Parapedobacter lycopersici sp. nov., isolated from the rhizosphere soil of tomato plants (Solanum lycopersicum L.).

A novel Gram-stain-negative bacterial strain, designated T16R-256T, was isolated from the rhizosphere soil of tomato plants grown in a greenhouse in Yecheon-gun, Gyeongsangbuk-do, Republic of Korea and characterized using polyphasic taxonomy. Cells were aerobic, non-flagellated and rod-shaped. Colonies were light yellow, convex and round. The strain grew in the temperature range of 15-37 °C (optimally at 28-30 °C) and pH range of 7.0-9.0 (optimally at 7.0-8.0) and in 4 % NaCl (w/v). A comparison of 16S rRNA gene sequences showed that strain T16R-256T is a member of the genus Parapedobacter, exhibiting high sequence similarity with Parapedobacter pyrenivorans P-4T (94.2 %), Parapedobacter indicus RK1T (93.7 %), Parapedobacter koreensis Jip14T (93.7 %), Parapedobacter luteus 4M29T (93.6 %) and Parapedobacter soli DCY14T (93.4 %). The major polar lipids were phosphatidylethanolamine, sphingolipid, one aminophospholipid, two aminolipids and three unknown lipids. The major fatty acids (>10 % of the total fatty acids) were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 0 2-OH/C16 : 1ω7c. Strain T16R-256T contained MK-7 as the predominant respiratory quinone and homospermidine as the major polyamine. The genomic DNA G+C content of the type strain was 55.5 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain T16R-256T should be designated as representative of a novel species of the genus Parapedobacter, for which the name Parapedobacter lycopersici sp. nov. is proposed. The type strain is T16R-256T (=KACC 18788T=JCM 31602T).

Arthrobacter silviterrae sp. nov., isolated from forest soil.

A novel actinomycete strain, designated KIS14-16T, was isolated from forest soil in Ongjin county, South Korea and characterized using polyphasic taxonomy. The cells are aerobic, Gram-stain-positive, non-flagellated and short rods. The strain grew in a temperature range of 4-33 °C (optimum, 28-30 °C) and pH range of 5.0-10.0 (optimum, 7.0) and in the presence of 0-5 % (w/v) NaCl (optimum, 0 %). Comparison of 16S rRNA gene sequences showed that strain KIS14-16T is a member of the genus Arthrobacter exhibiting high sequence similarity with A. livingstonensis LI2T (97.7 %), A. cryoconiti Cr6-08T (97.6 %), A. psychrochitiniphilus GP3T (97.4 %), A. stackebrandtii CCM 2783T (97.1 %) and A. globiformis DSM 20124T (96.3 %). DNA-DNA relatedness and phenotypic data distinguished strain KIS14-16T from phylogenetically related type strains. The peptidoglycan type of strain KIS14-16T was A3α, with an interpeptide bridge comprising l-Lys, l-Thr, Gly and l-Ala4. Strain KIS14-16T contained a large amount of MK-9(H2) and relatively small amounts of MK-10(H2) and MK-8(H2). The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and dimannosylglyceride. The major fatty acids (>10 %) were anteiso-C15 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 63.9 mol%. On the basis of these phenotypic, chemotaxonomic and phylogenetic data, strain KIS14-16T should be designated as a representative novel species of the genus Arthrobacter, for which the name Arthrobacter silviterrae sp. nov. is proposed. The type strain is KIS14-16T (=KACC 17303T=DSM 27180T=NBRC 109660T).

Survival and susceptibility of Burkholderia cepacia complex in chlorhexidine gluconate and benzalkonium chloride.

