Yumiko Kodama

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.

Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude oil storage cavities

ABSTRACT Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 106 microorganisms ml−1, a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the Proteobacteria; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to Thiomicrospira denitrificans (88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to Arcobacterspp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the Proteobacteria grow as major populations in the petroleum-contaminated cavity groundwater.

Isolation and Characterization of a Sulfur-Oxidizing Chemolithotroph Growing on Crude Oil under Anaerobic Conditions

ABSTRACT Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the ε subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S2− + 4NO3− → SO42− + 4NO2− (ΔG0 = −534 kJ mol−1). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.

Diversity, abundance, and activity of archaeal populations in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity.

ABSTRACT Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom Euryarchaeota. Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family Methanomicrobiaceae. KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for ∼50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.

Diversity and abundance of bacteria in an underground oil-storage cavity

BackgroundMicroorganisms inhabiting subterranean oil fields have recently attracted much attention. Since intact groundwater can easily be obtained from the bottom of underground oil-storage cavities without contamination by surface water, studies on such oil-storage cavities are expected to provide valuable information to understand microbial ecology of subterranean oil fields.ResultsDNA was extracted from the groundwater obtained from an oil-storage cavity situated at Kuji in Iwate, Japan, and 16S rRNA gene (16S rDNA) fragments were amplified by PCR using combinations of universal and Bacteria-specific primers. The sequence analysis of 154 clones produced 31 different bacterial sequence types (a unique clone or group of clones with sequence similarity of > 98). Major sequence types were related to Desulfotomaculum, Acetobacterium, Desulfovibrio, Desulfobacula, Zoogloea and Thiomicrospira denitrificans. The abundance in the groundwater of bacterial populations represented by these major sequence types was assessed by quantitative competitive PCR using specific primers, showing that five rDNA types except for that related to Desulfobacula shared significant proportions (more than 1%) of the total bacterial rDNA.ConclusionsBacteria inhabiting the oil-storage cavity were unexpectedly diverse. A phylogenetic affiliation of cloned 16S rDNA sequences suggests that bacteria exhibiting different types of energy metabolism coexist in the cavity.

Molecular characterization of resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in petroleum-contaminated soil.

ABSTRACT PCR assays for analyzing resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in soil were developed. Sequence analysis of amplicons showed that the PCR successfully retrieved transporter gene fragments from soil. Most of the genes retrieved from petroleum-contaminated soils formed a cluster (cluster PCS) that was distantly related to known transporter genes. Competitive PCR showed that the abundance of PCS genes is increased in petroleum-contaminated soil.

Dysgonomonas oryzarvi sp. nov., isolated from a microbial fuel cell

A Gram-stain-negative, non-motile and coccoid- to short-rod-shaped bacterium, designated strain Dy73(T), was isolated from a microbial fuel cell that had been inoculated with rice paddy field soil and fed starch, peptone and fish extract as fuels. On the basis of 16S rRNA gene sequence phylogeny, strain Dy73(T) was affiliated with the genus Dysgonomonas in the phylum Bacteroidetes, and most closely related to Dysgonomonas mossii CCUG 43457(T) with a 16S rRNA gene sequence similarity value of 99.7 %. However, the DNA-DNA relatedness value between strain Dy73(T) and Dysgonomonas mossii CCUG 43457(T) was 34.8%. In addition, strain Dy73(T) was found to be different from other recognized species of the genus Dysgonomonas in taxonomically important traits, including habitat, DNA G+C content, bile resistance and fatty-acid composition. Based on these characteristics, strain Dy73(T) represents a novel species of the genus Dysgonomonas for which the name Dysgonomonas oryzarvi sp. nov. is proposed. The type strain is Dy73(T) ( = JCM 16859(T) = KCTC 5936(T)).

Tropicibacter naphthalenivorans gen. nov., sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from Semarang Port in Indonesia

An aerobic, Gram-negative, motile bacterium, strain C02(T), was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain C02(T) were peritrichously flagellated and rod-shaped. Strain C02(T) was able to degrade naphthalene, alkylnaphthalenes and phenanthrene. 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Marinovum algicola FF3(T) (95.7 % similarity) and Thalassobius aestuarii JC2049(T) (95.2 %). The DNA G+C content of strain C02(T) was 64.6 mol%. The major cellular fatty acids were C(18 : 1)omega7c (50.9 % of the total), C(16 : 0) (17.9 %), 11 methyl C(18 : 1)omega7c (14.7 %), C(18 : 1)omega9c (2.9 %) and C(19 : 0) cyclo omega8c (2.4 %), and the predominant respiratory lipoquinone was ubiquinone-10. Based on physiological, chemotaxonomic and phylogenetic data, strain C02(T) is suggested to represent a novel species of a new genus, for which the name Tropicibacter naphthalenivorans gen. nov., sp. nov. is proposed. The type strain of Tropicibacter naphthalenivorans is C02(T) (=JCM 14838(T)=DSM 19561(T)).

Rhizomicrobium electricum sp. nov., a facultatively anaerobic, fermentative, prosthecate bacterium isolated from a cellulose-fed microbial fuel cell.

A facultatively anaerobic, prosthecate bacterium, strain Mfc52(T), was isolated from a microbial fuel cell inoculated with soil and fed with cellulose as the sole fuel. Cells were Gram-negative, non-spore-forming, straight or slightly curved rods, and some of them had one or two polar prosthecae (stalks). Cells reproduced by binary fission or by budding from mother cells having prosthecae. Strain Mfc52(T) fermented various sugars and produced lactate, acetate and fumarate. Ferric iron, nitrate, oxygen and fumarate served as electron acceptors, while sulfate and malate did not. Nitrate was reduced to nitrite. The DNA G+C content was 64.7 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain Mfc52(T) was affiliated with the genus Rhizomicrobium in the class Alphaproteobacteria and most closely related to Rhizomicrobium palustre with a sequence similarity of 97 %. Based on these physiological and phylogenetic characteristics, the name Rhizomicrobium electricum sp. nov. is proposed; the type strain is Mfc52(T) ( = JCM 15089(T)  = KCTC 5806(T)).

An electricity-generating prosthecate bacterium strain Mfc52 isolated from a microbial fuel cell

Rod-shaped Alphaproteobacteria possessing long prosthecae-like appendages have been detected abundantly in biofilms attaching onto anode graphite of cellulose-fed microbial fuel cells (MFCs). To identify their ecological roles, the present study isolated a corresponding bacterium (strain Mfc52) by direct plating of a biofilm suspension onto a solid medium containing glucose and ferric ion. Phylogenetic analysis revealed that this strain is deeply branched in the class Alphaproteobacteria and may represent a novel order. Strain Mfc52 fermented sugars and produced lactate, acetate, and fumarate, whereas ferric ion stimulated the growth on glucose. When an MFC was inoculated with this strain and supplemented with glucose, it fermented glucose and generated electricity by oxidizing organic acids produced from glucose. Electron micrographs showed that a fraction of cells in a liquid culture had prosthecae-like appendages that were abundantly observed in anode biofilm. These observations suggest that the bacterial population represented by strain Mfc52 shared an important niche in the cellulose-fed MFC, where it generated electricity by oxidizing intermediate metabolites from cellulose degradation.