systems in which the sigma and anti-sigma factors are two different proteins.

Identification of a novel anti-σE factor in Neisseria meningitidis

BackgroundFine tuning expression of genes is a prerequisite for the strictly human pathogen Neisseria meningitidis to survive hostile growth conditions and establish disease. Many bacterial species respond to stress by using alternative σ factors which, in complex with RNA polymerase holoenzyme, recognize specific promoter determinants. σE, encoded by rpoE (NMB2144) in meningococci, is known to be essential in mounting responses to environmental challenges in many pathogens. Here we identified genes belonging to the σE regulon of meningococci.ResultsWe show that meningococcal σE is part of the polycistronic operon NMB2140-NMB2145 and autoregulated. In addition we demonstrate that σE controls expression of methionine sulfoxide reductase (MsrA/MsrB). Moreover, we provide evidence that the activity of σE is under control of NMB2145, directly downstream of rpoE. The protein encoded by NMB2145 is structurally related to anti-sigma domain (ASD) proteins and characterized by a zinc containing anti-σ factor (ZAS) motif, a hall mark of a specific class of Zn2+-binding ASD proteins acting as anti-σ factors. We demonstrate that Cys residues in ZAS, as well as the Cys residue on position 4, are essential for anti-σE activity of NMB2145, as found for a minority of members of the ZAS family that are predicted to act in the cytoplasm and responding to oxidative stimuli. However, exposure of cells to oxidative stimuli did not result in altered expression of σE.ConclusionsTogether, our results demonstrate that meningococci express a functional transcriptionally autoregulated σE factor, the activity of which is controlled by a novel meningococcal anti-σ factor belonging to the ZAS family.

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.