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YlaD, a membrane-anchored anti-sigma (σ) factor of Bacillus subtilis , contains a HX 3 CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc (Zn)-co-ordinated anti-σ factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC-YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the P yla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained Zn and iron, while oxidized YlaD did not. Cysteine substitution in YlaD led to changes in its secondary structure; Cys3 had important structural functions in YlaD, and its mutation caused dissociation from YlaC, indicating the essential requirement of a HX 3 CXXC motif for regulating interactions of YlaC with YlaD. Analyses of the far-UV CD spectrum and metal content revealed that the addition of Mn ions to Zn-YlaD changed its secondary structure and that iron was substituted for manganese (Mn). The ylaC gene expression using βGlu activity from P yla : gusA was observed at the late-exponential and early-stationary phase, and the ylaC -overexpressing mutant constitutively expressed gene transcripts of clpP and sigH , an important alternative σ factor regulated by ClpXP. Collectively, our data demonstrated that YlaD senses redox changes and elicits increase in Mn ion concentrations and that, in turn, YlaD-mediated transcriptional activity of YlaC regulates sporulation initiation under oxidative stress and Mn-substituted conditions by regulating clpP gene transcripts. This is the first report of the involvement of oxidative stress-responsive B. subtilis extracytoplasmic function σ factors during sporulation via a Mn-dependent redox-sensing molecular switch., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch., Competing Interests: The authors declare no conflict of interest.

BldD regulates transcription of key developmental genes in Streptomyces coelicolor. While the N-terminal domain is responsible for both dimerization and DNA binding, the structural and functional roles of the C-terminal domain (CTD) remain largely unexplored. Here, the solution structure of the BldD-CTD shows a novel winged-helix domain fold not compatible with DNA binding, due to the negatively charged surface and presence of an additional helix. Meanwhile, a small elongated groove with conserved hydrophobic patches surrounded by charged residues suggests that the BldD-CTD could be involved in protein-protein interactions that provide transcriptional regulation., (© 2013 Wiley Periodicals, Inc.)

Lactosucrose (4(G)-beta-D-galactosylsucrose) is an oligosaccharide consisting of galactose, glucose, and fructose. In this study, we prepared lactosucrose from lactose and sucrose using a levansucrase derived from Zymomonas mobilis. Optimum conditions for lactosucrose formation were 23 degrees C, pH 7.0, 18.0% (w/v) lactose monohydrate, and 18% (w/v) sucrose as substrates, and 1 unit of enzyme/ml of reaction mixture. Under these conditions, the lactosucrose conversion efficiency was 28.5%. The product was purified and confirmed to be O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-glucopyranosyl- (1-->2)-beta-D-fructofuranoside, or lactosucrose. A mixed-enzyme system containing a levansucrase and a glucose oxidase was applied in order to increase the efficiency of lactose and sucrose conversion to lactosucrose, which rose to 43.2% as s result.

A homodimeric protein, BldD is a key regulator for developmental process of Streptomyces coelicolor and the bldD mutant exhibits severely pleiotropic defects in the antibiotic production and morphological differentiation of the bacterium. In the present work, we approached domain organization of BldD, to structurally and functionally characterize the protein as a DNA-binding protein. We first observed a proteolytic cleavage of BldD by the cytoplasmic extracts of S. coelicolor, which was highly dependent on the developmental stage of the bacterium. The resulting fragment of BldD was identified by mass spectrometry as the N-terminal domain resistant to the proteolysis. Recombinant proteins corresponding to the intact BldD, the N-terminal domain (residues 1-79) and the rest part (C-terminal domain; residues 80-167) were used for comparative analyses by several spectroscopic, thermodynamic, and biochemical experiments, respectively. The results of circular dichroism and nuclear magnetic resonance spectroscopies certified each of the two determined domains could be regarded as an individual folding unit possessing an independent thermodynamic cooperativity. Structural interaction between the two domains was little observed in the DNA-free and DNA-bound states. Strikingly, it was revealed by gel permeation chromatography, chemical crosslink, gel mobility shift, and NMR-monitored DNA-binding experiments, that only the N-terminal domain is responsible for the dimerization as well as DNA-binding of BldD. Detailed inspection of the present results suggests that BldD function in a unique and complicated mode to totally regulate the diverse developmental stages of S. coelicolor., (2007 Wiley-Liss, Inc.)

Background: Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1., Results: The 1.6 A crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im) but some disparity to an immunity protein against carnobacteriocin B2 (ImB2) in both the C-terminal conformation and the local structure constructed by alpha3, alpha4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity., Conclusion: The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.

BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.

In this study, we have attempted to characterize the functions of YlaC and YlaD encoded by ylaC and ylaD genes in Bacillus subtilis. The GUS reporter gene, driven by the yla operon promoter, was expressed primarily during the late exponential and early stationary phase, and its expression increased as the result of hydrogen peroxide treatment. Northern and Western blot analyses revealed that the level of ylaC transcripts and YlaC increased as the result of challenge with hydrogen peroxide. A YlaC-overexpressing strain evidenced hydrogen peroxide resistance and a three-fold higher peroxidase activity as compared with a deletion mutant. YlaC-overexpressing and YlaD-disrupted strains evidenced higher sporulation rates than were observed in the YlaC-disrupted and YlaD-overexpressing strains. Analyses of the results of native polyacrylamide gel electrophoresis of recombinant YlaC and YlaD indicated that interaction between YlaC and YlaD was regulated by the redox state of YlaD in vitro. Collectively, the results of this study appear to suggest that YlaC regulated by the YlaD redox state, contribute to oxidative stress resistance in B. subtilis.

