Biomedical subjects

M M Nakano

Publications and source records attributed to M M Nakano.

At least 19 recordsLinked to original sources

PubMed2001

Loss-of-function mutations in yjbD result in ClpX- and ClpP-independent competence development of Bacillus subtilis.

Mutations in clpP and clpX have pleiotropic effects on growth and developmentally regulated gene expression in Bacillus subtilis. ClpP and ClpX are needed for expression of comK, encoding the competence transcription factor required for the expression of genes within the competence regulon. ClpP, in combination with the ATPase ClpC, degrades the inhibitor of ComK, MecA. Proteolysis of MecA is stimulated by a small protein, ComS, which interacts with MecA. Suppressor mutations (cxs) were isolated that bypass the requirement for clpX for comK expression. These were found also to overcome the defect in comK expression conferred by a clpP mutation. These mutations were identified as missense mutations (cxs-5, -7 and -12) and a nonsense (UAG) codon substitution (cxs-10) in the yjbD coding sequence in a locus linked to mecA. That a yjbD disruption confers the cxs phenotype, together with its complementation by an ectopically expressed copy of yjbD, indicated that the suppressor alleles bear recessive, loss-of-function mutations of yjbD. ClpP- and ClpX-independent comK expression rendered by inactivation of yjbD was still medium-dependent and required ComS. MecA levels in a clpP-yjbD mutant were lower that those of clpP mutant cells and ComK protein concentration in the clpP mutant was restored to wild-type levels by the yjbD mutation. Consequently, the yjbD mutation bypasses the defect in competence development conferred by clpP and clpX. YjbD protein is barely detectable in wild-type cells, but is present in large amounts in the clpP mutant cells. The results suggest that the role of ClpP in competence development is to degrade YjbD protein so that ComS can productively interact with the MecA-ClpC-ComK complex. Alternatively, the result could suggest that YjbD has a negative effect on regulated proteolysis and that MecA is degraded independently of ClpP when YjbD is absent.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2001

Involvement of ResE phosphatase activity in down-regulation of ResD-controlled genes in Bacillus subtilis during aerobic growth.

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in Bacillus subtilis. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognate response regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD is postulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic conditions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defective in ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes. The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobic induction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaerobiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests that the ResE kinase activity is also subject to control in response to oxygen limitation.

Aerobiosis↗

PubMed2000

CcpC, a novel regulator of the LysR family required for glucose repression of the citB gene in Bacillus subtilis.

Synergistic carbon catabolite repression of the Bacillus subtilis aconitase (citB) gene by glucose and a source of 2-ketoglutarate is dependent on DNA sequences located upstream of the gene. Mutations in a dyad symmetry element centered at position -66 and in a repeat of the downstream arm of the dyad symmetry at position -27 cause derepressed citB expression. In this work, a protein able to bind to a DNA fragment containing these elements was purified and identified. This protein, named CcpC (Catabolite control protein C), shares sequence similarity with members of the LysR family of transcriptional regulators. In addition to binding to the citB promoter, CcpC bound to the promoter of the citZ gene, which encodes the cell's major citrate synthase and is subject to carbon catabolite repression. In a ccpC null mutant, expression of both citB and citZ was derepressed in glucose-glutamine minimal medium, indicating that CcpC is a negative regulator of citB and citZ gene expression. DNase I footprinting experiments showed that CcpC binds to two sites within the citB promoter region, corresponding to the dyad symmetry and -27 elements. In the presence of citrate, a putative inducer, only the dyad symmetry element was fully protected by CcpC. When the dyad symmetry element was mutated, CcpC was no longer able to bind to either the dyad symmetry or -27 elements. Repression of citB and citZ gene expression during anaerobiosis also proved to be mediated by CcpC.

Aconitate Hydratase↗

PubMed2000

Mutations conferring amino acid residue substitutions in the carboxy-terminal domain of RNA polymerase alpha can suppress clpX and clpP with respect to developmentally regulated transcription in Bacillus subtilis.

