Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa.

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.

Isolation, and biological properties of a new cell cycle inhibitor, curvularol, isolated from Curvularia sp. RK97-F166.

A new cell growth inhibitor, curvularol, was isolated from the fermentation broth of Curvularia sp. RK97-F166. Curvularol showed no antibacterial activity, and very weak antifungal activity. However, curvularol inhibited the cell cycle progression of normal rat kidney (NRK) cells in G1 phase at 150 ng/ml. Curvularol induced the morphological reversion of srcts-transformed NRK cells at 100 ng/ml, and inhibited protein synthesis same as cycloheximide.

Characterization of PBZ1, a probenazole-inducible gene, in suspension-cultured rice cells.

Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dose-dependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide.

Glutamine synthetase gene expression at elevated hydrostatic pressure in a deep-sea piezophilic Shewanella violacea.

A glutamine synthetase gene (glnA) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb SacI fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli. Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by sigma(70) and sigma(54) were identified in the region upstream of glnA. An electrophoretic mobility shift assay demonstrated that S. violacea sigma(54) specifically binds to the promoter region of glnA, suggesting that sigma(54) may play an important role in pressure-regulated transcription in this piezophilic bacterium.

Structural analysis of the ntrBC genes of deep-sea piezophilic Shewanella violacea.

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.

Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and parabasalian symbionts in the hindgut of termites.

The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Pressure regulation of soluble cytochromes c in a deep-Sea piezophilic bacterium, Shewanella violacea.

Two c-type cytochromes from the soluble fraction of a deep-sea moderately piezophilic bacterium, Shewanella violacea, were purified and characterized, and the genes coding for these cytochromes were cloned and sequenced. One of the cytochromes, designated cytochrome c(A), was found to have a molecular mass of approximately 8.3 kDa, and it contained one heme c per molecule. The other, designated cytochrome c(B), was found to have a molecular mass of approximately 23 kDa, and it contained two heme c molecules per protein molecule. The amount of cytochrome c(B) expressed in cells grown at high hydrostatic pressure (50 MPa) was less than that in cells grown at atmospheric pressure, whereas cytochrome c(A) was constitutively expressed under all pressure conditions examined. The results of Northern blotting analysis were consistent with the above-mentioned observations and suggested that the pressure regulation of cytochrome c(B) gene expression occurred at the transcriptional level. These results suggest that the components of the respiratory chain of moderately piezophilic S. violacea could be exchanged according to the growth pressure conditions.

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus.

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218-221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences.

Culture-independent characterization of a gene responsible for nitrogen fixation in the symbiotic microbial community in the gut of the termite Neotermes koshunensis.

Expression of the nitrogen fixation gene, nifH, in the gut of the termite Neotermes koshunensis was characterized without cultivation. nifH cDNA was directly amplified from mRNA of the mixed microbial population in the gut by reverse transcription (RT)-PCR. Analyses of the RT-PCR products revealed that, among the diverse nifH sequences, only a few corresponding to an alternative nitrogenase (encoded by the anf gene) were preferentially transcribed in the termite gut. Expression of the anf gene was further investigated quantitatively under several termite feeding conditions by competitive PCR. The levels of expression of the anf gene were largely congruent with the nitrogen fixation activity displayed by the termite. The amounts of the genomic anf gene in the population showed no significant change, indicating that the level of expression was critical for nitrogen fixation activity. Interestingly, no significant decrease in the expression level was observed when the diet contained molybdenum (Mo), which represses ordinary anf genes. A 3.6-kb DNA region downstream of the anf gene was isolated and found to contain reading frames homologous to anfH, anfD, and anfG of the Bacteria domain which encode subunits of an alternative nitrogenase having no Mo as a cofactor. This DNA region also contained reading frames encoding glnB-like proteins, which is a common feature of the nitrogenase genes of the Archaea domain. These results indicate that the anf group of nitrogenase genes is the most important group of genes responsible for nitrogen fixation in N. koshunensis and that the anf gene possesses novel features with respect to the regulation of its expression and its gene organization.

Outer membrane changes in a toluene-sensitive mutant of toluene-tolerant Pseudomonas putida IH-2000.

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant Pseudomonas putida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by insertion of Tn5 was identified as cyoC, which is one of the subunits of cytochrome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.

Production of Biodegradable Polyester by a Transgenic Tobacco.

The acetoacetyl-CoA reductase gene (phbB) of Ralstonia eutropha and the poly[(R)-(-)-3-hydroxyalkanoate] synthase gene (phaCAC) of Aeromonas caviae were introduced into tobacco plant by Agrobacterium mediated transformation method. The resulting transgenic tobacco expressed both introduced genes and the expression of these genes was confirmed by enzymatic analysis and western blotting. GC-MS analysis of the chloroform extract of tobacco leaves indicated that the transgenic plant produced biodegradable polyester, poly-[(R)-(-)-3-hydroxybutyrate]. GPC analysis indicated that the number-average molecular weights (Mn) and polydispersity (Mw/Mn) were 32,000 and 1.90, respectively.

