Biomedical subjects

M Asayama

Publications and source records attributed to M Asayama.

At least 19 recordsLinked to original sources

PubMed1999

Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells.

The psbA2 gene exhibits light-dependent and rhythmic expression in a unicellular cyanobacterium, Microcystis aeruginosa (Synechocystis) K-81. To further understand the psbA2 expression, biological analyses were performed in homologous and heterologous cyanobacterial cells. The results of the experiments using the K-81 cells revealed that (i) the light-dependent expression appeared on transcriptional and/or post-transcriptional level(s) under light/dark cycles, (ii) circadian-rhythmic transcripts were also observed under the control of an endogenous clock. To assess whether light-dependent and rhythmic psbA2 expression occurs in heterologous cyanobacterium, Synechococcus sp. strain PCC 7942, the K-81 psbA2 5'-upstream region of which the promoter and its around sequences share with those of PCC 7942 psbAII, was fused to the bacterial lacZ reporter gene, introduced into the genome of PCC 7942 and the psbA2 transcripts were directly investigated by primer extension. The K-81 psbA2 specific transcripts were also light-dependent and rhythmic in PCC 7942, strongly demonstrating that a common regulatory mechanism exists per se for the psbA2 expression in both strains. Furthermore, psbA2 expression in the recombinant PCC 7942 strain, AG400 in which the region from -404 to +111 of psbA2 is fused to lacZ, exhibited clear rhythmicity, while very little or no rhythmicity was observed in AG429 (-38 to +14, the only promoter region), suggesting that the region(s) around the promoter was essentially required for clear rhythmic expression.

Cyanobacteria↗

PubMed1999

An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

Bacterial Proteins↗

PubMed1999

Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.

Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.

Amino Acid Sequence↗

PubMed1998

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

Base Sequence↗

PubMed1998

Translational attenuation of the Bacillus subtilis spo0B cistron by an RNA structure encompassing the initiation region.

The spo0B gene, which exists as an operon with the obg gene, is required to initiate sporulation (stage 0) of Bacillus subtilis . This gene encodes a phosphotransferase in the multicomponent phosphorelay system. We here report the novel finding that a spo0B 5'-terminal SLR (stem-loop structure sequestering ribosome binding sequence; ACUCCUAA-X16-UUG GGAG U, Delta G = -8.71 kcal/mol) attenuated spo0B translation. The spo0B gene was efficiently transcribed but Spo0B protein was not overproduced in Escherichia coli when spo0B was induced using expression vectors carrying the SLR- spo0B region under control of the tac promoter. Deletion of the SLR from the vectors resulted in overexpression of spo0B . Therefore, to characterize expression of spo0B with a SLR in B.subtilis we constructed transcriptional and translational lacZ fusions combined with the spo0B 5'-terminal region with a deleted or mutagenized SLR. These constructs were subsequently introduced into B.subtilis as multiple and single copies, then beta-galactosidase activities were measured. The possible SLR also functioned as a negative cis element in B.subtilis. Furthermore, B.subtilis strain 1S16 (spo0B136) lysogenized straight phiCD0B-S and -W, harboring spo0B with mutagenized SLRs that were more (Delta G = -14.0 kcal/mol) and less-stable (Delta G = -1.31 kcal/mol) compared with the wild-type, exhibited null and wild-type sporulation respectively. These results indicate that the spo0B 5'-SLR affects spo0B gene expression for sporulation but that low expression of spo0B through the wild-type SLR was sufficient to initiate sporulation in B.subtilis.

Amino Acid Sequence↗

PubMed1997

A new sigma factor homolog in a cyanobacterium: cloning, sequencing, and light-responsive transcripts of rpoD2 from Microcystis aeruginosa K-81.

We isolated an rpoD2 gene encoding the potential sigma factor of RNA polymerase from the cyanobacterium Microcystis aeruginosa K-81, which can perform photosynthesis. The deduced amino acid sequence of RpoD2 (sigmaA2) exhibits extensive homology to other eubacterial RpoD proteins. This gene possessed multiple 5'-end transcripts, expressed specifically under light (P(L)), dark (P(D)), or constitutively light/dark (P(C)) conditions during exponential cell growth.

Amino Acid Sequence↗

PubMed1997

A novel bend of DNA CIT: changeable bending-center sites of an intrinsic curvature under temperature conditions.

We found a novel DNA curvature, which has changeable bending-center sites of an intrinsic curvature under temperature conditions (CIT) in the cyanobacterium strain Microcystis aeruginosa K-81. Circular permutation analyses (CPA) for CIT under different temperature conditions (4-50 degrees C) revealed that the changeable bending-center sites are located in the 5'-upstream region (-141 to -184) of the psbA2 gene, encoding the D1 protein homolog for photosynthesis. The nucleotide sequence around the bending center contains several dT (deoxy thymine) tracts, which seem to be a pivotal determinant for CIT.

Antiviral Agents↗

PubMed1997

A novel genetic organization: the leuA-rpoD1 locus in the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the region upstream of rpoD1, which encodes a principal sigma factor in the cyanobacterium Microcystis aeruginosa K-81. An open reading frame (orf1, 1599 bp) was discovered, the deduced amino-acid sequence of which (533 aa, 58, 016 Da) exhibits homology to another bacterial leuA gene product, 2-isopropylmalate synthase. The leuA (orf1) gene specifically complemented an E. coli leuA mutant. The 5'-upstream region of leuA did not contain possible leader peptide or stem-loop structures for attenuation. These findings indicate that the genetic structure of the leuA-rpoD1 locus in M. aeruginosa K-81 significantly differs from those of known leuA and rpoD loci found in other bacteria.