The Burkholderia cepacia complex (BCC) includes opportunistic pathogenic bacteria that have occasionally been recovered from various pharmaceutical products, including antiseptics and disinfectants. Plausible reasons for the contamination include intrinsic sources, such as inadequate process controls, especially for water or equipment used during product manufacture, or extrinsic sources, such as improper handling and dilution or distribution in contaminated containers. Because the survival of BCC in antiseptics is a concern to the public health and pharmaceutical industry, we determined minimum inhibitory concentrations (MICs) of 36 BCC strains against the antiseptics, following exposure to chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions (1-500 µg/ml for each chemical). Susceptibility to CHX and BZK varied across the BCC strains and was recorded as mean 90.3 and 111.1 µg/ml, respectively, at initial inoculation, which was significantly higher than the 46.4 and 61.1 µg/ml levels measured for BCC incubated in water for 40 days. After determining antiseptic MICs of individual BCC strains, BCC recovery was measured on Tryptic Soy Agar (TSA), Reasoner's Second Agar (R2A) and diluted preparations of these media under their sub-MICs. The survival of BCC was monitored for 14 days (336 h) in sub-MICs diluted to less than their antiseptic susceptible concentration value. Diluted TSA and R2A media exhibited greater efficiency of recovery for most BCC strains from the CHX and BZK solutions than full strength TSA or R2A. For BCC survival in antiseptic solutions, the cell number of BCC decreased rapidly within the first 20 min in both antiseptics, but after this, recovery remained constant in CHX and increased in BZK over the 14 day incubation period. The results indicate that BCC in water can remain viable with low susceptibility to antiseptics for 14 days, which suggests the necessity for improved detection methods and control measures to monitor BCC contamination in pharmaceutical products.

Pleiotropic and epistatic behavior of a ring-hydroxylating oxygenase system in the polycyclic aromatic hydrocarbon metabolic network from Mycobacterium vanbaalenii PYR-1.

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water.

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner's 2nd Agar or Broth (R2A or R2AB), Brain-Heart Infusion Broth (BHIB), Mueller-Hinton Broth (MHB), and Ashdown's (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.

Characterization of the denitrification-associated phosphorus uptake properties of "Candidatus Accumulibacter phosphatis" clades in sludge subjected to enhanced biological phosphorus removal.

To characterize the denitrifying phosphorus (P) uptake properties of "Candidatus Accumulibacter phosphatis," a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of "Ca. Accumulibacter" and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized "Ca. Accumulibacter" subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and "Candidatus Competibacter phosphatis" [from 16.4% to 20.0%]), while the overall "Ca. Accumulibacter" abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the "Ca. Accumulibacter" clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all "Ca. Accumulibacter" clades successfully took up phosphorus in the presence of nitrate. However, the "Ca. Accumulibacter" clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by "Ca. Accumulibacter" clades occurred when nitrite was added. These results suggest that the "Ca. Accumulibacter" cells lack nitrate reduction capabilities and that P uptake by "Ca. Accumulibacter" is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and "Ca. Competibacter."

Muricauda taeanensis sp. nov., isolated from a marine tidal flat.

A novel Gram-stain-negative, heterotrophic, moderate halophilic and strictly aerobic bacterium, strain 105(T), was isolated from a tidal flat of Taean in Korea. Cells were catalase- and oxidase-positive long rods that showed gliding motility. Optimum temperature, pH and salinity for the growth of strain 105(T) were observed at 30-37 °C, at pH 7.0-7.5, and in the presence of 2-4 % (w/v) NaCl, respectively. The major cellular fatty acids were iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH. Phosphatidylethanolamine and five unidentified lipids were identified as the major polar lipids. The genomic DNA G+C content of strain 105(T) was 42.4 mol% and MK-6 was detected as the predominant isoprenoid quinone. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 105(T) formed a phyletic lineage with members of the genus Muricauda. Strain 105(T) was most closely related to Muricauda aquimarina SW-63(T) (97.6 %), Muricauda beolgyonensis BB-My12(T) (97.5 %), Muricauda lutimaris SMK-108(T) (97.5 %), Muricauda ruestringensis B1(T) (97.3 %), Muricauda flavescens CL-SS4(T) (97.2 %) and Muricauda olearia (96.2 %). The DNA-DNA relatedness values of strain 105(T) with M. aquimarina JCM 11811(T), M. beolgyonensis KCTC 23501(T), M. lutimaris KCTC 22173(T), M. ruestringensis DSM 13258(T) and M. flavescens JCM 11812(T) were 17.2 ± 6.0, 8.7 ± 2.2, 3.7 ± 0.5, 11.0 ± 1.9 and 7.1 ± 1.3 %, respectively. On the basis of phenotypic and molecular features, strain 105(T) represents a novel species of the genus Muricauda, for which the name Muricauda taeanensis sp. nov. is proposed. The type strain is 105(T) ( = KACC 16195(T) = JCM 17757(T)).

