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.

Functional analysis of the stability determinant AlfB of pBET131, a miniplasmid derivative of bacillus subtilis (natto) plasmid pLS32.

Bacillus subtilis plasmid pBET131 is a derivative of pLS32, which was isolated from a natto strain of Bacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of which alfA, encoding an actin-like ATPase, and alfB are essential for plasmid stability. In this work, the alfB gene product and its target DNA region were studied in detail. Transcription of the alf operon initiated from a sigma(A)-type promoter was repressed by the alfB gene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of the alf operon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designated parN), which partially overlaps the -35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression of alfA-lacZ expression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA and parN, the mode of which is typified by the type II partition mechanism.

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.

The Bacillus subtilis late competence operon comE is transcriptionally regulated by yutB and under post-transcription initiation control by comN (yrzD).

The Bacillus subtilis genome has been sequenced, and disruptants with disruptions in genes that were not characterized previously were systematically generated. We screened these gene disruptants for decreased transformation frequency and identified two genes, yrzD and yutB, whose disruption resulted in severely reduced transformation frequency and modestly reduced transformation frequency, respectively. In the regulation of competence development, various signals affect the expression of comK, which encodes a master regulator of genetic competence that drives late competence gene transcription. Epistatic analyses of both the yrzD and yutB genes revealed no significant differences in the expression of comK. Further analysis of the expression of late competence genes in the yrzD disruptant revealed that yrzD is specifically required for regulation of the comE operon, which is one of the late competence operons, and thus was renamed comN. An analysis of various comE-lacZ fusions revealed that the target cis element for comN action is in the large (approximately 1-kb) 5' untranslated region of comE, while the activity of the comE promoter was not affected by disruption of comN. These results suggested that there is post-transcription initiation control of comE by comN. A sequential deletion analysis of this region revealed the 35-bp region required for comN action. The yutB gene encodes a putative lipoic acid synthetase and yet is specifically required for transcription of comE, based on the results of lacZ fusion analyses. Therefore, yutB and comN regulate comE at the transcription and post-transcription initiation levels, respectively. These results demonstrate that a comE-specific regulatory mechanism is involved in development of genetic competence.

Regulation of Bacillus subtilis aprE expression by glnA through inhibition of scoC and sigma(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.

TRPV2 expression in rat oral mucosa.

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Distribution of substance P and neurokinin-1 receptors in the peri-implant epithelium around titanium dental implants in rats.

We examined the distribution of substance P and neurokinin-1 (NK1) receptors and substance-P-containing nerve fibers in the peri-implant mucosa around titanium dental implants in rats. Immunohistochemistry and immunocytochemistry revealed that substance-P-immunoreactive nerve fibers abundantly innervated the peri-implant epithelium (PIE) compared with other epithelia of the peri-implant mucosa. NK1 receptor mRNA and protein expression in the peri-implant mucosa were confirmed by reverse transcription with the polymerase chain reaction and immunoblotting. Immunoelectron microscopy revealed that NK1 receptor immunoreactivity was preferentially localized in peri-implant epithelial cells. NK1-receptor-positive products were found on the plasma membrane and in vesicles and granules in PIE cells. Neutrophils and intraepithelial nerve axons in the PIE were positive for the NK1 receptor. NK1 receptor immunoreactivity was also detected in endothelial cells, fibroblasts, and nerve fibers in the connective tissue beneath the PIE. These findings suggest that peri-implant tissue receives sensory information through regenerated nerves expressing substance P and the NK1 receptor. In the peri-implant mucosa, the substance P/NK1 receptor system may play a role in pain transmission, the endocytosis of neutrophils, the extravasation of crevicular fluid, and the migration of macrophages and neutrophils in response to neurogenic inflammation, as in healthy gingiva.

Involvement of nitrogen regulation in Bacillus subtilis degU expression.

