Norio Wakao

Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria : its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. nov

Phylogenetic analyses based on 16S rDNA sequences and genomic DNA-DNA relatedness showed that the sulphur-oxidizing facultative chemolithotroph Thiobacillus acidophilus was closely related to members of the genus Acidiphilium, which is a group of strictly aerobic, heterotrophic acidophiles now categorized into aerobic photosynthetic bacteria. Lipophilic pigment analyses revealed that zinc-chelated bacteriochlorophyll a and carotenoids occurred in appreciable amounts in T. acidophilus and all established species of the genus Acidiphilium. PCR experiments showed that T. acidophilus as well as Acidiphilium species contained puf genes, encoding the photosynthetic reaction centre proteins and the core light-harvesting complex of the purple bacteria. There were high similarities between T. acidophilus and Acidiphilium species in the primary structure of their reaction centre proteins deduced from the nucleotide sequence data. The phylogenetic tree of the reaction centre proteins was in agreement with the 16S rDNA sequence-based phylogenetic tree in the relationship between T. acidophilus and Acidiphilium species and between the Acidiphilium cluster and other purple photosynthetic bacteria. Based on these results, together with previous phylogenetic and phenotypic information, it is proposed to reclassify T. acidophilus (Guay and Silver) Harrison 1983 as Acidiphilium acidophilum comb. nov. The type strain is ATCC 27807T (= DSM 700T).

Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments.

Four strains of aerobic, mesophilic, acidophilic bacteria that produced bacteriochlorophyll (BChl) a were isolated from acidic hot springs and mine drainage. The characteristics of the four isolates were almost identical. The isolates were strictly aerobic and chemo-organotrophic. They were gram-negative, non-motile cocci and coccobacilli, formed salmon-pink colonies on solidified media and produced BChl a and carotenoids only under aerobic growth conditions. The cells also produced small amounts of zinc-substituted BChl a when grown in the presence of 1 mM zinc sulfate. Anaerobic growth in the light was not found, but aerobic growth was stimulated by continuous incandescent illumination. The isolates grew in a pH range of 3.5-6.0, with pH optima of 4.5-5.0. A phylogenetic analysis based on 16S rDNA sequences showed that the isolates clustered in the major acidophilic group of the class Proteobacteria, which includes species of the genera Acidiphilium and Rhodopila. The anaerobic phototrophic bacterium Rhodopila globiformis was the closest relative to the new isolates (95% level of sequence similarity). The G+C content of the genomic DNA of the isolates was 69.1-69.8 mol%. On the basis of these results, it was concluded that the four isolates should be classified into a new genus and a new species, for which the name Acidisphaera rubrifaciens is proposed. The type strain is strain HS-AP3T (= JCM 10600T).

Microbial oxidation of arsenite and occurrence of arsenite‐oxidizing bacteria in acid mine water from a sulfur‐pyrite mine

Abstract The acid mine waters (pH 2.0–2.4) discharged from the Matsuo sul‐fur‐pyrite mine contained high concentrations of dissolved inorganic arsenic (2–13 ppm). Arsenic in the superficial acid mine waters was predominantly in the (V) state (arsenate); however, the element in the water from a deep mine drift was almost in the (III) state (arsenite). Microbial arsenite oxidation occurred in the acid mine waters and along the stream of the river, which was contaminated with a large volume of the mine drift water. Arsenite (500 ppm As)‐resistant bacteria (0–27 colonies/ml) were detected in the water samples and 208 slant cultures were obtained. Arsenite‐oxidizing activities of all the cultures were determined and six strains with strong arsenite‐oxidizing activity were isolated. These bacteria were acidophilic (optimum growth pH, 3—4), gram‐negative, aerobic, and rod‐shaped. They could not oxidize ferrous iron and elemental sulfur as a sole energy source and not derive the energy for chemoautotrophic growth...

Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the Genus Acidocella gen. nov., and Emendation of the Genus Acidiphilium

Summary Phylogenetic relationships among members of the genus Acidiphilium and related genera were elucidated by studying 16S rRNA gene sequence information. Approximately 1.5 kbp fragments of 16S rDNAs from the test organisms were amplified by the polymerase chain reaction and sequenced directly by the combined method consisting of cycle sequencing and automated fluorescence detection. Sequence comparisons and evolutionary distance analyses with the sequence from some reference species showed that all members of the genus Acidiphilium formed a cluster within the alpha- I subclass of the Proteobacteria, with Acidomonas methanolica, Acetobacter aceti, and Rhodopila globiformis as the nearest phylogenetic neighbors. The Acidiphilium cluster was divided into two subclusters whose monophyly was supported by high levels of bootstrap confidence: one included A. facilis and A. aminolytica, and the other consisted of all other Acidiphilium species. The levels of sequence similarity between species of the different subclusters were less than 94.2%. The A. facilis-A. aminolytica group is also distinguishable from the other group in a number of phenotypic and chemotaxonomic characteristics. Thus we propose to transfer A. aminolytica and A. facilis to a new genus, Acidocella gen. nov., with Acidocella facilis as the type species. Following this proposal, the description of the genus Acidiphilium was emended.

Advances in soil microbial ecology and the biodiversity

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection. Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections (EC). A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.

Distribution of bacteriochlorophylla in species of the genusAcidiphilium

Bacteriochlorophyll (Bchl)a was found in strains of the strictly aerobic acidophilic heterotrophic bacteria:Acidiphilium rubrum, A. angustum, andA. cryptum. Absorption spectra of the cell-free extracts showed a large peak at 865 nm and a small peak around 802 nm. Anaerobic growth was not observed in either the light or the dark. Bchla was not detected in eitherA. organovorum orA. facilis. Bchla contents were less than ca. 0.7 nmol/mg dry cell weight, being variable among the species.

Purification and some properties of cholesterol oxidase stable in detergents from γ-proteobacterium Y-134

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the gamma-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30 degrees C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3beta-ol (cholesterol) and 5alpha-cholestan-3beta-ol (dihydrocholesterol) at a high reaction rate, and the K(m) value for cholesterol was 65 microM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.

Distribution of sulfate-reducing bacteria in paddy-field soil

Abstract The distribution of sulfate-reducing bacteria in a paddy-field soil was investigated by enumerating the viable number of the bacteria in minute loci. A small area having 20 mm or 40 mm square was divided into 100 sections; a small amount of soil was sampled form each section and the bacterial number was estimated by the anaerobic agar plate method. The distribution patterns of the bacteria were represented by distribution maps. The numbers of the bacteria in the sections showed distinct variations among the sections. The sections having large bacterial numbers showed high concentrations in some parts of an area. The aggregated distribution pattern was common in the soil, whether it was under drained or waterlogged condition, and indicates that there seem to be some centers of multiplication of sulfate-reducing bacteria in paddy-field soil.

SEASONAL VARIATIONS IN NUMBER OF ACIDOPHILIC IRON-OXIDIZING BACTERIA AND IRON OXIDATION IN THE RIVER CONTAMINATED WITH ACID MINE WATER

Abstract Number of acidophilic iron-oxidizing bacteria, soluble iron, pH, and water temperature were measured seasonally at five sampling stations in the Akagawa River which is highly polluted by strongly acid mine water discharged from the abandoned Matsuo sulfur and iron-sulfide mine area. Other environmental variables of the river water were also determined (titratable acidity. dissolved oxygen, electrical conductivity, sulfate, aluminum, ammonia and nitrate-nitrogen, and phosphate). Distinct seasonal variations in the number of iron-oxidizing bacteria and ferric iron concentration were observed of the river water during the flow and were correlated with seasonal change in water temperature. Hydrogen ion concentration, dissolved oxygen, nutrients for microorganisms, such as, nitrogen and phosphate in the water of the main stream were judged to be not limited to iron oxidation or bacterial growth. Thus, water temperature was found to be a major environmental factor influencing on the activity of iron ox...

Microbial oxidation of ferrous iron in acid mine water at sulfur and iron-sulfide mine

Abstract The microbial oxidation of ferrous iron in the acid mine water from the abandoned Matsuo sulfur and iron-sulfide mine area was investigated. The acid mine water had an extremely low pH value and contained a high concentration of ferrous iron (about 1,000 ppm). The ferrous iron was oxidized rapidly to the ferric form in the acid mine water. The oxidation of ferrous iron in the acid mine water was completely inhibited by millipore filtration, toluene addition or incubation at a low temperature. These facts indicate that the oxidation of ferrous iron is not a spotaneous oxidation by atmospheric oxygen but a biological oxidation. Iron-oxidizing bacteria were ubiquitously found in the acid mine water samples taken from the mine area. The number of iron-oxidizing bacteria and the iron oxidation ratio along the Akagawa River were found to increase gradually as the stream ran down. The degree of iron oxidation was well correlated with the number of iron-oxidizing bacteria. From these results it is conclu...