Hideyuki Tamegai

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.

A denitrifying bacterium from the deep sea at 11 000-m depth

Abstract The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30°C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4°C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.

Purification, and some molecular and enzymatic features of a novel ccb-type cytochrome c oxidase from a microaerobic denitrifier, Magnetospirillum magnetotacticum

A novel ccb‐type cytochrome c oxidase was purified from the magnetic bacterium, Magnetospirillum magnetotacticum MS‐1. The enzyme was composed of three subunits with M rs of 43,000, 34,000 and 28,000, respectively, and contained 0.91 mol of protoheme, 2.0 mol of heme c and 0.70 g atom of copper per mol of minimal structural unit. One mol of enzyme oxidized 187 mol of horse heart ferrocytochrome c and 34.4 mol of M. magnetotacticum ferrocytochrome c 550/s. The cytochrome c oxidase activity of the enzyme was 50% inhibited by 12 μM KCN. The enzyme seems to function as the terminal oxidase in microaerobic respiration.

Pressure-regulated biosynthesis of cytochrome bd in piezo- and psychrophilic deep-sea bacterium Shewanella violacea DSS12

The genes of cytochrome bd-encoding cydAB were identified from a deep-sea bacterium Shewanella violacea DSS12. These showed significant homologies with known cydAB gene sequences from various organisms. Additionally, highly conserved regions that are important for the enzymatic function were also conserved in cydA of S. violacea. Based on the results, transcriptional analysis of cydAB operon and cydDC operon (required for assembly of cytochrome bd) of S. violacea in microaerobic condition was performed under the growth condition of various pressures. The gene of cydA was expressed even under the condition of atmospheric pressure and its expression was enhanced with pressurization. On the other hand, the expression of cydC was strongly depressed under the condition of atmospheric pressure compared with the case under high pressure. It appeared spectrophotometrically that loss of cytochrome bd in S. violacea under atmospheric pressure shown in previous study is caused mainly by the loss of cydDC. Further, under the growth condition of atmospheric pressure, either less amount or no d-type cytochrome was expressed compared with the case of high-pressure condition even if the organism was grown under alkaline condition or in the presence of uncoupler, which are the inducible condition of d-type cytochrome in Escherichia coli. These results suggested that the significant amount of d-type cytochrome expression is specific event under the growth condition of high pressure.

Molecular analysis of the nitrogen cycle in deep-sea microorganisms from the Nankai Trough: genes for nitrification and denitrification from deep-sea environmental DNA

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.

Extended Sequence and Functional Analysis of the Butirosin Biosynthetic Gene Cluster in Bacillus circulans SANK 72073

Butirosin produced by Bacillus circulans is among the clinically important 2-deoxystreptamine containing aminoglycoside antibiotics and its unique structure is found in (S)-4-amino-2-hydroxyburyric acid substituted at C-1 of 2-deoxystreptamine. Recently, the key part of the butirosin biosynthetic gene cluster has been identified from Bacillus circulans SANK 72073, however the whole gene for the biosynthesis awaited for identification. In the present study, we undertook extended analysis of the butirosin biosynthetic gene cluster and found nine additional open reading flames (ORFs), btrQ, btrR1, btrR2, btrT, btrU, btrV, btrW, btrX and orf1 in the cluster. In addition, we constructed disruption mutants of btrR1 and btrP-V, and found that the btr genes (ca. 24 Kb) between btrR1 and btrP-V are at least required for the butirosin biosynthesis.

Piezo-Adapted 3-Isopropylmalate Dehydrogenase of the Obligate Piezophile Shewanella benthica DB21MT-2 Isolated from the 11,000-m Depth of the Mariana Trench

3-Isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures.

The Respiratory System of the Piezophile Photobacterium profundum SS9 Grown under Various Pressures

It is known that the facultative piezophile Shewanella violacea DSS12 alters its respiratory components under the influence of hydrostatic pressure during growth. This can be considered one of the mechanisms of bacterial adaptation to high pressure. In this study, we investigated the respiratory system of another well-studied piezophile, Photobacterium profundum SS9. We analyzed cytochrome contents, the expression of genes encoding respiratory components in P. profundum SS9 grown under various conditions, and the pressure dependency of the terminal oxidase activities. Activity was more tolerant of relatively high pressures, such as 125 MPa when the cells were grown under high pressure as compared with cells grown under atmospheric pressure. Such properties observed are similar to the case of S. violacea. However, the contents of the cytochromes and expression of the respiratory genes were not influenced by growth pressure in P. profundum SS9, inconsistent with the case of S. violacea. We suggest that the mechanism of the piezoadaptation of the respiratory system of P. profundum SS9 differs from that of S. violacea, as described above, and that each strain chooses its own strategy.

Biosynthesis of 2-Deoxystreptamine-containing Antibiotics in Streptoalloteichus hindustanus JCM 3268: Characterization of 2-Deoxy-scyllo-inosose Synthase

A part of the new biosynthetic gene cluster for 2-deoxystreptamine-containing antibiotics was identified from Streptoalloteichus hindustanus. The alloH gene in the gene cluster was deduced to encode 2-deoxy-scyllo-inosose synthase and the expressed protein AlloH was confirmed to have this enzyme activity. Furthermore, biochemical properties of AlloH were studied.

Identification of Aminotransferase Genes for Biosynthesis of Aminoglycoside Antibiotics from Soil DNA

Aminoglycoside has been known as a clinically important antibiotic for a long time, but genetic information for the biosynthesis of aminoglycoside is still insufficient. In this study, we tried to clone aminoglycoside-biosynthetic genes from soil DNA for accumulation of genetic information. We chose the genes encoding L-glutamine:(2-deoxy-)scyllo-inosose aminotransferase as the target, because it is specific for all types of aminoglycoside biosynthesis. By degenerate PCR, we obtained 33 individual clones that were homologous with aminotransferase genes in aminoglycoside biosynthesis. Phylogenetic analysis and alignment of these genes showed that horizontal gene transfer has occurred in the soil. Among these, several quite interesting genes were obtained. Some genes probably originated from non-actinomycetes, and some were far from the known homologs. These genes can be useful markers for the isolation of entire gene clusters and originating organisms.