Bacillus subtilis DegS-DegU belongs to a bacterial two-component system that controls many processes, including the production of exocellular proteases and competence development. It was found that when the glutamine synthetase gene glnA, which is involved in nitrogen regulation, was disrupted, the expression of the response regulator degU gene was increased. Deletion analysis and 5'-end mapping of the degU transcripts showed that the increase was caused by induction of a promoter (P2) located before the degU gene. Disruption of tnrA, a global regulator of nitrogen regulation, eliminated the P2 promoter induction by the glnA mutation. The fact that the P2 promoter is under nitrogen regulation was demonstrated by an increase in P2 expression with nitrogen-limited growth. It was also found by primer extension analysis that degU was transcribed by another promoter, P3, that is located downstream of P2. Efficient expression of P3 was dependent on phosphorylated DegU, as inactivation of the sensor kinase gene, degS, resulted in the loss of degU expression, although less efficient stimulation of degU expression was also observed with an enhanced level of DegU in a degS-deficient mutant. The promoter located upstream of the degSU operon, designated the P1 promoter here, was insensitive to glnA and degU mutations. These results suggest that degU expression is controlled by the three promoters under different growth conditions.

Participation of intercellular adhesion molecule-2 (CD102) in B lymphopoiesis.

The survival and fate of blood cell precursors is dependent on their communication with stromal cells of various types within bone marrow. Monoclonal antibodies have proven to be powerful tools for identifying molecules responsible for such interactions and we now describe one that selectively blocks B lymphopoiesis. The BF/32 antibody inhibited the establishment, but not the maintenance of long-term bone marrow cultures capable of lymphocyte production. However, there was no obvious effect on lymphocyte-stromal cell adhesion or responsiveness of pre-B cells to intereleukin-7. Furthermore, the reagent had no influence on myeloid precursors or myeloid bone marrow cultures. Injection of adult mice with BF/32 reduced B lineage precursors within bone marrow, but spared mature B cells. Moreover, the reagent did not alter responsiveness of mature B cells to activating stimuli. The 60 kDa protein recognized by this antibody was widely expressed on lymphocytes. Amino terminal protein sequencing and transfection experiments identified it as the murine homologue of ICAM-2 (CD102).

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

The Bacillus subtilis yrkP gene encodes a response regulator of a two-component regulatory system of unknown function. A previous DNA microarray experiment suggested that multicopy yrkP greatly enhanced the expression of yrkN, the ykcBC operon, and yrkO, which encodes a putative transporter. Here, lacZ fusion analysis confirmed these results and also revealed that YrkP autoregulates the putative yrkPQR operon, indicating that yrkPQR and yrkO form a divergon structure. In addition, real-time PCR analysis revealed that transcription of yrkO, yrkN, and ykcBC was significantly reduced in the yrkP strain. Hence, YrkP positively regulates the expression of these genes. Gel retardation analyses showed that YrkP bound to the promoter regions of yrkO, yrkN, and ykcB, albeit with lower binding affinities to the latter two promoters. The in vitro binding of YrkP to the promoter region of the yrkPQR and yrkO divergon was then analyzed by DNase I footprinting analysis. This revealed that YrkP recognizes three regions containing single-motifs or a direct repeat of the ten-base sequence [T/G]TCA[T/C]AAATT. lacZ fusion analysis of deleted and mutagenized promoter regions of yrkO and yrkPQR divergon confirmed that the three YrkP-binding regions are needed for the YrkP-mediated activation of yrkO and/or yrkPQR.

Controlled release of simvastatin acid using cyclodextrin inclusion system.

Simvastatin acid (SVA) has been reported to stimulate bone formation by increasing expression of BMP-2 in osteoblasts. Due to their multi-functional characteristics and bioadaptability, cyclodextrins (CDs) are capable of forming inclusion complexes with many drugs by including a whole drug molecule inside their cavity. In the present study, we prepared SVA/CD inclusion complex solutions with different pH values. These were then used to determine their SVA release behavior after coating on titanium substrates, as well as to clarify the characteristics of SVA/CD complexes per se. Results showed that the lower the pH value of the solution, the lower the release kinetics of SVA. Besides, the amount of crystalline complexes in the coatings increased with decrease in pH. These results suggested that the release rate of SVA depended on two factors: pH of the solution and concomitant crystallinity of the coating.

