Glucose controls manganese homeostasis through transcription factors regulating known and newly identified manganese transporter genes in Bacillus subtilis.

Mn2+ is an essential nutrient whose concentration is tightly controlled in bacteria. In Bacillus subtilis, the Mn2+-activated transcription factor MntR controls Mn2+ transporter genes. However, factors regulating intracellular Mn2+ concentration are incompletely understood. Here, we found that glucose addition induces an increase in intracellular Mn2+ concentration. We determined this upshift was mediated by glucose induction of the major Mn2+ importer gene mntH by the transcription factor AhrC, which is known to be involved in arginine metabolism and to be indirectly induced by glucose. In addition, we identified novel AhrC-regulated genes encoding the Mn2+ importer YcsG and the ABC-type exporter YknUV. We found the expression of these genes was also regulated by glucose and contributes to the glucose induction of Mn2+ concentrations. ycsG expression is regulated by MntR as well. Furthermore, we analyzed the interaction of AhrC and MntR with the promoter driving ycsG expression and examined the Mn2+-dependent induction of this promoter to identify the transcription factors responsible for the Mn2+ induction. RNA-Seq revealed that disruption of ahrC and mntR affected the expression of 502 and 478 genes, respectively (false discovery rate, <0.001, log2[fold change] ≥ |2|. The AhrC- and/or MntR-dependent expression of twenty promoters was confirmed by LacZ analysis, and AhrC or MntR binding to some of these promoters was observed via EMSA. The finding that glucose promotes an increase in intracellular Mn2+ levels without changes in extracellular Mn2+ concentrations is reasonable for the bacterium, as intracellular Mn2+ is required for enzymes and pathways mediating glucose metabolism.

Identification of transposon-inserted mutations including rnpB::Tn that abolished glucose induction of sigX encoding extracytoplasmic function-sigma factor in Bacillus subtilis.

We investigated the regulators of the glucose induction (GI) of the ECF-sigma genes sigX/M. During further screening of transposon-inserted mutants, we identified several regulators including an RNA component of RNase P (rnpB), which is required for tRNA maturation. A depletion of rnpB is known to trigger the stringent response. We showed evidence that the stringent response inhibited GI of sigX/M.

RNA-seq analysis identified glucose-responsive genes and YqfO as a global regulator in Bacillus subtilis.

OBJECTIVE: We observed that the addition of glucose enhanced the expression of sigX and sigM, encoding extra-cytoplasmic function sigma factors in Bacillus subtilis. Several regulatory factors were identified for this phenomenon, including YqfO, CshA (RNA helicase), and YlxR (nucleoid-associated protein). Subsequently, the relationships among these regulators were analyzed. Among them, YqfO is conserved in many bacterial genomes and may function as a metal ion insertase or metal chaperone, but has been poorly characterized. Thus, to further characterize YqfO, we performed RNA sequencing (RNA-seq) analysis of YqfO in addition to CshA and YlxR. RESULTS: We first performed comparative RNA-seq to detect the glucose-responsive genes. Next, to determine the regulatory effects of YqfO in addition to CshA and YlxR, three pairs of comparative RNA-seq analyses were performed (yqfO/wt, cshA/wt, and ylxR/wt). We observed relatively large regulons (approximately 420, 780, and 180 for YqfO, CshA, and YlxR, respectively) and significant overlaps, indicating close relationships among the three regulators. This study is the first to reveal that YqfO functions as a global regulator in B. subtilis.

Glucose-Mediated Protein Arginine Phosphorylation/Dephosphorylation Regulates ylxR Encoding Nucleoid-Associated Protein and Cell Growth in Bacillus subtilis.