Mechanisms of gene expression controlled by pressure in deep-sea microorganisms.

A pressure-regulated operon has been cloned and sequenced from deep-sea barophilic Shewanella strains. To understand pressure-regulated mechanisms of gene expression, a regulatory element upstream of the pressure-regulated operon from Shewanella sp. strain DSS12 was studied. Regions A and B were classified by sequence analysis. A unique octamer motif, AAGGTAAG, was found to be repeated in tandem 13 times in region B. An electrophoretic mobility shift assay demonstrated that a O54-like factor recognizes region A and other unknown factors recognize region B. Different shift patterns of the protein-DNA complexes were observed when extracts of cells cultured at 0.1 MPa or 50 MPa were incubated with a DNA probe specific for region B. These results indicate that the deep-sea strain DSS12 expresses different DNA-binding factors under different pressure conditions.

Physiological Properties of a Neutralo-sensitive Mutant Derived from Facultative Alkaliphilic Bacillus sp. C-125.

A neutralo-sensitive mutant (M-12) was isolated from the facultative alkaliphilic Bacillus sp. C-125. This mutant strain was able to grow to the same extent as did the parent strain above pH 8, but did not grow below pH 7.5. The same extent of oxygen uptake was shown by the cells of the parent and mutant strains at pH 10.3. On the other hand, the oxygen uptake rate was about one-fifth of that of the parent strain at pH 7. NADH-dependent oxygen uptake by everted vesicles of the mutant was lower than that of the parent strain at pH 7-7.5, while the rate at pH 8-9 was almost identical in both strains. The activity at pH 7 of cytochrome c oxidase of right-side-out membrane vesicles of the mutant strain was lower than that of the parent strain at pH 7, while both samples had almost the same enzymatic activity at pH 8.5. These results suggest that poor respiratory activities of the mutant strain at pH 7 are the reason why this mutant strain was unable to grow at neutral pH.

RKS-1778, a new mammalian cell-cycle inhibitor and a key intermediate of the [11]cytochalasin group.

In the course of screening for the mammalian cell-cycle inhibitors, we have isolated a new [11]cytochalasin, RKS-1778 (1), and epoxycytochalasin H (2) from a fungus, Phoma sp. SNF-1778. The structure of 1 was determined to be 21-acetoxy-18-hydroxy-10-phenyl-5,6,16,18- tetramethyl[11]cytochalasa-6,13,19-trien-1-one, one the basis of spectroscopic methods, including 1H- and 13C-2D NMR techniques, RKS-1778 (1) may be a precursor of 2 and the key direct product of a proposed biosynthetic intramolecular Diels-Alder reaction. Both 1 and 2 completely arrested the cell-cycle progression of tsFT210 cells in the M phase at concentrations of 2.1 and 2.0 microM, respectively.

Isolation and characterization of the gene encoding an aminopeptidase involved in the selective toxicity of ascamycin toward Xanthomonas campestris pv. citri.

An aminopeptidase gene named XAP has been isolated from Xanthomonas campestris pv. citri, a plant pathogenic bacterium. The bacterium is one of the rare micro-organisms susceptible to ascamycin, an aminoacyl nucleoside antibiotic that inhibits protein synthesis. Sequence analysis reveals that the gene encodes a 311 amino acid protein with a calculated molecular mass of 35134 Da and approx. 50% identity for amino acids to the proline iminopeptidase from Neisseria gonorrhoeae. The XAP gene product, Xap, expressed in Escherichia coli has proline iminopeptidase activity as well as ascamycin dealanylating activity in vitro.

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus.

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.

Open reading frame 3 of the barotolerant bacterium strain DSS12 is complementary with cydD in Escherichia coli: cydD functions are required for cell stability at high pressure.

Escherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth. The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions. Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain. Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria.

Characterization of malate dehydrogenase from deep-sea psychrophilic Vibrio sp. strain no. 5710 and cloning of its gene.

A metabolic key enzyme malate dehydrogenase (MDH) was purified from a deep-sea psychrophilic bacterium, Vibrio sp. strain no. 5710. The enzyme displayed an optimal activity shifted toward lower temperature and a pronounced heat lability. A gene encoding this enzyme was isolated and cloned. Recombinant Escherichia coli cells harboring the isolated clone expressed MDH activity with temperature stability identical to that of the parental psychrophile. Nucleotide sequencing of the gene revealed that its primary sequence was similar to that of a mesophile E. coli MDH (78% amino acid identity), for which the three-dimensional structure is known. The enzyme is thus suitable for the analysis of molecular adaptations to low temperatures.