Oxygen plasma surface modification enhances immobilization of simvastatin acid.

Simvastatin acid (SVA) has been reported to stimulate bone formation with increased expression of BMP-2. Therefore, immobilization of SVA onto dental implants is expected to promote osteogenesis at the bone tissue/implant interface. The aim of this study was to evaluate the immobilization behavior of SVA onto titanium (Ti), O(2)-plasma treated titanium (Ti + O(2)), thin-film coatings of hexamethyldisiloxane (HMDSO), and O(2)-plasma treated HMDSO (HMDSO + O(2)) by using the quartz crystal microbalance-dissipation (QCM-D) technique. HMDSO surfaces were activated by the introduction of an OH group and/or O(2)-functional groups by O(2)-plasma treatment. In contrast, titanium surfaces showed no appreciable compositional changes by O(2)-plasma treatment. The QCM-D technique enabled evaluation even at the adsorption behavior of a substance with a low molecular weight such as simvastatin. The largest amount of SVA was adsorbed on O(2)-plasma treated HMDSO surfaces compared to untreated titanium, HMDSO-coated titanium, and O(2)-plasma treated titanium. These findings suggested that the adsorption of SVA was enhanced on more hydrophilic surfaces concomitant with the presence of an OH group and/or O(2)-functional group resulting from the O(2)-plasma treatment, and that an organic film of HMDSO followed by O(2)-plasma treatment is a promising method for the adsorption of SVA in dental implant systems.

The bisphosphonate pamidronate on the surface of titanium stimulates bone formation around tibial implants in rats.

Many materials with differing surfaces have been developed for clinical implant therapy in dentistry and orthopedics. We analyzed the quantity of new bone formed in vivo around calcium-immobilized titanium implants with surfaces modified using pamidronate (PAM), a nitrogen-containing bisphosphonate (N-BP), implants of pure titanium, and titanium implants immobilized with calcium ions. New bone formation was visualized using fluorescent labeling (calcein blue and alizarin complexone) with intravenous injection at 1 and 3 weeks after implantation. After 4 weeks, undecalcified sections were prepared, and new bone formation around the implants was examined by morphometry using confocal laser scanning microscopy images. After 1 week, more new bone formed around the PAM-immobilized implant than around the calcium-immobilized and pure titanium implants. This was also seen with the new bone formation after 3 weeks. After 4 weeks, significantly more new bones were formed around the BP-immobilized implant than around the calcium ion-implanted and pure titanium implants. The new N-BP-modified titanium surface stimulates new bone formation around the implant, which might contribute to the success of implant therapy.

Changes in the distribution of laminin-5 during peri-implant epithelium formation after immediate titanium implantation in rats.

Laminin-5 (Ln-5), a component of the basement membrane (BM), regulates epithelial cell migration and adhesion. This study used anti-Ln-5 (gamma2chain) antibody to investigate the distribution of Ln-5 during the formation of peri-implant epithelium (PIE) in rats, and compared it to the distribution of Ln-5 during oral mucosa formation after tooth extraction. One day after extraction, the junctional epithelium (JE) had disappeared. After 3 days, new epithelium formed from the oral sulcular epithelium (OSE) and extended horizontally over the wound with Ln-5-positive cells at the leading edge. After 5 days, the epithelium extending from the OSE on each side of the wound joined and formed additional new epithelium. The new epithelium expressed Ln-5 in the BM. After 1-2 weeks, the oral epithelium (OE) extending from the sides of the wound joined in the center. Thereafter, OSE and new epithelium disappeared, and only OE remained covering the wound. Three days after implantation (titanium), no JE remained. New epithelium formed from the keratinized OSE extending apically with Ln-5-positive cells. After 1-2 weeks, the new epithelium became the PIE and spread further apically facing the implant surface. Ln-5 was expressed at the PIE-connective tissue interface, but not at the implant-PIE interface. Finally, after 4 weeks, Ln-5 was expressed at the implant-PIE interface, and the PIE was non-keratinized epithelium. These findings suggest that Ln-5 induces cell migration during PIE formation, and that PIE originates from OSE. Furthermore, they support the hypothesis that Ln-5 contributes to the attachment of PIE to titanium, regardless of the delay in the synthesis and deposition of Ln-5 at the titanium-PIE interface.