Aquamicrobium aestuarii sp. nov., a marine bacterium isolated from a tidal flat.

A Gram-negative, strictly aerobic, motile bacterium with flagella, designated strain G210(T), was isolated from a crude-oil-contaminated tidal flat of the Taean coast in South Korea. Cells were non-spore-forming, ovoid rods showing catalase- and oxidase-positive reactions. Growth of strain G210(T) was observed between 15 and 45 °C (optimum, 30-35 °C) and between pH 5.5 and 9.0 (optimum, pH 6.5-7.5). Strain G210(T) contained Q-10 as the sole isoprenoid quinone and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C19 : 0 cyclo ω8c as the major fatty acids. Putrescine and spermidine were identified as the major polyamines. Strain G210(T) contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, an unidentified phospholipid and an unidentified aminolipid as polar lipids. The G+C content of the genomic DNA was 56.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G210(T) formed a phyletic lineage with members of the genus Aquamicrobium. Strain G210(T) was most closely related to Aquamicrobium ahrensii 905/1(T) (97.9 % similarity). The DNA-DNA relatedness between strain G210(T) and the type strain of A. ahrensii was 24±0.5 %. On the basis of phenotypic, chemotaxonomic and molecular properties, strain G210(T) represents a novel species within the genus Aquamicrobium, for which the name Aquamicrobium aestuarii sp. nov. is proposed. The type strain is G210(T) ( = KACC 14931(T) = JCM 16876(T)).

Arenibacter hampyeongensis sp. nov., a marine bacterium isolated from a tidal flat.

A Gram-staining-negative, dark orange, strictly aerobic bacterium, designated strain HP12(T), was isolated from a tidal flat at Hampyeong in South Korea. Cells were moderately halotolerant, catalase- and oxidase-positive, non-motile rods. Growth was observed at 5-35 °C (optimum, 25 °C), at pH 6.0-8.5 (optimum, pH 7.0-7.5), and in the presence of 1-6 % (w/v) NaCl (optimum, 1-2 %). The major cellular fatty acids were summed feature 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH), iso-C(17 : 0) 3-OH, C(15 : 0), iso-C(15 : 1) G and iso-C(15 : 0). The polar lipid pattern indicated the presence of phosphatidylethanolamine and two unidentified lipids. The G+C content of the genomic DNA was 37.1 mol% and the predominant respiratory quinone was menaquinone-6. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain formed a tight phyletic lineage with members of the genus Arenibacter and was most closely related to Arenibacter palladensis KMM 3961(T), Arenibacter troitsensis KMM 3674(T) and Arenibacter echinorum KMM 6032(T), with 16S rRNA gene sequence similarities of 98.1 %, 98.0 % and 97.8 %, respectively. However, the DNA-DNA relatedness values between strain HP12(T) and A. palladensis JCM 13509(T), A. troitsensis KCTC 12362(T) and A. echinorum KCTC 22013(T) were only 20.2±0.3 %, 22.6±0.6 % and 9.1±2.6 %, respectively. On the basis of phenotypic and molecular features, strain HP12(T) represents a novel species of the genus Arenibacter, for which the name Arenibacter hampyeongensis sp. nov. is proposed. The type strain is HP12(T) ( = KACC 16193(T) = JCM 17788(T)).

Roseovarius lutimaris sp. nov., isolated from a marine tidal flat.

