sigD Expression Research Articles

The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis.

Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1β, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4+CD69+, CD4+CD154+) and activated B cell subsets (CD19+CD23+, CD19+CD21+, CD19+IgD+ and CD19-CD138+) were increased by IgD. The percentage of unactivated T cell subset (CD4+CD62L+) and immature B cell subset (CD19+IgM+IgD-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA.

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla/che Operon.

Exponentially growing cultures of Bacillus subtilis (PY79) are composed primarily of nonmotile, chained cells. The alternative sigma factor, SigD, promotes the phenotypic switch from nonmotile, chained cells to unchained, motile cells. In the present work, we investigated the role of the GTP-sensing protein CodY in the regulation of SigD. Deletion of codY resulted in a significant increase in SigD accumulation and activity and shifted the proportion of unchained cells up from ∼15% to ∼75%, suggesting that CodY is an important regulator of SigD. CodY was previously shown to bind to the PD3 and Pfla/che promoters located upstream of the first gene in the sigD-containing fla/che operon. Using electrophoretic mobility shift assays, we found that CodY also binds to two other previously uncharacterized sites within the fla/che operon. Mutations in any one of the three binding sites resulted in SigD levels similar to those seen with the ΔcodY mutant, suggesting that each site is sufficient to tip cells toward a maximal level of CodY-dependent SigD accumulation. However, mutations in all three sites were required to phenocopy the ΔcodY mutant's reduced level of cell chaining, consistent with the idea that CodY binding in the fla/che operon is also important for posttranslational control of SigD activity. One way that bacteria adapt quickly and efficiently to changes in environmental quality is to employ global transcriptional regulators capable of responding allosterically to key cellular metabolites. In this study, we found that the conserved GTP-sensing protein CodY directly regulates cell motility and chaining in B. subtilis by controlling expression and activity of SigD. Our results suggest that B. subtilis becomes poised for cell dispersal as intracellular GTP levels are depleted.

IL-4 Exerts Opposing Effects on Surface-IgM and CXCR4 Mediated Signalling in Chronic Lymphocytic Leukaemia

The B cell receptor (BCR) is critical for survival and proliferation of chronic lymphocytic leukemia (CLL) cells and is activated following antigen/autoantigen engagement. BCR engagement in vivo triggers a partial receptor down modulation, phosphorylation of tyrosine residues in CD79A and CD79B, signalosome formation and subsequent calcium mobilization and ERK phosphorylation (ERK-P). Interestingly CLL cells incubated in vitro recover surface(s)IgM receptor expression and subsequently their ability to signal (calcium flux and ERK-P). During this recovery of sIgM, expression of a fully glycosylated “mature” form of sIgM increases. Interestingly, sIgD is not downregulated in vivo and expression does not increase in vitro. Downregulation by antigen engagement therefore appears only to affect sIgM. IL-4 appears to be present in CLL tissue sites and has a known role in protecting CLL cells from apoptosis however its effect on BCR expression and subsequent downstream signalling has not previously been demonstrated. Therefore we investigated the effects of IL-4 on BCR signalling and on the expression of surface receptors including chemokine receptors CXCR4 and MHC-class II (MHCII).CLL cells were pretreated with and without IL-4 for 24h and sIgM and sIgD expression measured by flow cytometry. sIgM naturally recovers with time and this is significantly enhanced further by IL-4 (p=0.006, n=33), whereas sIgD does not (p=0.48, n=30). The same CLL samples pre-treated with IL-4 for 24 hours followed by treatment with anti-IgM (n=22) for 15 minutes showed enhanced calcium flux in 77% (p=0.0005) of samples compared to the control. The increase in sIgM expression correlated strongly with the ability to induce calcium mobilization in IL-4 treated samples (p=0.0007, r=0.7). The fold increase in sIgM expression following pre-treatment with IL-4 was significantly greater in U-CLL compared with M-CLL (p=0.012) and in ZAP70+ve samples (p=0.02). However, analysis of IL-4R expression did not show a significant difference between M-CLL and U-CLL samples (p=0.18, n=20). The increase in sIgM expression by IL-4 coincided with an increase in CD79b protein expression (p<0.0001, n=26). Futhermore IL-4 pretreatment significantly augmented expression of the fully glycosylated µ-chain of sIgM on the surface of the CLL cells (p=0.005), as confirmed by resistance to EndoH cleavage, indicating a role of IL-4 in IgM assembly and transport to the cytoplasmic membrane. These IL-4-specific effects were blocked with either a JAK3 or STAT6 inhibitor (Tofacitinib or Axon 1992, respectively) which specifically inhibit IL-4 signalling.We next identified whether the effects observed with IL-4 was limited to sIgM. Interestingly, in contrast to the spontaneous recovery of CXCR4 expression which occurs in vitro and parallels the recovery of sIgM expression, IL-4 significantly inhibited CXCR4 recovery in vitro (p<0.0001, n=21) and prevented migration towards CXCL12 in transwell migration assays (p=0.03, n=6). This indicates that IL-4 is not simply amplifying the natural process of recovery of these receptors but is having a differential effect. For other molecules, a significant increase in MHCII expression in IL-4 pre-treated samples (p=0.0011, n=12) was observed mimicking the changes with sIgM, whilst other negative or positive BCR regulator such as CD5 (p=0.47, n=25) and CD19 (p=0.93, n=20) respectively, remained unchanged. Indicating the effect of IL-4 on receptor expression was receptor dependent.These data suggest a role for IL-4 in augmenting sIgM expression and subsequent downstream signalling, increasing MHCII expression, whilst down modulating chemokine receptors to retain CLL cells within the lymph node. Thus IL-4 may play an important biological role when CLL cells encounter antigen/autoantigen and therefore inhibition of this pathway with JAK3 inhibitors may be a potential clinical target for the treatment of CLL. DisclosuresNo relevant conflicts of interest to declare.

