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The ResD-ResE signal transduction system is essential for aerobic and anaerobic respiration in Bacillus subtilis. ResDE-dependent gene expression is induced by oxygen limitation, but full induction under anaerobic conditions requires nitrite or nitric oxide (NO). Here we report that NsrR (formerly YhdE) is responsible for the NO-dependent up-regulation of the ResDE regulon. The null mutation of nsrR led to aerobic derepression of hmp (flavohemoglobin gene) partly in a ResDE-independent manner. In addition to its negative role in aerobic hmp expression, NsrR plays an important role under anaerobic conditions for regulation of ResDE-controlled genes, including hmp. ResDE-dependent gene expression was increased by the nsrR mutation in the absence of NO, but the expression was decreased by the mutation when NO was present. Consequently, B. subtilis cells lacking NsrR no longer sense and respond to NO (and nitrite) to up-regulate the ResDE regulon. Exposure to NO did not significantly change the cellular concentration of NsrR, suggesting that NO likely modulates the activity of NsrR. NsrR is similar to the recently described nitrite- or NO-sensitive transcription repressors present in various bacteria. NsrR likely has an Fe-S cluster, and interaction of NO with the Fe-S center is proposed to modulate NsrR activity.

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit ([alpha]CTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-[alpha]CTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the [alpha]l region of [alpha]CTD. Alanine-scanning mutagenesis of B. subtilis [alpha]CTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analvsis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-[alpha]CTD interaction and that residues Y263, C265, and K267 of the [alpha]1 region lie within overlapping ComA-and Spx-binding sites for [alpha]CTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.

Spx, a global transcription regulator in Bacillus subtilis, interacts with the C-terminal domain of the [alpha] subunit ([alpha]CTD) of RNA polymerase to control gene expression under conditions of disulfide stress, which is sensed by disulfide bond formation between Spx residues C10 and C13. Here, we describe the crystal structure of the B. subtilis [alpha]CTD bound to oxidized Spx. Analysis of the complex reveals interactions between three regions of 'antialpha' Spx and helix [alpha]1 and the '261' determinant of [alpha]CTD. The former contact could disrupt the interaction between [alpha]CTD and activator proteins or alter the DNA-bound conformation of [alpha]CTD, thereby repressing activator-stimulated transcription. Binding to the 261 determinant would prevent interaction between [alpha]CTD and region 4 of [[sigma].sup.A]. Intriguingly, the Spx disulfide bond is far from the [alpha]CTD--Spx interface, suggesting that Spx regulates transcription allosterically or through the redox-dependent creation or destruction of binding sites for additional components of the transcription machinery. ArsC family | global transcription regulation | oxidative/disulfide stress

In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse [C.sub.2][C.sub.12] myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix. sciatic neurectomy; Gpnmb family; [C.sub.2][C.sub.12] cells; NIH-3T3 cells; osteoactivin-transgenic mice

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions.

The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redoxrelated signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the -41 to -83 region of hmp and the -46 to -92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

Research has been conducted on Bacillus subtilis. The involvement of the ATP-dependent protease members in regulation and degradation of Spx is discussed.