Biomedical subjects

Kozo Ochi

Publications and source records attributed to Kozo Ochi.

At least 37 records · Page 2Linked to original sources

PubMed2003

An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2).

Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.

Amino Acid Substitution↗

PubMed2003

Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus.

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str(r)), gentamicin, or rifampin (Rif(r)). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str(r) mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif(r) mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase beta subunit). Increased productivity of salinomycin in the Str(r) mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg(2+) concentrations. We conclude that this aberrant protein synthesis ability in the Str(r) mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.

Anti-Bacterial Agents↗

PubMed2003

Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans.

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.

Amino Acid Sequence↗

PubMed2003

Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2).

We found that a 46-kDa protein is highly expressed in an actinorhodin-overproducing Streptomyces coelicolor A3(2) mutant (KO-179), which exhibited a low-level resistance to streptomycin. The protein was identified as S-adenosylmethionine (SAM) synthetase, which is a product of the metK gene. Enzyme assay revealed that SAM synthetase activity in strain KO-179 was 5- to 10-fold higher than in wild-type cells. The elevation of SAM synthetase activity was found to be associated with an increase in the level of intracellular SAM. RNase protection assay revealed that the metK gene was transcribed from two distinct promoters (p1 and p2) and that enhanced expression of the MetK protein in the mutant strain KO-179 was attributed to elevated transcription from metKp2. Strikingly, the introduction of a high-copy-number plasmid containing the metK gene into wild-type cells resulted in a precocious hyperproduction of actinorhodin. Furthermore, the addition of SAM to the culture medium induced Act biosynthesis in wild-type cells. Overexpression of metK stimulated the expression of the pathway-specific regulatory gene actII-ORF4, as demonstrated by the RNase protection assay. The addition of SAM also caused hyperproduction of streptomycin in Streptomyces griseus. These findings implicate the significant involvement of intracellular SAM in initiating the onset of secondary metabolism in STREPTOMYCES:

Amino Acid Sequence↗

PubMed2003

RNase ES of Streptomyces coelicolor A3(2) can complement the rne and rng mutations in Escherichia coli.

The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.

Endoribonucleases↗

PubMed2003

Expression profiling of translation-associated genes in sporulating Bacillus subtilis and consequence of sporulation by gene inactivation.

A DNA microarray technique was used to demonstrate global changes in the transcription pattern of translation-associated genes that encode fifty-four ribosomal proteins including a putative ribosomal gene, and eleven translation factors in sporulating B. subtilis. We found that the mRNA levels of nine genes involved in the translation system, which include the genes for three ribosomal proteins (rpmA, rpmGB, and ctc) and two translation factors (efp, and frr), were maintained at a high level at the onset of sporulation. The ypfD gene, which encodes the ribosomal protein S1 homologue, was also found to be expressed significantly during the early sporulation stage. In order to demonstrate the significance of these genes for sporulation, mutants were constructed using the pMutinT3 disruption vector. We detected an impaired sporulation in the mutants of rpmA (gene for the ribosomal protein L27), efp (elongation factor P), frr (ribosome recycling factor), and ypfD. The effect was especially pronounced in the efp mutant, sporulation of which was entirely abolished without affecting growth. The reduced expression of rpmGB (ribosomal protein L33) resulted in an impaired sporulation only at a high temperature (47 degrees C). Only the rplI mutant, which encodes the ribosomal protein L9, could not be obtained, implying that its function is essential for viability. Thus, we successfully demonstrated the significance of several translation-associated genes in sporulation by using the results of the gene expression profiling.

Bacillus subtilis↗

PubMed2003

In vitro susceptibility and a new point mutation associated with tylosin-resistance in Japanese canine intestinal spirochetes.

The in vitro susceptibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 microg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.

Animals↗

PubMed2002

Genetic and biochemical characterization of EshA, a protein that forms large multimers and affects developmental processes in Streptomyces griseus.

The 52-kDa protein, EshA, whose expression is controlled developmentally, is produced during the late growth phase of Streptomyces spp. We found that disruption of the eshA gene, which encodes the EshA protein, abolishes the aerial mycelium formation and streptomycin production in Streptomyces griseus when grown on an agar plate. The eshA disruptant KO-390 demonstrated a reduced amount of expression of the transcriptional activator strR, thus accounting for the failure to produce streptomycin. KO-390 was found to accumulate deoxynucleoside triphosphates at high levels, including dGTP, at late growth phase. The accumulation of dGTP was a cause for the impaired ability of KO-390 to produce aerial mycelium, because the ability to form aerial mycelium was completely repaired by addition of decoyinine, an inhibitor of GMP synthetase. The accumulation of dNTP in KO-390 coincided with a reduced rate of DNA synthesis. The developmental time frame of these phenomena in KO-390 matched a burst of EshA expression in the wild-type strain. In contrast to S. griseus, the eshA disruption did not affect the ability for Streptomyces coelicolor to form aerial mycelium and did not result in the aberrant accumulation of dNTP accompanied by arrest of DNA synthesis, implying qualitative differences in addition to quantitative differences between the two EshA proteins. We propose that the S. griseus EshA protein somehow positively affects (or regulates) the replication of DNA in wild-type cells at late growth phase but leads to aberrant phenotypes in mutant cells due to the disturbed DNA replication. The EshA protein was found to exist as a multimer ( approximately 20-mers) creating a cubic-like structure with a diameter of 27 nm and located predominantly in cytoplasm.