The Bacillus subtilis clpX and clpP genes are the sites of pleiotropic mutations that adversely affect growth on a variety of media and impair developmental processes such as sporulation and competence development. ClpX is necessary for the post-exponential induction of genes that require the sigmaH form of RNA polymerase for transcription. Both ClpX and ClpP are required for the activation of sigmaA-dependent transcription of the srf operon that encodes surfactin synthetase and the regulatory peptide ComS, required for the development of genetic competence. Transcription of srf is activated by the two-component regulatory system ComPA in response to the peptide pheromone, ComX, which mediates cell density-dependent control. A clpX mutant, although able to produce ComX, is unable to respond to the pheromone. A mutant allele of comP, encoding a product whose activity is independent of ComX, is not able to suppress clpX with respect to srf expression, suggesting that ClpXP acts at the level of ComA-dependent activation of srf transcription initiation. Suppressor mutations of clpX (cxs-1 and cxs-2) were isolated in screens for pseudorevertants exhibiting high levels of srf expression and sigmaH-dependent transcription respectively. One mutation, cxs-1, suppressed a clpP null mutation with respect to srf transcription, but did not overcome the block conferred by clpP on competence development and sporulation. Both cxs-1 and cxs-2 mutations map to the region of the rpoA gene encoding the RNA polymerase alpha C-terminal domain (alphaCTD). The reconstruction of the cxs-1 and cxs-2 alleles of rpoA confirmed that these mutations confer the suppressor phenotype. These findings provide further support for the hypothesis that ClpX and ClpP might be intimately associated with transcription initiation in B. subtilis.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2000

Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis.

The two-component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE-dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis-acting sequences required for ResDE-dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE-dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.

ATP-Binding Cassette Transporters↗

PubMed2000

Dual control of sbo-alb operon expression by the Spo0 and ResDE systems of signal transduction under anaerobic conditions in Bacillus subtilis.

The Bacillus subtilis sbo-alb operon contains sboA, the structural gene for the bacteriocin subtilosin, and the alb genes required for subtilosin production. Transcription from the sbo-alb promoter is highly induced by oxygen limitation. The transcriptional regulation of the sbo-alb operon is under dual control involving the transition state regulator AbrB and the two-component regulatory proteins ResD and ResE.

Anaerobiosis↗

PubMed2000

[Presence of larva migrans in sand boxes of public elementary schools, Araçatuba, Brazil].

There are sandboxes in public elementary school playground areas in Brazil, which can be harmful to children. They are at risk of cutaneous and visceral larva migrans infection caused by Ancylostoma spp. and Toxocara spp., respectively. The study was designed to investigate contamination by Toxocara spp. and/or their eggs and Ancylostoma spp. larvae in sand samples collected from the schools' sandboxes. Five hundred and thirty-five sand samples from 28 public elementary schools were collected during summer and winter and analyzed by both Baerman's method and centrifugal flotation technique. Ancylostoma spp. larvae were found in 35.7% (10/28) schools in summer time and in 46.4% (13/28) schools in the winter time. Eggs of Toxocara spp. could not be recovered from the samples analyzed and eggs from Ancylostoma spp. were seen in 0.56% (3/535) of the samples.

Ancylostoma↗

PubMed1999

Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis.

