Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3.

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells.

Functional up-regulation of heat shock factor 1 (HSF1) activity through different posttranslational modifications has been implicated in the survival and proliferation of various cancers. It is increasingly recognized that the HSF1 gene is also up-regulated at the transcriptional level, a phenomenon correlated with poor prognosis for patients with different cancers, including breast cancer. Here, we analyzed the transcriptional up-regulation of HSF1 in human cells upon arsenite- or peroxide-induced oxidative stress. Sequential promoter truncation coupled with bioinformatics analysis revealed that this activation is mediated by two antioxidant response elements (AREs) located between 1707 and 1530 bp upstream of the transcription start site of the HSF1 gene. Using shRNA-mediated down-regulation, ChIP of NRF2, site-directed mutagenesis of the AREs, and DNase I footprinting of the HSF1 promoter, we confirmed that nuclear factor erythroid-derived 2-like 2 (NRF2, also known as NFE2L2) interacts with these AREs and up-regulates HSF1 expression. We also found that BRM/SWI2-related gene 1 (BRG1), a catalytic subunit of SWI2/SNF2-like chromatin remodeler, is involved in this process. We further show that NRF2-dependent HSF1 gene regulation plays a crucial role in cancer cell biology, as interference with NRF2-mediated HSF1 activation compromised survival, migration potential, and the epithelial-to-mesenchymal transition and autophagy in MCF7 breast cancer cells exposed to oxidative stress. Taken together, our findings unravel the mechanistic basis of HSF1 gene regulation in cancer cells and provide molecular evidence supporting a direct interaction between HSF1 and NRF2, critical regulators of two cytoprotective mechanisms exploited by cancer cells.

The cofactors and domains of a staphylococcal capsule-producing enzyme preserve its structure, stability, shape and dimerization ability.

CapF, a staphylococcal capsule-producing enzyme, binds Zn2+ ion and NADPH using its C-terminal domain (CTD) and N-terminal domain (NTD), respectively. To elucidate the roles of cofactors and domains, we have systematically investigated the related recombinant proteins, rCapF, rCTD, recombinant NTD (rNTD) and the Zn2+-free rCapF/rCTD, Apo-rCapF/Apo-rCTD. The results show that the secondary structure, tertiary structure, shape and surface hydrophobicity of Apo-rCapF and Apo-rCTD are different from those of rCapF and rCTD. The removal of Zn2+ made rCapF thermo-sensitive, whereas both rCTD and Apo-rCTD are thermo-resistant proteins. Further, Apo-rCapF and rCapF existed as the dimers, whereas rCTD and Apo-rCTD formed a mixture of dimers and tetramers in the aqueous solution. Zn2+ maintained the structure of NTD as well. The NADPH binding activity and Cys accessibility of rNTD, rCapF and Apo-rCapF were significantly different from each other. The binding of NADPH to the above three proteins freely occurred, liberated heat at 25°C and increased their diameters. In addition, the structure, stability, shape and oligomerization ability of rNTD, rCTD and rCapF little resembled each other. Collectively, the domains and cofactors of CapF contribute to preserving its conformation, stability, shape and dimerization ability.

Modeling and monitoring the effects of three partly conserved Ile residues in the dimerization domain of a Mip-like virulence factor from Escherichia coli.

FKBP22, an Escherichia coli-made peptidyl-prolyl cis-trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

Serine 106 preserves the tertiary structure, function, and stability of a cyclophilin from Staphylococcus aureus.

SaCyp, a staphylococcal cyclophilin involved in both protein folding and pathogenesis, has a Ser residue at position 106 and a Trp residue at position 136. While Ser 106 of SaCyp aligned with a cyclosporin A (CsA) binding Ala residue, its Trp 136 aligned with a Trp or a Phe residue of most other cyclophilins. To demonstrate the exact roles of Ser 106 and Trp 136 in SaCyp, we have elaborately studied rCyp[S106A] and rCyp[W136A], two-point mutants of a recombinant SaCyp (rCyp) harboring an Ala substitution at positions 106 and 136, respectively. Of the mutants, rCyp[W136A] showed the rCyp-like CsA binding affinity and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Conversely, the PPIase activity, CsA binding affinity, stability, tertiary structure, surface hydrophobicity, and Trp accessibility of rCyp[S106A] notably differed from those of rCyp. The computational experiments also reveal that the structure, dimension, and fluctuation of SaCyp are not identical to those of SaCyp[S106A]. Furthermore, Ser at position 106 of SaCyp, compared to Ala at the same position, formed a higher number of non-covalent bonds with CsA. Collectively, Ser 106 is an indispensable residue for SaCyp that keeps its tertiary structure, function, and stability intact.Communicated by Ramaswamy H. Sarma.

The helix located between the two domains of a mip-like peptidyl-prolyl cis-trans isomerase is crucial for its structure, stability, and protein folding ability.