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of σD in Bacillus subtilis

Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for σ(d) -dependent gene expression: a pattern suggestive of bistability. The gene encoding σ(D) , sigD, is part of the 31-gene fla/che operon where its location at the 3' end, 25 kb away from the strong P(fla/che) promoter, determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the chromosome inhibited σ(d) -dependent gene expression. SlrA together with SinR and SlrR reduced sigD transcript by potentiating a distance-dependent decrease in fla/che operon transcript abundance that was not mediated by changes in expression from the P(fla/che) promoter. Consistent with acting upstream of σ(D) , SlrA/SinR/SlrR was bypassed by artificial ectopic expression of sigD and hysteretically maintained for 20 generations by engaging the sigD gene at the native locus. SlrA/SinR/SlrR was also bypassed by increasing fla/che transcription and resulted in a hypersensitive output in flagellin expression. Thus, flagellin gene expression demonstrated hypersensitivity and hysteresis and we conclude that σ(d) -dependent gene expression is bistable.

Gene position in a long operon governs motility development in Bacillus subtilis

Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor sigma(D). Here we show that chaining cells were OFF for sigma(D)-dependent gene expression because sigma(D) levels fell below a threshold and sigma(D) activity was inhibited by the anti-sigma factor FlgM. The probability that sigma(D) exceeded the threshold was governed by the position of the sigD gene. The proportion of ON cells increased when sigD was artificially moved forward in the 27 kb fla/che operon. In addition, we identified a new sigma(D)-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension.

B cells compartement in centenarian offspring and old people

Immunosenescence is considered a major contributory factor to the increased frequency of morbidity and mortality among elderly. Immunosenescence studies focus mainly on T cells, although B cells are affected too. In our studies we analyzed B cells in two classes of individuals: old people (O), (age range 70‐85 years) and centenarian offspring (CA). Our data showed difference in number of B cells from O and CA. B cells were analyzed according to the expression of sIgD and CD27. B cells were classified as follows: IgD+CD27−(naïve),IgD+CD27+(unswitched‐memory),IgD−CD27+ (switched ‐memory) and IgD−CD27−(double negative, DN). As expected, in both cohorts we observed a marked decreased of B cell count although within the B cell population, CA do not behave as O subjects. Indeed, in CA , naïve B cells are more abundant whereas DN B cells don't show the typical increase that we have previously demonstrated in healthy elderly donors. So, B cells of the CA seem to be more similar to that of young respect to the old one. B cell subsets changes could represent a hallmark of immunosenescence and could be used as a biomarker of human life span, potentially useful for the evaluation of anti‐ageing treatment.This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR, ex 40%)

Regulation of Bacillus subtilis aprE Expression by glnA through Inhibition of scoC and σ D -Dependent degR Expression

Expression of the gene for the extracellular alkaline protease (aprE) of Bacillus subtilis is subject to regulation by many positive and negative regulators. We have found that aprE expression was increased by disruption of the glutamine synthetase gene glnA. The increase in aprE expression was attributed to a decreased in expression of scoC, which encodes a negative regulator of aprE expression. The glnA effect on scoC expression was abolished by further disruption of tnrA, indicating that aprE expression is under global regulation through TnrA. In the scoC background, however, aprE expression was decreased by glnA deletion, and it was shown that the decrease was due to a defect in positive regulation by DegU. Among the genes that affect aprE expression through DegU, the expression of degR, encoding a protein that stabilizes phosphorylated DegU, was inhibited by glnA deletion. It was further shown that the decrease in degR expression by glnA deletion was caused by inhibition of the expression of sigD, encoding the sigma(D) factor, which is required for degR expression. In accordance with these findings, the expression levels of aprE-lacZ in glnA scoC degR and scoC degR strains were identical. These results led us to conclude that glnA deletion brings about two effects on aprE expression, i.e., a positive effect through inhibition of scoC expression and a negative effect through inhibition of degR expression, with the former predominating over the latter.

