Yuki Kasai

Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment

ABSTRACT To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 103 cells/g of grains before crude oil was added to the tanks and increased to 3 × 106 cells/g of grains after crude oil was added. The number increased further after 14 days to 108 cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 × 106 cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.

RNA-Based Stable Isotope Probing and Isolation of Anaerobic Benzene-Degrading Bacteria from Gasoline-Contaminated Groundwater

ABSTRACT Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [13C6]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the 13C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [14C]benzene showed that the labeled carbon was mostly incorporated into 14CO2 within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

ABSTRACT Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m-Xylene Uptake

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation.

AbstractBenzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10–40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.

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)).

Tropicimonas isoalkanivorans gen. nov., sp. nov., a branched-alkane-degrading bacterium isolated from Semarang Port in Indonesia

An aerobic, Gram-negative, motile bacterium, strain B51(T), was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain B51(T) were peritrichously flagellated and rod-shaped. Strain B51(T) was able to degrade alkanes, branched alkanes and alkylnaphthalenes. 16S rRNA gene sequence analysis revealed that strain B51(T) was affiliated with the family Rhodobacteraceae, and was related most closely to Thioclava pacifica TL 2(T) (94.6 % similarity). The DNA G+C content of strain B51(T) was 66.5 mol%. The major cellular fatty acids were C(18 : 1)omega7c (84.9 %), C(18 : 1)omega9c (13.8 %), C(16 : 0) (8.7 %), C(18 : 0) (6.4 %) and anteiso-C(15 : 0) (5.8 %) and the major quinone was ubiquinone-10. Based on its phenotypic and phylogenetic characteristics, strain B51(T) is considered to represent a novel species of a new genus, for which the name Tropicimonas isoalcanivorans gen. nov., sp. nov. is proposed. The type strain of the type species is B51(T) (=JCM 14837(T)=DSM 19548(T)).

Catabolism of PAHS

Polycyclic aromatic hydrocarbons (PAHs) refer to hydrocarbons containing two or more fused benzene rings in a linear, angular, and cluster arrangement (Figure 1). PAHs are mainly formed as products from the combustion of fossil fuels, as by-products of industrial processing and during the cooking of foods91. PAHs enter the environment from a multiplicity of sources which include direct aerial fallout, chronic leakage of industrial or sewage effluent, accidental discharge during transport, use and disposal of petroleum products, and from natural sources such as oil seepage and surface water run-off from forest and prairie fire sites. More specifically, industrial effluent from coal gasification and liquefaction processes, waste incineration, coke, carbon black, and other petroleum-derived products releases high quantities of PAHs into the environment. PAH contamination, particularly from the high-molecular-weight types, in soil and aquifers is a cause of great environmental concern because of their toxic, mutagenic, and carcinogenic effects on experimental animals and their potential health risk to humans19, 38, 95. Some PAHs are classified as priority pollutants to be monitored in aquatic and terrestrial ecosystems by the U.S.

Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa

We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura−) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura− strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker–gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.