PedB, a bacterial immunity protein conferring immunity to a newly identified pediocin (pediocin PP-1), was crystallized by the hanging-drop vapor diffusion method at 296 K. A 1.35 A data set has been collected from a single crystal at 100 K using synchrotron-radiation source. The PedB crystals belong to the hexagonal space group P6(2) or P6(4), with unit cell parameters a = b = 62.2, c = 39.9 A. Analysis of the packing density shows that the asymmetric unit probably contains one molecule with a solvent content of 33.8%.

The effect of cytochrome c550 encoded by cccA in Bacillus subtilis during the event of sporulation was investigated. The sporulation of cccA-overexpressing mutant was significantly accelerated, while disruptant strain showed delayed sporulation in spite of the same growth rate. Activity of sporulation stage-0-specific enzyme, extracellular alpha-amylase of mutant strains was similar to that of the control strain, but cccA-overexpressing mutant exhibited higher activity of stage-II-specific alkaline phosphatase and stage-III-specific glucose dehydrogenase when compared to deletion mutant and control strain. Northern blot analysis also revealed that cccA-overexpressing mutant showed high level of spo0A transcripts, while the disruptant rarely expressed spo0A. These results suggested that although cytochrome c550 is dispensable for growth and sporulation, expression of cccA may play an important role for initiation of sporulation through regulation of spo0A expression.

The N-terminal DNA-binding domain of BldD from Streptomyces coelicolor A3(2) was crystallized by the hanging-drop vapour-diffusion method at 296 K. A 1.8 angstroms data set has been collected using synchrotron radiation at Pohang Light Source, South Korea. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 77.2, b = 31.8, c = 33.6 angstroms, beta = 105.1 degrees. Analysis of the packing density shows that the asymmetric unit probably contains one molecule, with a solvent content of 43.6%., (Copyright 2004 International Union of Crystallography)

PpsR from Rhodobacter sphaeroides is involved in the repression of photosystem gene expression. The PpsR protein was heterologously overexpressed and purified to homogeneity. Gel mobility shift assay showed that the purified PpsR has DNA-binding activity. SDS-PAGE analysis showed that some portions of PpsR were oxidized, indicating that intramolecular or intermolecular disulphide bonds were formed between the two cysteines in each subunit. When the disulphide bond of PpsR was reduced by DTT, the binding activity of PpsR to the puc promoter region distinctly increased. The changes in protein level and DNA-binding activity of PpsR were observed in a conjugant with an extra copy of the ppsR gene and in a PpsR-null mutant (PPS1), respectively. Both cysteines in PpsR existed in their reduced form under aerobic, anaerobic-dark and anaerobic-light growth conditions, as determined using thiol-specific chemical modification. In an AppA-null mutant (APP11), the binding activity and the amount of PpsR decreased compared to those of the wild-type and an appA-complemented strain, and decreased even more under anaerobic-dark conditions than under aerobic conditions. PpsR had a redox-sensitive property but retained its reduced state in the cell, and its amount was reduced by disruption of AppA.

To find the accessory proteins participating in expression and maturation of nickel-containing superoxide dismutase (NiSOD), a metal-binding protein (CbiXhp) homologous to cobaltochelatase (CbiX) of Bacillus megaterium was isolated by nickel-nitrilotriacetic acid resin from Streptomyces seoulensis. The deduced amino acid sequence of cbiXhp showed 87% and 39% identity to CbiX of Streptomyces coelicolor and that of B. megaterium, respectively. Overexpression of CbiXhp increased the activity and the expression of NiSOD in the presence and absence of nickel, but to a lesser extent in its absence. This result indicates that CbiXhp is involved in the expression of NiSOD.

The bldB gene from Streptomyces lividans was cloned, and its product was overexpressed in Escherichia coli using a T7 expression system. Gel mobility shift assays showed that the BldB protein was functionally expressed in the E. coli system and may negatively regulate its own expression. The comparative analyses by mass spectrometry, Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical ultracentrifuge established that BldB is a dimeric protein with 24 kDa molecular mass, of which monomers do not covalently interact with each other. Gel filtration result implied that the protein shape would not be globular. More detailed structural investigations by CD and NMR spectroscopy confirmed that the majority of the BldB structure is not only disordered but also highly flexible. The highly reversible, but hardly cooperative, property of the thermal denaturation also supported the idea that the protein structure is not compact. However, the existence of a structural nucleus, of which the ordered conformation remains stabilized even at more than 80 degrees C, was evidenced. The overall structure and the thermal stability of BldB were sensitive to pH, suggesting a proton-induced conformation change. Altogether, the results provide the first detailed characterization on the biophysical and structural property of the putative DNA-binding protein, BldB., (Copyright 2003 Wiley Periodicals, Inc. Biopolymers 69: 343-350, 2003)

The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR-reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE-rseABC operon. In-frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re-reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.

The DDB_G0291732 gene product from Dictyostelium discoideum, which is a NmrA-like protein that belongs to the short-chain dehydrogenase/reductase superfamily but shows deviations in conserved sequence regions, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.65 Å resolution data set was collected using synchrotron radiation. The crystals of DDB_G0291732 protein belonged to space group P21, with unit-cell parameters a = 38.5, b = 63.7, c = 56.0 Å, β = 91.7°. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 38.1%. [ABSTRACT FROM AUTHOR]