A novel Gram-staining-negative, moderately halophilic and strictly aerobic bacterium, designated strain 112(T), was isolated from a tidal flat at Taean, Korea. Cells were catalase- and oxidase-positive ovoids to rods and did not produce bacteriochlorophyll a. Optimum growth of strain 112(T) was observed at 30 °C, at pH 6.5-7.5 and in the presence of 2-4 % (w/v) NaCl. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0 and Q-10 was detected as the predominant ubiquinone. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and three unidentified lipids. The genomic DNA G+C content of strain 112(T) was 58.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 112(T) formed a phyletic lineage within the genus Roseovarius of the class Alphaproteobacteria. Strain 112(T) was most closely related to Roseovarius mucosus DFL-24(T), with 96.52 % similarity. On the basis of phenotypic, chemotaxonomic and molecular properties, strain 112(T) represents a novel species of the genus Roseovarius, for which the name Roseovarius lutimaris sp. nov. is proposed. The type strain is 112(T) ( = KACC 16185(T) = JCM 17743(T)).

Microbulbifer gwangyangensis sp. nov. and Microbulbifer pacificus sp. nov., isolated from marine environments.

Two novel Gram-stain-negative, chemoheterotrophic and strictly aerobic bacteria, strains GY2(T) and SPO729(T), were isolated from a tidal flat at Gwangyang Bay in Korea and a marine sponge sample from the Pacific Ocean, respectively. The two strains were halotolerant, catalase- and oxidase-positive, and non-motile rods. Optimum temperature and pH for growth of both strains were observed to be 35 °C and pH 7.0-7.5, but optimum salinity for strain SPO729(T) [2-3 % (w/v)] was slightly higher than that for strain GY2(T) (1-2 %). The major cellular fatty acids of both strains were C16 : 0, iso-C15 : 0, iso-C17 : 0, iso-C17 : 1ω9c, C18 : 1ω7c, iso-C11 : 0 and iso-C11 : 0 3-OH. The genomic DNA G+C contents of strains GY2(T) and SPO729(T) were 55.1 and 57.9 mol%, respectively, and ubiquinone 8 (Q-8) was detected as the sole respiratory quinone from the two strains. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains GY2(T) and SPO729(T) formed tight phyletic lineages with members of the genus Microbulbifer. Strain GY2(T) was closely related to Microbulbifer okinawensis ABABA23(T) (98.2 %), strain SPO729(T) (98.0 %) and Microbulbifer donghaiensis CN85(T) (97.0 %); strain SPO729(T) was closely related to M. okinawensis ABABA23(T) (98.3 %) and M. donghaiensis CN85(T) (98.2 %). The DNA-DNA relatedness values of strain GY2(T) with M. okinawensis ABABA23(T), strain SPO729(T) and M. donghaiensis CN85(T) were 40.0±2.1 %, 13.1±3.9 % and 16.2±5.8 %, respectively, whereas those of strain SPO729(T) with M. okinawensis ABABA23(T) and M. donghaiensis CN85(T) were 48.0±4.0 % and 34.6±9.3 %, respectively. On the basis of phenotypic and molecular features, it is concluded that the two strains GY2(T) and SPO729(T) represent two novel species of the genus Microbulbifer, for which the names Microbulbifer gwangyangensis sp. nov. and Microbulbifer pacificus are proposed; the type strains are GY2(T) ( = KACC 16189(T) = JCM 17800(T)) and SPO729(T) ( = KCCM 42667(T) = JCM 14507(T)), respectively.

Paenibacillus hordei sp. nov., isolated from naked barley in Korea.