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1α. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10−/− B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells—perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis—identifying a strategy that appears to be conserved between at least EBV and MHV68.

Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium

Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigD(R468A), was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigD(R468A) in yeast. First, expression of SigD(R468A) caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigD(R468A) was mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigD(R468A) rather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigD(R468A)-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigD(R468A) was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.

Signaling through CD70 regulates B cell activation and IgG production.

CD70, the cellular ligand of the TNF receptor family member CD27, is expressed transiently on activated T and B cells and constitutively on a subset of B cell chronic lymphocytic leukemia and large B cell lymphomas. In the present study, we used B cells constitutively expressing CD70 to study the functional consequences of signaling through CD70. In vitro, CD70 ligation with anti-CD70 mAbs strongly supported proliferation and cell cycle entry of B cells submitogenically stimulated with either anti-CD40 mAb, LPS, or IL-4. In this process, the cell surface receptors CD25, CD44, CD69, CD95, and GL7 were up-regulated, whereas the expression of CD21, CD62L, surface IgM (sIgM), and sIgD was decreased. Addition of CD70 mAb to low dose LPS-stimulated CD70-positive B cells strongly diminished IgG secretion and enhanced production of IgM. Signaling through CD70 on B cells was dependent on the initiation of both PI3K and MEK pathways. In vivo exposure to either CD70 mAb or the CD70 counterreceptor CD27 down-regulated CD62L and sIgM on CD70-positive B cells. CD70 signaling during T cell-dependent immune responses also decreased IgG-specific Ab titers. Together, the in vitro and in vivo data demonstrate that CD70 has potent reverse signaling properties in B cells, initiating a signaling cascade that regulates expansion and differentiation.

Dictyostelium discoideum developmentally regulated genes whose expression is dependent on MADS box transcription factor SrfA.

The MADS box transcription factor SrfA is required for spore differentiation in Dictyostelium discoideum. srfA null strains form rounded spores that do not resist adverse environmental conditions. Five genes whose expression is dependent on SrfA have been isolated by differential hybridization. One of these genes, sigC, is identical to phg1b, previously characterized in mutants with altered adhesive properties and found to encode a nine-transmembrane-domain protein. This gene is transcribed into two mRNAs as the result of alternative splicing of two internal exons. The slower-migrating mRNA codes for a shorter protein that lacks the first transmembrane fragment and is not expressed in srfA null strains. The other four genes (sigA, sigB, sigD, and 45D) are expressed only during late developmental stages. In situ hybridization experiments showed that expression of sigA, sigB, and sigD is restricted to the sorus of developing structures. sigA codes for a homologue of malate dehydrogenase that converts pyruvate to malate to replenish the tricarboxylic acid cycle. sigB encodes a protein with significant similarity to the GP63 metalloproteinase of Leishmania, leishmanolysin. The sequence of SigD is highly similar to that of several spore coat proteins of D. discoideum, and it may play a role in that structure. The gene 45D codes for an RNA-binding protein homologue whose expression is also dependent on the GATA transcription factor stalky (StkA). The expression of sigB is also dependent on both SrfA and StkA. The expression of 45D, but not of sigA, sigB, sigC, and sigD, can be induced in srfA null cells by constitutive protein kinase A activation. Strains in which either sigA, sigB, or sigD is disrupted were isolated and found to form spores that are not detectably different from those of wild-type strains.

Purification, Characterization, and Gene Expression of All Sigma Factors of RNA Polymerase in a Cyanobacterium

The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp. PCC 6803, which can perform photosynthesis. All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared. Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per microgram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed. Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability. Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them. In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA. On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed. These findings clearly indicated that SigB is a heat-shock responsive sigma factor. The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein.

Elimination of host cell PtdIns(4,5)P2 by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.

Relative roles of the fla/che P(A), P(D-3), and P(sigD) promoters in regulating motility and sigD expression in Bacillus subtilis.

Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3' end of the operon. Two of these promoters, fla/che P(A) and P(D-3), lie upstream of the >26-kb fla/che operon. The third promoter, P(sigD), lies within the operon, immediately upstream of sigD. fla/che P(A), transcribed by E sigma(A), lies >/=24 kb upstream of sigD and appears to be largely responsible for sigD expression. P(D-3), transcribed by E sigma(D), has been proposed to participate in an autoregulatory positive feedback loop. P(sigD), a minor sigma(A)-dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P(A), P(D-3), or P(sigD). Our analysis of these strains indicates that fla/che P(A) is absolutely essential for motility, that P(D-3) does not function in positive feedback regulation of sigD expression, and that P(sigD) is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.

Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression.

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.

The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella typhimurium virulence genes.

SigD and SigE (Salmonella invasion gene) are proteins needed for optimal invasion of Salmonella typhimurium into eukaryotic cells in vitro. SigD is a secreted protein and SigE is a putative chaperone required for SigD stability and/or secretion. SigD is secreted by a type III secretion apparatus encoded within a pathogenicity island on the Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI1). The expression of sigDE, which is not linked to SPI1, is co-ordinately regulated with the SPI1 genes and is dependent on the transcriptional regulators SirA, HilA and InvF. These three proteins alone are unable to activate transcription from the sigD promoter in Escherichia coli, therefore it is likely that other factors are needed for expression. A screen for genes required for the expression of a sigD-lacZYA reporter fusion found a mutant with a transposon insertion in spaS, an SPI1 gene which encodes a putative inner-membrane component of the type III secretion system. The expression of a SPI1 operon encoding a putative chaperone (SicA) and several secreted proteins (Sips B, C, D and A) was also reduced in this mutant. The regulation defect of the spaS mutant was complemented by sicA and not by spaS. Because sicA is encoded immediately downstream of spaS, the mutation in spaS was likely to be polar on the expression of sicA. In addition, a sicA disruption mutant was as defective as an invF deletion mutant for the expression of sigD, sicA and sipC reporter fusions. The introduction of plasmids encoding invF and sicA into a non-pathogenic E. coli K-12 strain stimulated the transcription of both a sicA- and a sigD-lacZYA promoter fusion. This result suggests that InvF and SicA are sufficient for the expression of these genes. This is the first demonstration of a positive regulatory role for a putative type III secretion system chaperone in the expression of virulence genes.

Positive regulation of Bacillus subtilis sigD by C-terminal truncated LacR at translational level

DegR is a positive regulator for degradative enzyme synthesis in Bacillus subtilis. The degR gene is transcribed by RNA polymerase containing σ D, and the level of its expression is low in a mecA-deficient mutant. In a search for suppressors of the mecA effect through mini-Tn 10 transposon mutagenesis, a lacR mutation designated lacR288 was discovered. The B. subtilis lacR gene encodes the repressor for lacA which specifies β-galactosidase, and therefore, inactivation of the lacR gene results in overproduction of the enzyme. In the lacR288 mutant, however, the expression of lacA was at a negligible level, indicating that the repressor activity was not destroyed by the mutation. The putative gene product of the lacR288-containing gene is a 288-amino acid protein lacking the C-terminal 42 amino acids of intact LacR and carries no extra amino acids derived from the transposon sequence. The suppression by lacR288 of the decreased degR expression in the mecA background was found to be caused by an increase in the σ D level as shown by Western blot analysis. Furthermore, the increase was due to post-transcriptional regulation of sigD, the gene encoding σ D, as revealed by using both transcriptional and translational sigD-lacZ fusions. The lacR288 mutation had no effect on the stability of the σ D protein. Based on these results we conclude that the lacR288 mutation stimulates sigD expression at the translational level.

A new sigma factor, SigD, essential for stationary phase is also required for multicellular differentiation in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that undergoes spectacular development to form multicellular fruiting bodies under nutrient deprivation. Inside a fruiting body, vegetative cells differentiate into spores. A number of sigma factors have been shown to play roles in the regulation of gene expression in the M. xanthus life cycle. Additional sigma factors were searched to further explore the M. xanthus life cycle. A new sigma factor was identified, SigD, which consists of 297 amino acid residues. Two transcription initiation sites for the sigD gene were detected by primer extension analysis using total RNA from the vegetative and developmental cells, one of which was specific for development. The characterization of sigD-lacZ fusion strains demonstrated that sigD expression increased during entry into stationary phase of vegetative growth and during early development. A deletion mutant of sigD exhibited growth defects during the late-log phase and stationary phase, with dramatically reduced cell viability. The patterns of protein synthesis at late log phase of vegetative growth and at early development on CF agar plates were significantly different between the deletion mutant and the wild-type strain. The deletion mutant was more sensitive to various stresses when compared with the wild-type strain and did not accumulate trehalose in response to osmotic stress. The deletion mutant also showed a significant delay in fruiting body formation and sporulation and yielded fewer spores than the wild-type strain. SigD shows characteristic features of the stationary phase sigma factors and also plays important roles in multicellular differentiation of M. xanthus.