The development of Bacillus subtilis genetic competence is a highly regulated adaptive response to stationary-phase stress. A key step in competence development is the activation of the transcriptional regulator ComK, which is required for the expression of genes encoding the products that function in DNA uptake. In log-phase cultures, ComK is trapped in a complex composed of MecA and ClpC, in which it is rendered inactive. The comS gene, contained within the srf operon, is induced in response to high culture cell density and nutritional stress. Its product functions to release active ComK from the complex, allowing ComK to stimulate the transcription initiation of its own gene as well as that of the late competence operons. Western analysis showed that ComS accumulates to maximal levels between T3 and T4, mirroring the pattern of competence cell development and late competence gene expression. Experiments to examine the target of ComS activity in vitro showed that ComS binds to MecA. This is further supported by coimmunoprecipitation using anti-MecA antiserum. To clarify the role of ComS in competence regulation, a system for evaluating the effect of comS and mutant derivatives on the expression of comG, one of the late competence operons, was constructed. comS mutations, created by alanine-scanning mutagenesis, that significantly reduced comG-lacZ expression were clustered within two regions, one at the N-terminus and the other at the C-terminus of ComS. ComSI13 --> A and ComSW43 --> A were selected for further analysis as representative mutants for both regions required for ComS activity. We observed that ComSI13 --> A showed significantly reduced affinity for MecA, whereas ComSW43 --> A showed near normal binding affinity for MecA. The results show that binding to MecA is critical for ComS function, but do not rule out the possibility that ComS possesses other activities.

Alleles↗

PubMed1999

A mutation in the 3-phosphoglycerate kinase gene allows anaerobic growth of Bacillus subtilis in the absence of ResE kinase.

The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1, 3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.

Aerobiosis↗

PubMed1998

Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

Amino Acid Sequence↗

PubMed1998

Anaerobic regulation of Bacillus subtilis Krebs cycle genes.

Krebs cycle enzyme activity in Bacillus subtilis was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions. The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures. These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription. This repression was not dependent on either the fnr or resDE gene products, which have been shown to regulate expression of other B. subtilis genes in response to anaerobic conditions. Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression. A dyad symmetry element located between positions -73 and -59 relative to the transcription start site of the aconitase gene (citB) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth. The deletion of the upstream arm of the dyad symmetry region abolished the citB repression observed during anaerobic growth. Furthermore, neither citZ or citB was repressed in an anaerobically grown citB mutant, an effect that was very likely the result of citrate accumulation. These results suggest that catabolite repression and anaerobic repression of citZ and citB are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.

Aconitate Hydratase↗

PubMed1998

Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.

The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.

Aerobiosis↗

PubMed1998

Anaerobic growth of a "strict aerobe" (Bacillus subtilis).

There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

Anaerobiosis↗

PubMed1997

Adaptation of Bacillus subtilis to oxygen limitation.

Bacillus subtilis grows anaerobically by at least two different pathways, respiration using nitrate as an electron acceptor and fermentation in the absence of electron acceptors. Regulatory mechanisms have evolved allowing cells to shift to these metabolic capabilities in response to changes in oxygen availability. These include transcriptional activation of fnr upon oxygen limitation, a process requiring the ResD-ResE two-component signal transduction system that also regulates aerobic respiration. FNR then activates transcription of other anaerobically induced genes including the narGHJI operon which encodes a respiratory nitrate reductase. Genes involved in fermentative growth are controlled by an unidentified FNR-independent regulatory pathway.

Adaptation, Physiological↗

PubMed1997

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.

ATP-Dependent Proteases↗

PubMed1996

Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.

Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor. The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis. Unlike Escherichia coli fnr, B. subtilis fnr transcription was shown to be highly induced by oxygen limitation. fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon. DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon. ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis. In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac. The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B. subtilis anaerobic gene regulation.

Anaerobiosis↗

PubMed1996

Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis.

A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions. The hmp protein belongs to the family of two-domain flavohemoproteins, homologs of which have been isolated from various organisms such as Escherichia coli, Alcaligenes eutrophus, and Saccharomyces cerevisiae. These proteins consist of an amino-terminal hemoglobin domain and a carboxy-terminal redox active site domain with potential binding sites for NAD(P)H and flavin adenine dinucleotide. The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI. The requirement of FNR and NarGHJI for hmp expression is completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for transcriptional activation of narGHJI, which produces nitrite, leading to induction of hmp expression. In contrast, induction of hmp was still dependent on resDE in the presence of nitrite. A defect in hmp in B. subtilis has no significant effect on anaerobic growth.

Anaerobiosis↗

PubMed1996

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Bacillus subtilis↗