Glucose is the most favorable carbon source for many bacteria, and these bacteria have several glucose-responsive networks. We proposed new glucose responsive system, which includes protein acetylation and probable translation control through TsaEBD, which is a tRNA modification enzyme required for the synthesis of threonylcarbamoyl adenosine (t6A)-tRNA. The system also includes nucleoid-associated protein YlxR, regulating more than 400 genes including many metabolic genes and the ylxR-containing operon driven by the PylxS promoter is induced by glucose. Thus, transposon mutagenesis was performed for searching regulatory factors for PylxS expression. As a result, ywlE was identified. The McsB kinase phosphorylates arginine (Arg) residues of proteins and the YwlE phosphatase counteracts against McsB through Arg-dephosphorylation. Phosphorylated Arg has been known to function as a tag for ClpCP-dependent protein degradation. The previous analysis identified TsaD as an Arg-phosphorylated protein. Our results showed that the McsB/YwlE system regulates PylxS expression through ClpCP-mediated protein degradation of TsaD. In addition, we observed that glucose induced ywlE expression and repressed mcsB expression. It was concluded that these phenomena would cause glucose induction (GI) of PylxS, based on the Western blot analyses of TsaD-FLAG. These observations and the previous those that many glycolytic enzymes are Arg-phosphorylated suggested that the McsB/YwlE system might be involved in cell growth in glucose-containing medium. We observed that the disruption of mcsB and ywlE resulted in an increase of cell mass and delayed growth, respectively, in semi-synthetic medium. These results provide us broader insights to the physiological roles of the McsB/YwlE system and protein Arg-phosphorylation.

Bacillus subtilis Nucleoid-Associated Protein YlxR Is Involved in Bimodal Expression of the Fructoselysine Utilization Operon (frlBONMD-yurJ) Promoter.

Bacteria must survive harsh environmental fluctuations at times and have evolved several strategies. "Collective" behaviors have been identified due to recent progress in single-cell analysis. Since most bacteria exist as single cells, bacterial populations are often considered clonal. However, accumulated evidence suggests this is not the case. Gene expression and protein expression are often not homogeneous, resulting in phenotypic heterogeneity. In extreme cases, this leads to bistability, the existence of two stable states. In many cases, expression of key master regulators is bimodal via positive feedback loops causing bimodal expression of the target genes. We observed bimodal expression of metabolic genes for alternative carbon sources. Expression profiles of the frlBONMD-yurJ operon driven by the frlB promoter (PfrlB), which encodes degradation enzymes and a transporter for amino sugars including fructoselysine, were investigated using transcriptional lacZ and gfp, and translational fluorescence reporter mCherry fusions. Disruption effects of genes encoding CodY, FrlR, RNaseY, and nucleoid-associated protein YlxR, four known regulatory factors for PfrlB, were examined for expression of each fusion construct. Expression of PfrlB-gfp and PfrlB-mCherry, which were located at amyE and its original locus, respectively, was bimodal; and disruption of ylxR resulted in the disappearance of the clear bimodal expression pattern in flow cytometric analyses. This suggested a role for YlxR on the bimodal expression of PfrlB. The data indicated that YlxR acted on the bimodal expression of PfrlB through both transcription and translation. YlxR regulates many genes, including those related to translation, supporting the above notion. Depletion of RNaseY abolished heterogenous expression of transcriptional PfrlB-gfp but not bimodal expression of translational PfrlB-mCherry, suggesting the role of RNaseY in regulation of the operon through mRNA stability control and regulatory mechanism for PfrlB-mCherry at the translational level. Based on these results, we discuss the meaning and possible cause of bimodal PfrlB expression.

Bacillus subtilis YlxR, Which Is Involved in Glucose-Responsive Metabolic Changes, Regulates Expression of tsaD for Protein Quality Control of Pyruvate Dehydrogenase.

