Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10-2 to 10' when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The trapsformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70°C. T. thermophilus HB27 and four other typical' Thermus strains, T. thermophilus HBS, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants.A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro-at a frequency of 10-2.Thermus spp. are extremely thermophilic bacteria which can grow at temperatures over 75°C. They are aerobic, rod-shaped, nonsporulating, gram-negative bacteria. These organisms synthesize macromolecules which are not only heat stable but also resistant to many other drastic conditions, including organic solvents and high concentrations of urea and detergents. For these reasons, a number of enzymes (3, 4, 9) and tRNAs (20) from Thermus spp. have been purified and extensively characterized. Because of the lack of mutant strains and DNA transfer systems, genetic studies on Thermus spp. are limited, an exception being that the leucine gene from Thermus thermophilus was cloned in Escherichia coli and sequenced (7,8,18). Several plasmids of Thermus spp. have been reported (6, 19), but their functions are unknown. We have found natural DNA transformation events in Thermus spp., and moreover we have demonstrated introduction of a cryptic plasmid, pTT8, into T. thermophilus by using the same DNA transformation system. We report here some characteristics of natural transformation by using chromosomal and plasmid DNA in these extre,mely thermophilic organisms.Mutants auxotrophic for proline (K102 Pro-), leucine (K103 Leu-), methionine (K104 Met-), lysine (K105 Lys-), and tryptophan (K106 Trp-) were obtained after exposure of wild-type T. thermophilus HB27 cells (16) to N-methyl-N'-nitro-N-nitrosoguanidine by the method of Adelberg et al.(1). Auxotrophic mutants of T. thermophilus HB27 were transformed to prototrophy as follows. The TM broth medium was used for transformation experiments and routine cultivations. It consisted of 0.4% Polypeptone (Daigo-Eiyo Chemical Co. Ltd., Osaka, Japan), 0.2% yeast extract (Difco Laboratorie4, Detroit, Mich.), 0.1% NaCl, and basal salt (13), and the pH was adjusted to 7.5 with NaOH at room temperature. An overnight TM broth culture of the auxotrophic mutant was diluted 1:100 into fresh TM broth and incubated with shaking at 70°C for 2 h. Then, a 0.45-ml po...
Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.
In previous studies we determined the nucleotide sequence of the gene cluster containing lys20, hacA (lys4A), hacB (lys4B), orfE, orfF, rimK, argC, and argB of Thermus thermophilus, an extremely thermophilic bacterium. In this study, we characterized the role of each gene in the cluster by gene disruption and examined auxotrophy in the disruptants. All disruptants except for the orfE disruption showed a lysine auxotrophic phenotype. This was surprising because this cluster consists of genes coding for unrelated proteins based on their names, which had been tentatively designated by homology analysis. Although the newly found pathway contains ␣-aminoadipic acid as a lysine biosynthetic intermediate, this pathway is not the same as the eukaryotic one. When each of the gene products was phylogenetically analyzed, we found that genes evolutionarily-related to the lysine biosynthetic genes in T. thermophilus were all present in a hyperthermophilic and anaerobic archaeon, Pyrococcus horikoshii, and formed a gene cluster in a manner similar to that in T. thermophilus. Furthermore, this gene cluster was analogous in part to the present leucine and arginine biosyntheses pathways. This lysine biosynthesis cluster is assumed to be one of the origins of lysine biosynthesis and could therefore become a key to the evolution of amino acid biosynthesis.
The Morpho-butterfly wing reflects interfered brilliant blue, which originates from nanostructures on its scales, for any incidence angle of white light. We have, for the first time, fabricated a Morpho-butterfly-scale quasi-structure using focused-ion-beam chemical-vapor-deposition (FIB-CVD) and observed brilliant blue reflection from this quasi-structure with an optical microscope. We measured the reflection from real Morpho-butterfly scales and from the quasi-structure with a photonic multi-channel spectral analyzer system. The reflection spectra of the quasi-structure were very similar to those of Morpho-butterfly scales.
Members of the CarA/LitR family are MerR-type transcriptional regulators that contain a C-terminal cobalamin-binding domain. They are thought to be involved in light-induced transcriptional regulation in a wide variety of nonphototrophic bacteria. Based on the distribution of this kind of regulator, the current study examined carotenoid production in Thermus thermophilus, and it was found to occur in a light-induced manner. litR and carotenoid and cobalamin biosynthesis genes were all located on the large plasmid of this organism. litR or cobalamin biosynthesis gene knockout mutants were unable to switch off carotenoid production under dark conditions, while a mutant with a mutation in the downstream gene adjacent to litR (TT_P0055), which encodes a CRP/FNR family transcriptional regulator, was unable to produce carotenoids, irrespective of light conditions. Overall, genetic and biochemical evidence indicates that LitR is bound by cobalamin and associates with the intergenic promoter region between litR and crtB (phytoene synthase gene), repressing the bidirectional transcription of litR and crtB. It is probable that derepression of LitR caused by some photodependent mechanism induces the expression of TT_P0055 protein, which serves as a transcriptional activator for the crtB operon and hence causes the expression of carotenoid biosynthesis and the DNA repair system under light condition.
The tripeptide glutathione is involved in cellular defense mechanisms for xenobiotics and reactive oxygen species. This study investigated glutathione-dependent mechanisms in the model organism Aspergillus nidulans. A recombinant dimeric protein of A. nidulans glutathione reductase (GR) contained FAD and reduced oxidized glutathione (GSSG) using NADPH as an electron donor. A deletion strain of the GR gene (glrA) accumulated less intracellular reduced glutathione (GSH), indicating that the fungal GR contributes to GSSG reduction in vivo. Growth of the deletion strain of glrA was temperature-sensitive, and this phenotype was suppressed by adding GSH to the medium. The strain subsequently accumulated more intracellular superoxide, and cell-free respiration activity was partly defective. Growth of the strain decreased in the presence of oxidants, which induced glrA expression 1.5-6-fold. These results indicated that the fungal glutathione system functions as an antioxidant mechanism in A. nidulans. Our findings further revealed an initial proteomic differential display on GR-depleted and wild type strains. Up-regulation of thioredoxin reductase, peroxiredoxins, catalases, and cytochrome c peroxidase in the glrA-deletion strain revealed interplay between the glutathione system and both the thioredoxin system and hydrogen peroxide defense mechanisms. We also identified a hypothetical, up-regulated protein in the GR-depleted strains as glutathione S-transferase, which is unique among Ascomycetes fungi.
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