FKBP22, a PPIase (peptidyl-prolyl cis-trans isomerase) produced by Escherichia coli, binds FK506 and rapamycin (both immunosuppressive drugs), shares significant homology with the Mip-like virulence factors, and has been thought to carry a long α-helix (namely α3) between its two domains. To understand whether the length of helix α3 plays any role in the structure, function, and stability of FKBP22-like proteins, we studied a recombinant E. coli FKBP22 (rFKBP22) and its four helix α3 mutant variants by various in vitro probes. Of the helix α3 mutants, two were deletion mutants (rFKBP22D5 and rFKBP22D30), whereas the two others were insertion mutants (rFKBP22I3 and rFKBP22I6). Our investigations revealed that the molecular dimensions, dimerization efficiencies, secondary structures, tertiary structures, stabilities, and protein folding abilities of all mutant proteins are different from those of rFKBP22. Conversely, the rapamycin binding affinities of the mutant proteins were affected very little. Urea-induced unfolding of each protein followed a two-state mechanism and was reversible in nature. Interestingly, rFKBP22D30 was the least stable, whereas rFKBP22I3 appeared to be the most stable of the five proteins. The data together suggest that length of helix α3 contributes significantly to the preservation of the structure, function, and stability of E. coli FKBP22.

Domain structure and denaturation of a dimeric Mip-like peptidyl-prolyl cis-trans isomerase from Escherichia coli.

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

A conserved arginine residue in a staphylococcal anti-sigma factor is required to preserve its kinase activity, structure, and stability.

RsbW, σB, and RsbV, encoded by Staphylococcus aureus and related bacteria, act as an anti-sigma factor, an sigma factor, and an anti-anti-sigma factor, respectively. The interaction between RsbW and σB blocks the transcription initiation activity of the latter protein. RsbW also functions as a serine kinase and phosphorylates RsbV in the presence of ATP. Our modeling study indicates that the RsbW-RsbV complex is stabilized by twenty-four intermolecular non-covalent bonds. Of the bond-forming RsbW residues, Arg 23, and Glu 49 are conserved residues. To understand the roles of Arg 23 in RsbW, rRsbW[R23A], a recombinant S. aureus RsbW (rRsbW) harboring Arg to Ala change at position 23, was investigated using various probes. The results reveal that rRsbW[R23A], like rRsbW, exists as the dimers in the aqueous solution. However, rRsbW[R23A], unlike rRsbW, neither interacted with a chimeric RsbV (rRsbV) nor formed the phosphorylated rRsbV in the presence of ATP. Furthermore, the tertiary structure and hydrophobic surface area of rRsbW[R23A] matched little with those of rRsbW. Conversely, both rRsbW[R23A] and rRsbW showed interaction with a recombinant σB (rσB). rRsbW and rRsbW[R23A] were also unfolded via the formation of at least one intermediate in the presence of urea. However, the thermodynamic stability of rRsbW significantly differed from that of rRsbW[R23A]. Our molecular dynamics (MD) simulation study also reveals the substantial change of structure, dimension, and stability of RsbW due to the above mutation. The ways side chain of critical Arg 23 contributes to maintaining the tertiary structure, and stability of RsbW was elaborately discussed.Communicated by Ramaswamy H. Sarma.

Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity.

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations.Communicated by Ramaswamy H. Sarma.

Staphylococcus aureus ClpC divergently regulates capsule via sae and codY in strain newman but activates capsule via codY in strain UAMS-1 and in strain Newman with repaired saeS.

ClpC is an ATPase chaperone found in most Gram-positive low-GC bacteria. It has been recently reported that ClpC affected virulence gene expression in Staphylococcus aureus. Here we report that ClpC regulates transcription of the cap operon and accumulation of capsule, a major virulence factor for S. aureus. As virulence genes are regulated by a complex regulatory network in S. aureus, we have used capsule as a model to understand this regulation. By microarray analyses of strain Newman, we found that ClpC strongly activates transcription of the sae operon, whose products are known to negatively regulate capsule synthesis in this strain. Further studies indicated that ClpC repressed capsule production by activating the sae operon in strain Newman. Interestingly, the clpC gene cloned into a multiple-copy plasmid vector exhibited an activation phenotype, suggesting that ClpC overexpression has a net positive effect. In the absence of sae function, by either deletion or correction of a native mutation within saeS, we found that ClpC had a positive effect on capsule production. Indeed, in the UAMS-1 strain, which does not have the saeS mutation, ClpC functioned as an activator of capsule production. Our microarray analyses of strain Newman also revealed that CodY, a repressor of capsule production, was repressed by ClpC. Using genetic approaches, we showed that CodY functioned downstream of ClpC, leading to capsule activation both in Newman and in UAMS-1. Thus, ClpC functions in two opposite pathways in capsule regulation in strain Newman but functions as a positive activator in strain UAMS-1.

Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus.

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σB, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed ß-sheet composed of five ß-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a Kd of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σB (rσB). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°-35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor.Communicated by Ramaswamy H. Sarma.

Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1.

Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.

Characterization of an unusual cold shock protein from Staphylococcus aureus.