Glucose is the most favorable carbon source for many bacteria, which have several glucose-responsive gene networks. Recently, we found that in Bacillus subtilis glucose induces the expression of the extracellular sigma factor genes sigX and sigM through the acetylation of CshA (RNA helicase), which associates with RNA polymerase (RNAP). We performed a transposon mutagenesis screen for mutants with no glucose induction (GI) of sigX-lacZ. While screening for such mutants, we recently found that the GI of sigX/M involves YlxR, a nucleoid-associated protein (NAP) that regulates nearly 400 genes, including metabolic genes. It has been shown that acetylated CshA positively regulates expression of ylxR-containing operon. Here, we report additional mutations in yqfO or tsaD required for the GI of sigX. YqfO contains a universally conserved domain with unknown function. YqfO and YlxR were found to regulate expression of the tsaEBD-containing operon. Mutational analysis using lacZ fusions revealed the adenine-rich cis-element for YlxR. TsaD is a component of the TsaEBD enzyme required for the synthesis of threonylcarbamoyl adenosine (t6A). The t6A modification of tRNA is universal across the three domains of life. Western blot analysis showed that the tsaD mutation in the presence of glucose reduced levels of soluble PdhA, PdhB, and PdhD, which are subunits of the pyruvate dehydrogenase complex (PDHc). This resulted in severely defective PDHc function and thus reduced concentrations of cellular acetyl-CoA, a reaction product of PDHc and plausible source for CshA acetylation. Thus, we discuss a suggested glucose-responsive system (GRS) involving self-reinforcing CshA acetylation. This self-reinforcing pathway may contribute to the maintenance of the acetyl-CoA pool for protein acetylation.

Newly Identified Nucleoid-Associated-Like Protein YlxR Regulates Metabolic Gene Expression in Bacillus subtilis.

Glucose is the most favorable carbon source for the majority of bacteria, which have several glucose-responsive gene networks. Recently, we found that in Bacillus subtilis, glucose induces expression of the extracellular sigma factor genes sigX/M To explore the factors affecting this phenomenon, we performed a transposon mutagenesis screen for mutants with no glucose induction (GI) of sigX-lacZ and identified ylxR YlxR is widely conserved in eubacteria. Further analysis revealed that ylxR is induced by glucose addition. In vitro DNA-binding and cytological studies suggested that YlxR is a nucleoid-associated protein (NAP) in B. subtilis In many cases, NAPs influence transcription, recombination, and genome stability. Thus, we performed transcriptome sequencing (RNA-Seq) analysis to evaluate the impact of ylxR disruption on the transcriptome in the presence of glucose and observed that YlxR has a profound impact on metabolic gene expression in addition to that of four sigma factor genes. The wide fluctuations of gene expression may result in abolition of GI of sigX/M in the ylxR disruptant.IMPORTANCE Expression of genes encoding NAPs is often temporally regulated. According to results from single-cell analysis, the ylxR gene is induced by glucose and expressed in a bistable mode. These characteristics have not previously been reported for NAP gene expression. Transcriptional profiling of the ylxR disruptant revealed a change in the expression levels of approximately 400 genes, including genes for synthesis of 12 amino acids and 4 nucleotides, in addition to the SigX/M regulons. Thus, YlxR is a critical regulator of glucose response in B. subtilis.

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

Glucose Induces ECF Sigma Factor Genes, sigX and sigM, Independent of Cognate Anti-sigma Factors through Acetylation of CshA in Bacillus subtilis.

Extracytoplasmic function (ECF) σ factors have roles related to cell envelope and/or cell membrane functions, in addition to other cellular functions. Without cell-surface stresses, ECF σ factors are sequestered by the cognate anti-σ factor, leading to inactivation and the resultant repression of regulons due to the inhibition of transcription of their own genes. Bacillus subtilis has seven ECF σ factors including σX and σM that transcribe their own structural genes. Here, we report that glucose addition to the medium induced sigX and sigM transcription independent of their anti-σ factors. This induction was dependent on an intracellular acetyl-CoA pool. Transposon mutagenesis searching for the mutants showing no induction of sigX and sigM revealed that the cshA gene encoding DEAD-box RNA helicase is required for gene induction. Global analysis of the acetylome in B. subtilis showed CshA has two acetylated lysine residues. We found that in a cshA mutant with acetylation-abolishing K to R exchange mutations, glucose induction of sigX and sigM was abolished and that glucose addition stimulated acetylation of CshA in the wild type strain. Thus, we present a model wherein glucose addition results in a larger acetyl-CoA pool, probably leading to increased levels of acetylated CshA. CshA is known to associate with RNA polymerase (RNAP), and thus RNAP with acetylated CshA could stimulate the autoregulation of sigX and sigM. This is a unique model showing a functional link between nutritional signals and the basal transcription machinery.