Amino Acid Sequence↗

PubMed1996

Cloning, sequencing and characterization of the gene encoding a principal sigma factor homolog from the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the rpoD1 gene of Microcystis aeruginosa K-81, a unicellular colony-forming cyanobacterium that can perform photosynthesis involving light-responsive gene expression. The deduced amino acid sequence of RpoD1 exhibited extensive homology to the other eubacterial principal sigma factors. Primer extension and Western blot analyses revealed that the rpoD1 gene, which encodes a principle sigma factor homolog, had two transcription start points, P1 and P2. These transcripts, and the corresponding protein, constitutively appeared in M. aeruginosa, irrespective of light or dark conditions.

Amino Acid Sequence↗

PubMed1996

Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp.

The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonucleases. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, MaeK81I and MaeK81II, which were isoschizomers of SplI and Sau96I, respectively.

Bacterial Proteins↗

PubMed1996

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains.

Microcystis aeruginosa (Synechocystis) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.

Amino Acid Sequence↗

PubMed1996

The rpoD1 gene product is a principal sigma factor of RNA polymerase in Microcystis aeruginosa K-81.

We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs). We overproduced and purified RpoD1 (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M. aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth.

Amino Acid Sequence↗

PubMed1996

The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome.

The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.

Bacteriophages↗

PubMed1995

Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.

Bacillus subtilis↗

PubMed1993

Signal transduction and sporulation in Bacillus subtilis: autophosphorylation of Spo0A, a sporulation initiation gene product.

Spo0A is a positive/negative transcriptional regulator that plays a very important role in sporulation initiation in Bacillus subtilis. The N-terminal amino acid sequence of Spo0A is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria. Phosphorylation of SpooA through a phosphorelay has been reported recently. In this study, we found that Spo0A is autophosphorylated in the presence of ATP and that an autophosphorylation-deficient Spo0A mutant is completely defective in initiating sporulation. These results suggest that Spo0A autophosphorylation is an essential event in the signal transduction process that controls sporulation in B. subtilis.

Amino Acid Sequence↗

PubMed1993

Signal transduction and sporulation in Bacillus subtilis: heterologous phosphorylation of Spo0A, a sporulation initiation gene product.

Spo0A is both a positive and a negative transcriptional regulator which plays a very important role in sporulation initiation in Bacillus subtilis. Its N-terminal amino acid sequence is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria. Phosphorylation of Spo0A through phosphorelay has been reported by Burbulys et al. (1991). In this study, we found that (i) Spo0A is phosphorylated effectively with phospho-EnvZ* (N-terminal truncated EnvZ), which is a heterologous osmotic sensor protein in Escherichia coli, and (ii) a phosphorylation deficient mutant of Spo0A protein is completely defective in initiating sporulation. These results suggest that Spo0A phosphorylation may be an essential event in signal transduction of sporulation in B. subtilis and the signal transduction mechanism has a common feature in Gram-positive and Gram-negative bacteria.

Bacillus subtilis↗

PubMed1993

Characteristics of DNA and multiple rpoD homologs of Microcystis (Synechocystis) strains.

The base compositions of DNAs from nine Microcystis strains, as determined by high-performance liquid chromatography, were 41 to 42 mol% G+C. Chromosomal DNAs derived from these strains were found to be extremely resistant to many restriction endonucleases, and a restriction analysis revealed the presence of a dam-like methylase or both dam- and dcm-like methylases in all of the strains examined. Genomic Southern hybridization in which a synthetic oligonucleotide probe (rpoD probe) was used showed that members of the genus Microcystis might have multiple rpoD homologs, and the hybridization signal patterns observed with the DNAs of Microcystis aeruginosa strains were different from each other.

Bacterial Proteins↗

PubMed1988

Effect of facial cooling during heat acclimation process on adaptive changes in sweating activity.

On assumptions that tympanic temperature (Tty) reflects brain temperature and that the latter can be lowered by cooling of the face, effect of facial cooling during acclimation process on adaptive changes in sweating activity was examined, in comparison with the results of our previous studies on heat acclimation with controlled hyperthermia. Face fanning, by which Tty was clamped at approximately 37.1 degrees C, was combined with either of the following 9-day acclimation procedures: 90-min heating in a "Sauna box," keeping mean skin temperature slightly above 40 C, or 90-min exercise on a bicycle, clamping rectal temperature (Tre) at approximately 38 degrees C. Each procedure was imposed on the same four male subjects on different occasions, two of whom had participated in our previous experiments. Sweat tests, carried out before and immediately after the completion of the procedure, consisted of measurements of local sweat rates, whole body sweat rate, Tre, Tty, and skin temperatures on 5 areas, and of calculations of mean body temperature (Tb) and the rate of sweat expulsions (Fsw, as an indicator of central sudomotor activity). No or only a slight increase in sweating activity was observed following the acclimation procedures with face fanning, whereas similar procedures without face fanning had resulted in substantial enhancement of sweating activity in most of the cases, which had been attributed mainly to adaptive changes in central sudomotor activity (as indicated by a shift of the regression line relating Fsw to Tb). Similar results were obtained in an additional series of experiments, where the effects of 9-day 90-min exercise in heat, clamping Tre at approximately 38.2 degrees C, with and without facial cooling, were compared with each other in a subject. From the above results it is inferred that Tty reflects brain temperature and that enhancement of sweating activity induced by repeated heat load is strongly impeded, if not accompanied, by an elevation of brain temperature.

Acclimatization↗