Ultrastructural localization of laminin-5 (gamma2 chain) in the rat peri-implant oral mucosa around a titanium-dental implant by immuno-electron microscopy.

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (gamma2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.

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.

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease.

ISG20 is an interferon-induced antiviral exoribonuclease that acts on single-stranded RNA and also has minor activity towards single-stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5'-monophosphate (UMP) at 1.9 A resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh-group DNases, the epsilon subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2'-OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.

Recent progress in Bacillus subtilis two-component regulation.

Two-component regulatory systems serve to control gene expression in response to environmental and physiological changes. They are widespread among a variety of organisms and most often found in prokaryotes. One of the gram-positive microorganisms Bacillus subtilis is a well-studied bacterium whose complete nucleotide sequence has been determined. Thus, it is now possible to study transcription of the whole genome with microarray analysis. In this review we summarize the recent progress in B. subtilis two-component regulatory systems by describing the known systems and those for which the function was recently assigned. Also included is an attempt to construct a partial transcriptional network involving several two-component systems. The studies described here are based on the data from traditional genetics and biochemistry, and from microarray analysis of 29 two-component systems.

In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue.

Many studies have examined mineralized-tissue formation on titanium in vivo and in vitro; however, no precise method for measuring the mineralized tissue produced by cultured osteoblastic cells on titanium in vitro has been established. This study developed a method for measuring mineralized-tissue formation by cultured rat osteoblastic cells on titanium in vitro, and re-evaluated the effects of modifying the titanium surface. We used calcein blue, which binds to mineralized tissue, and measured the resulting fluorescence under ultraviolet light. A 1-h incubation with 3.1x10(-3)M calcein blue resulted in sufficient fluorescence of bone-like nodules on the titanium. Consequently, we found that osteoblastic cells produced larger bone-like nodules on titanium than in culture dishes. Fewer bone-like nodules formed on calcium-ion impregnated titanium than on pure titanium. However, when bisphosphonate was immobilized on the calcium-ion impregnated titanium, more bone-like nodules formed than on pure titanium. The results suggest that bisphosphonate immobilization on titanium is useful for stimulating mineralized-tissue formation on titanium implants.

Inhibition of Bacillus subtilis aprE expression by lincomycin at the posttranscriptional level through inhibition of ppGpp synthesis.

Expression of the Bacillus subtilis alkaline protease gene aprE is controlled by many positive and negative regulators at the transcriptional level. During the course of screening for organic compounds that affect the expression of a translational aprE'-'lacZ fusion, we found that lincomycin (Lm), erythromycin and chloramphenicol exhibited an inhibitory effect in concentrations that hardly affected cell growth. The antibiotics are known to inhibit protein synthesis by binding to ribosomes. We chose one of them, Lm, for further study. We have previously shown that aprE expression requires guanosine 3',5'-bisdiphosphate (ppGpp) synthesized on the ribosome by the stringent factor RelA. An examination of Lm-treated cells showed that the levels of ppGpp were greatly reduced in these cells, and the inhibitory effect of the antibiotic was not seen in relA-disruption mutants. Transcriptional levels of aprE, however, were not influenced by Lm treatment as shown by using a transcriptional aprE-lacZ fusion as well as quantitative RT-PCR. Furthermore, disruption of relA did not affect the expression of transcriptional aprE-lacZ. From these results, we conclude that aprE expression is controlled by the stringent control at the posttranscriptional level, and that Lm inhibits this process by inhibiting ppGpp synthesis on the ribosome.