Post-transcriptionally generated cell heterogeneity regulates biofilm formation in Bacillus subtilis.

Bacillus subtilis forms biofilms in appropriate environments by producing extracellular matrices. Genes required for matrix formation, for example tapA, are regulated by the SinI/SinR/SlrR system. SinR is the repressor for tapA. SinI and SlrR inhibit DNA-binding of SinR. sinI and sinR constitute two-gene operon, and sinR has its own promoter. During biofilm formation, a portion of the population differentiates into matrix-producing cells. This is thought to be caused by Spo0A-dependent, heterogeneous expression of the PsinI promoter, whereas the PsinR promoter is expressed homogeneously. However, we observed that at its original locus, overall sinI transcription was almost homogeneous, because upstream read-through transcription from PyqHG would overcome expression of PsinI. When we used translational sinI-gfp and sinR-mCherry reporters at their original loci, their fluorescence distribution patterns in the cell population were clearly bimodal. This bimodal expression might be caused by cell-to-cell variations of mRNA stability. This study shows that the post-transcriptionally regulated bimodal expression of SinI and SinR is important for bacterial cell-fate determination.

Bacillus subtilis degSU operon is regulated by the ClpXP-Spx regulated proteolysis system.

The DegS-DegU two-component regulatory system regulates many cellular events in Bacillus subtilis. Genes for DegSU constitutes an operon directed by the P1 promoter and downstream degU is autoregulated via the P3 promoter activated by phosphorylated DegU. In the Gram-positive bacteria, Spx plays a major role in the protection system against oxidative stresses as a transcriptional regulator. Spx is a substrate of the ATP-dependent ClpXP protease. It regulates diamide-stress regulon in addition to many genes with unknown functions. We have found that null mutations for clpX and clpP, which encode the subunits for the protease ClpXP, enhanced the DegU level through activation of the P1 promoter. We isolated four suppressors for the clpP-enhancing effect. Whole-genome sequencing of the suppressors revealed that two have a point mutation in spx and the rest have a deletion of spx. The clpP-enhancing effect on degS-lacZ expression was abolished in the spx disruptant. These results show that the degSU operon is a new target of Spx-mediated positive regulation. Furthermore, we found that the P1 promoter was induced by glucose and that this induction was greatly reduced in the spx mutant. These results suggested that Spx-mediated glucose induction at the P1 promoter.

Regulation of the response regulator gene degU through the binding of SinR/SlrR and exclusion of SinR/SlrR by DegU in Bacillus subtilis.

Bacillus subtilis DegU is a response regulator of the DegS-DegU two-component regulatory system. Phosphorylated DegU (DegU-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the degU gene, by binding to target gene promoters. Nonphosphorylated DegU and low levels of DegU-P are required for swarming motility and genetic competence. The DNA-binding repressors SinR and SlrR are part of a double-negative feedback loop and comprise the epigenetic switch governing biofilm formation. In this study, we found that SinR repressed degU. Furthermore, SlrR, which interacts with SinR through protein-protein interaction, seems to have an active role in degU expression in in vivo lacZ analysis. An in vitro transcription assay supported this observation. An electrophoretic mobility shift assay (EMSA) showed that SinR bound to the degU promoter and that SlrR formed a complex with SinR on the degU promoter. In EMSA, DegU-P excluded the SinR/SlrR complex but not SinR from the degU promoter in the presence of RNA polymerase. These findings suggest that DegU-P interacts with SlrR. In support of this hypothesis, disruption of the slrR gene resulted in decreased degU expression. This newly identified regulatory mechanism for degU is considered to be sequential transcription factor replacement.