Adenosine↗

PubMed2002

A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro.

A gene encoding a putative guanosine 3',5'-bispyrophosphate (ppGpp) synthase-degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH2-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA-SpoT family of proteins. Expression of an NH2-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of 32P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase-degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.

Amino Acid Sequence↗

PubMed2002

Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis.

We found that a polycistronic operon (ywfBCDEFG) and a monocistronic gene (ywfH) are required for the biosynthesis of bacilysin in Bacillus subtilis. The disruption of these genes by plasmid integration caused loss of the ability to produce bacilysin, accompanied by a lack of bacilysin synthetase activity in the crude extract. We investigated the regulatory mechanism for bacilysin biosynthesis using the transcriptional lacZ fusion system. The transcription of these genes was found to be induced at the transition from exponential to stationary phase. Induction of transcription was accelerated by depleting a required amino acid, which was done by transferring the wild-type (rel(+)) cells to an amino acid-limited medium. In contrast, no enhancement of the gene expression was detected in relA mutant cells. In wild-type (rel(+)) cells, a forced reduction of intracellular GTP, brought about by addition of decoyinine, which is a GMP synthetase inhibitor, enhanced the expression of both the ywfBCDEFG operon and the ywfH gene, resulting in a 2.5-fold increase in bacilysin production. Disruption of the codY gene, which regulates stationary phase genes by detecting the level of GTP, also induced transcription of these genes. In contrast, the expression of ywfBCDEFG in relA cells was not activated either by decoyinine addition or codY disruption, although the expression of ywfH was induced. Moreover, the codY disruption resulted in an increase of bacilysin production only in rel(+) cells. These results indicate that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) plays a crucial role in transcription of the ywfBCDEFG operon and that the transcription of these genes are dependent upon the level of intracellular GTP which is transmitted as a signal via the CodY-mediated repression system. We propose that, unlike antibiotic production in Streptomyces spp., bacilysin production in B. subtilis is controlled by a dual regulation system composed of the guanine nucleotides ppGpp and GTP.

Adenosine↗

PubMed2002

Streptomycin-resistant (rpsL) or rifampicin-resistant (rpoB) mutation in Pseudomonas putida KH146-2 confers enhanced tolerance to organic chemicals.

We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA). The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene. The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12. The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit. Mutation points in Gen mutants still remain unknown. Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA. Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance. Uptake experiments using [14C]-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA. Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.

Adaptation, Physiological↗

PubMed2002

Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans.

Antibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like 'stringent' RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.

Amino Acids↗

PubMed2002

Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168.

We have found a novel phospholipid antibiotic (named bacilysocin) which accumulates within (or associates with) the cells of Bacillus subtilis 168 and determined the structure by nuclear magnetic resonance and mass spectrometry analyses. The structure of bacilysocin elucidated was 1-(12-methyltetradecanoyl)-3-phosphoglyceroglycerol. Bacilysocin demonstrated antimicrobial activity, especially against certain fungi. Production of bacilysocin commenced immediately after growth ceased and before the formation of heat-resistant spores. The production of bacilysocin was completely blocked when the ytpA gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the ytpA gene did not significantly affect growth. Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected. Since the ytpA disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin.

Anti-Bacterial Agents↗

PubMed2002

RelA protein is involved in induction of genetic competence in certain Bacillus subtilis strains by moderating the level of intracellular GTP.

We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA+) strain. Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain. We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography. The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase). Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development. Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression. Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation.

Adenosine↗

PubMed2002

Activation of antibiotic biosynthesis by specified mutations in the rpoB gene (encoding the RNA polymerase beta subunit) of Streptomyces lividans.

We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the rpoB gene that confer resistance to rifampin to Streptomyces lividans 66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the rpoB gene was shown to be dependent on the mutation's position and the amino acid species substituted in the beta-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the rpoB gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of actII-ORF4 and redD, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the rpoB gene of S. lividans, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes, actII-ORF4 and redD. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in STREPTOMYCES:

Amino Acid Sequence↗

PubMed2002

Isolation and identification of novel ADP-ribosylated proteins from Streptomyces coelicolor A3(2).

An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.

Adenosine Diphosphate Ribose↗

PubMed2002

OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase.

A rice gene, OsRALyase1, encoding a product similar to wheat ribosomal RNA apurinic site specific lyase (RALyase), was isolated and expressed in vitro. An open reading frame of the gene predicted a protein of 476 amino acid residues with 75% identity to RALyase and contained an F-box-like motif in its amino terminal region. The rice gene product expressed in a wheat-germ protein expression system had the same characteristics as its wheat counterpart, cleaving a specific depurinated site of the 28S rRNA sarcin-ricin domain.

Amino Acid Motifs↗

PubMed1999

Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.

4-Butyrolactone↗