Of the three cold shock proteins expressed by Staphylococcus aureus, CspC is induced poorly by cold but strongly by various antibiotics and toxic chemicals. Using a purified CspC, here we demonstrate that it exists as a monomer in solution, possesses primarily ß-sheets, and bears substantial structural similarity with other bacterial Csps. Aggregation of CspC was initiated rapidly at temperatures above 40 °C, whereas, the Gibbs free energy of stabilization of CspC at 0 M GdmCl was estimated to be +1.6 kcal mol(-1), indicating a less stable protein. Surprisingly, CspC showed stable binding with ssDNA carrying a stretch of more than three thymine bases and binding with such ssDNA had not only stabilized CspC against proteolytic degradation but also quenched the fluorescence intensity from its exposed Trp residue. Analysis of quenching data indicates that each CspC molecule binds with ∼5 contiguous thymine bases of the above ssDNA and binding is cooperative in nature.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Antibiotics, arsenate and H2O2 induce the promoter of Staphylococcus aureus cspC gene more strongly than cold.

Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (P(c)) activity even at 37 degrees C. A reporter S. aureus strain CHANDA2, constructed by inserting the P(c)-lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of beta-galactosidase after cold shock, indicating that low temperature induces P(c) very weakly. Interestingly, transcription from P(c ) was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately.

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity.

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.

Dimerization ability, denaturation mechanism, and the stability of a staphylococcal phage repressor and its two domains.

The lysogenic growth of phage ф11 in Staphylococcus aureus is controlled by a repressor (CI) that harbors an N-terminal domain (NTD), and a C-terminal domain (CTD). Previously, NTD, like CI, showed DNA binding activity and dimerized in the aqueous solution. To precisely understand the folding mechanism, function, and the stability of CI, NTD, and CTD, we have investigated their recombinant forms, rCI, rNTD, and rCTD, using various probes. The data reveal that rCTD, like rCI and rNTD, is a well-structured protein and produces dimers in the aqueous environment. However, the stability order of the dimers appears to be rCI > rCTD > rNTD. Interestingly, the stability of rNTD or rCTD looks slightly higher than that of rCI. The urea-induced equilibrium unfolding of these proteins proceeded via the production of two intermediates. The structure, surface hydrophobicity, and the dimeric status of one intermediate mostly differed from those of another intermediate or the native protein. Our MD simulation study on the representative NTD shows the substantial change in its structure and stability at the urea concentrations, which formed rNTD intermediates. Collectively, the computational data have supported the experimental data and indicated that the CI and its domains are folded by a similar multiphasic pathway.

The delayed early gene G23 of temperate mycobacteriophage L1 regulates the expression of deoxyribonuclease, the product of another delayed early gene of the phage.

To get clues about the genes as well as the gene regulatory circuit controlling the lytic development of temperate mycobacteriophage L1, previously we screened several conditional lethal mutants of L1 and characterized some of them to an extent. One of the mutants, L1 G23ts23, was found defective in both growth and late gene transcription at 42 degrees C but not at 32 degrees C. Here we show that the above phage mutant is also defective in the expression of phage-coded deoxyribonuclease (DNase) at 42 degrees C but not at 32 degrees C. The G23 gene however does not code for the above enzyme. Further analyses using the L1 G23ts23 mutant suggest that synthesis of DNase is also not regulated by G23 at transcriptional level. Expression of functional DNase in fact requires de novo protein synthesis. Among the 25 revertants isolated from the L1 G23ts23 mutant, which are capable of growing at 42 degrees C (by overcoming the ts defect in late transcription), two, R4 and R22, have been shown to retain the ts defect in the expression of the above enzyme and R4, to retain also the G23ts23 mutation. This suggests that R4 (R22 was not tested for the presence of G23ts23 mutation) carries an extragenic suppressor of G23ts23 mutation in a different gene (we call this putative gene as Gx), which now helps bypass the requirement of G23 for late gene transcription. Possible role of G23 on the regulation of L1-coded Gx and deoxyribonuclease has been discussed at length.

A staphylococcal anti-sigma factor possesses a single-domain, carries different denaturant-sensitive regions and unfolds via two intermediates.

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme.

Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus.

SarA, a pleiotropic transcription regulator, is encoded by Staphylococcus aureus, a pathogenic bacterium. The expression of many virulence and non-virulence genes in S. aureus is modulated by this regulator. Structural studies have shown it to be a winged-helix DNA-binding protein carrying two monomers. Each SarA monomer is composed of five α-helices (α1-α5), three ß-strands (ß1-ß3) and multiple loops. The putative DNA binding region of SarA is constituted with α3, α4, ß2, and ß3, whereas, its dimerization seems to occur using α1, α2, and α5. Interestingly, many SarA-like proteins are dimeric and use three or more helices for their dimerization. To clearly understand the roles of helix α1 in the dimerization, we have constructed and purified a SarA mutant (Δα1) that lacks helix α1. Our in-depth studies with Δα1 indicate that the helix α1 is critical for preserving the structure, DNA binding activity and thermodynamic stability of SarA. However, the helix has little affected its dimerization ability. Possible reasons for such anomaly have been discussed at length.