The Bacillus subtilis response regulator gene degU is positively regulated by CcpA and by catabolite-repressed synthesis of ClpC.

In Bacillus subtilis, the response regulator DegU and its cognate kinase, DegS, constitute a two-component system that regulates many cellular processes, including exoprotease production and genetic competence. Phosphorylated DegU (DegU-P) activates its own promoter and is degraded by the ClpCP protease. We observed induction of degU by glucose in sporulation medium. This was abolished in two mutants: the ccpA (catabolite control protein A) and clpC disruptants. Transcription of the promoter of the operon containing clpC (PclpC) decreased in the presence of glucose, and the disruption of ccpA resulted in derepression of PclpC. However, this was not directly mediated by CcpA, because we failed to detect binding of CcpA to PclpC. Glucose decreased the expression of clpC, leading to low cellular concentrations of the ClpCP protease. Thus, degU is induced through activation of autoregulation by a decrease in ClpCP-dependent proteolysis of DegU-P. An electrophoretic mobility shift assay showed that CcpA bound directly to the degU upstream region, indicating that CcpA activates degU through binding. The bound region was narrowed down to 27 bases, which contained a cre (catabolite-responsive element) sequence with a low match to the cre consensus sequence. In a footprint analysis, CcpA specifically protected a region containing the cre sequence from DNase I digestion. The induction of degU by glucose showed complex regulation of the degU gene.

SwrA regulates assembly of Bacillus subtilis DegU via its interaction with N-terminal domain of DegU.

The Bacillus subtilis response regulator DegU controls many physiological events including swarming motility and exoprotease production. Swarming motility is a multicellular movement of hyper-flagellated cells on a surface. The swarming motility regulator SwrA and DegU cooperatively drive transcription of fla/che encoding flagella components, chemotaxis constituents and motility-specific sigma factor, which is regarded as the primary event in the development of motility. We have identified ycdA involved in swarming motility, encoding a putative lipoprotein. We showed that the ycdA gene is positively regulated by DegU and SwrA. Mutational analysis of ycdA-lacZ revealed that SwrA changes the use of cis-acting sites for DegU. This suggested that SwrA operates the DegU-regulation mode through changes in the DegU assembly state. DegU binding to the ycdA-promoter region carrying an unusual arrangement of DegU-recognition sequences with low affinity was found to be stimulated by SwrA in electrophoretic mobility shift assay and DNase I footprinting. Yeast two- and three-hybrid analyses revealed that the N-terminal domain of DegU interacts with whole DegU, which is facilitated by SwrA. Together, these results demonstrate that SwrA can stabilize the binding of DegU to the ycdA promoter with low affinity. Thus, SwrA is a novel type of bacterial transcription factor in this regard.

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion.

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.

ZnuABC and ZosA zinc transporters are differently involved in competence development in Bacillus subtilis.

Disruptants of genes encoding the ZnuABC high-affinity zinc incorporator and zosA encoding a P-type ATPase for zinc incorporation were identified to show low transformability. The low transformability of the znuB cells was rescued by excess zinc addition and epistatic analysis of the mutation revealed no effect on the expression of comK, which encodes a master regulator for late com operons. We further examined the expression of each late com operon in the znuA mutant and found that the znuA mutation specifically inhibited the expression of comF, but not the other late com operons. The addition of zinc also rescued the low transformability of the zosA cells. In zosA cells, transcription of comK was severely repressed. Using a strain carrying comK driven by a xylose-inducible promoter, we showed that the zosA mutation inhibited the post-transcriptional control of comK. The addition of zinc also rescued the defect of xylose-inducible comK expression in zosA cells, suggesting that post-transcriptional control of comK requires zinc incorporation. Taken together, we propose that the both ZnuABC- and ZosA-mediated zinc incorporation is involved in competence development, although the two zinc transporters are differently implicated in this developmental process.

Identification of the sequences recognized by the Bacillus subtilis response regulator YclJ.

The Bacillus subtilis yclJ gene encodes an OmpR-type response regulator of a two-component regulatory system with unknown function. A previous DNA microarray experiment suggested that multicopy yclJ greatly enhances the expression of several operons in a cognate kinase (YclK)-deficient strain. To confirm this, lacZ fusion analysis was performed in the yclK background with overexpressed yclJ. As a result, yclHI, ykcBC, and yngABC were indeed positively regulated by YclJ. Gel retardation and DNase I footprint analyses revealed that YclJ binds to the promoter regions of yclHI, ykcBC, and yngABC. Nucleotide sequence analysis of the binding regions suggested that YclJ recognizes a direct repeat of the consensus sequence TTCATANTTT, the upstream half of which has close similarity to the consensus binding sequence of the other OmpR family response regulator PhoP. LacZ fusion analysis of the control region of yngA with deletion or point mutation confirmed that the YclJ-binding sequence is required for the YclJ-mediated activation of yngA. Furthermore, we identified two more YclJ-regulated genes, yycA and yfjR, using bioinformatic analysis of the B. subtilis genome, and it was shown that YclJ binds to those promoters and controls the expression of those genes.

Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU-P by ClpCP.

The response regulator DegU and its cognate kinase DegS constitute a two-component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU-lacZ fusions, we showed that phosphorylated DegU (DegU-P) binds to two direct repeats (DR1 and DR2) of the consensus DegU-binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP-dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild-type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU-P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU-P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N-terminal region of DegU is important for proteolysis.

[Three adult cases of severe acute glomerulonephritis with acute renal failure requiring hemodialysis].

Acute poststreptococcal glomerulonephritis (APSGN) typically recovers within 2 weeks with conservative therapy, but severe cases are known to develop acute renal failure and or nephrotic syndrome. We experienced 3 adult cases of APSGN with acute renal failure. All 3 cases required hemodialysis, 2 cases received double filtration plasmapheresis, and 2 cases received steroid therapy. In all cases, renal biopsy specimens showed endocapillary proliferative glomerulonephritis. One case had 25% cellular crescents and the others showed wide subendothelial deposits. There were no tubulointerstitial lesions. These 3 cases of severe APSGN with acute renal failure showed the benefits of combination therapy, hemodialysis, double filtration plasmapheresis and steroid therapy. Further clinical study is required to determine which therapy, double filtration plasmapheresis or steroid therapy, is useful.

Bacillus subtilis response regulator DegU is a direct activator of pgsB transcription involved in gamma-poly-glutamic acid synthesis.

pgsB encodes gamma-poly glutamic acid (gamma-PGA) synthetase and constitutes an operon with pgsC, pgsAA, and pgsE. Genetic analysis revealed that degQ and swrA, the known regulators of pgsB, are not required for pgsB expression when high cellular concentrations of phosphorylated form of the response regulator DegU (DegU-P) are present. However, swrA appeared still to be required for gamma-PGA synthesis under the conditions we tested. Since genetic analysis suggested that DegU-P activates pgsB directly, we performed gel retardation and footprint analyses using purified His-tagged DegU and the pgsB promoter. The in vitro experiments revealed that His-tagged DegU bound to the immediate upstream region of the -35 region of the pgsB promoter. A six-base deletion within the sequence (the -44 to -39 region) abolished DegU-binding to the pgsB promoter and pgsB transcription, confirming the importance of the sequence for DegU-dependent regulation of pgsB. Hence we conclude that DegU is a direct activator of the pgsB operon.