A Gram-staining positive, facultative aerobic bacterium, designated strain RH-N24(T), was isolated from naked barley in South Korea. Cells of the isolate were observed to be motile rods by means of peritrichous flagella and showed catalase-positive and oxidase-negative reactions. Growth of strain RH-N24(T) was observed at 4-40 °C (optimum: 35-37 °C) and at pH 5.0-9.0 (optimum: pH 6.0-7.0). Chemotaxonomic data (major isoprenoid quinone: MK-7; DNA G + C content: 53.5 mol %; cell wall type: A1γ-meso-diaminopimelic acid; major fatty acids: anteiso-CB(15:0) and CB(16:0B)) supported the affiliation of the isolate to the genus Paenibacillus. The major cellular polar lipids were identified as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. Phylogenetic analysis based on 16S rRNA gene sequences also supported the conclusion that strain RH-N24(T) belonged to the genus Paenibacillus. Comparative 16S rRNA gene sequence analysis showed that strain RH-N24(T) was most closely related to Paenibacillus hunanensis FeL05(T) and Paenibacillus illinoisensis NRRL NRS-1356(T) with similarities of 94.64 and 94.54 %, respectively. On the basis of phenotypic and molecular properties, strain RH-N24(T) represents a novel species within the genus Paenibacillus for which the name Paenibacillus hordei sp. nov. is proposed. The type strain is RH-N24(T) (=KACC 15511(T) = JCM 17570(T)).

Complete genome sequence of the BTEX-degrading bacterium Pseudoxanthomonas spadix BD-a59.

Pseudoxanthomonas spadix BD-a59, able to metabolize all six BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylene) compounds, was isolated from gasoline-contaminated sediment. Here, we report the complete 3.45-Mb genome sequence and annotation of strain BD-a59. These advance the understanding of strain BD-a59's genomic properties and pollutant metabolic versatility.

Kordiimonas aestuarii sp. nov., a marine bacterium isolated from a tidal flat.

A Gram-staining negative, strictly aerobic bacterium, designated 101-1(T), was isolated from a sea tidal flat, Taean, Korea. The strain formed small light-yellow, smooth, and circular colonies on marine agar. Cells were weakly halophilic, motile rods showing catalase- and oxidase-positive reactions. Growth of strain 101-1(T) was observed at 15-40 °C (optimum, 30 °C), pH 5.0-8.0 (optimum, pH 6.5-7.0) and 1.0-9.0% (w/v) NaCl (optimum, 2.0-3.5%). The G+C content of the genomic DNA was 53.3 mol%. Strain 101-1(T) contained ubiquinone-10 (Q-10) as the respiratory quinone and iso-C(17:1)ω9c, iso-C(15:0) and iso-C(17:0) as major fatty acids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 101-1(T) formed a tight phylogenetic lineage with members of the genus Kordiimonas and was most closely related to Kordiimonas gwangyangensis GW14-5(T) and Kordiimonas lacus S3-22(T) with 97.3% and 96.3% 16S rRNA gene sequence similarities, respectively. The DNA-DNA relatedness values between strain 101-1(T) and K. gwangyangensis GW14-5(T) and K. lacus S3-22(T) were 24.8 ± 4.4% and 32.2 ± 3.6%, respectively. Based on the data from the phenotypic and genotypic studies, strain 101-1(T) represents a novel species of the genus Kordiimonas, for which the name Kordiimonas aestuarii sp. nov. is proposed. The type strain is 101-1(T) ( = KACC 16184(T) = JCM 17742(T)).

Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment.

Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated Alteromonas (among others) were active. Alteromonas sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: Alteromonas and Cycloclasticus, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed in situ expression of only the Alteromonas-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 µM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring Alteromonas-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments.

Identification of a novel subgroup of uncultured gammaproteobacterial glycogen-accumulating organisms in enhanced biological phosphorus removal sludge.

The presence of glycogen-accumulating organisms (GAO) has been hypothesized to be a cause of deterioration in enhanced biological phosphorus removal (EBPR) processes due to their abilities to out-compete polyphosphate-accumulating organisms (PAO). Based on 16S rRNA gene sequences, new members of uncultured gammaproteobacterial GAO (GB) were identified from sludge samples of a lab-scale sequencing batch reactor used for EBPR. The new GB formed a phylogenetic lineage (GB8) clearly distinct from the previously reported seven GB subgroups. Because the new GB8 members were not targeted by the known fluorescence in situ hybridization (FISH) oligonucleotide probes, a GB8-specific FISH probe (GB429) and a new FISH probe (GB742) targeting all eight GB subgroups were designed, and the phenotypic properties of the new GB8 members were investigated. FISH and microautoradiography approaches showed that GB429-targeted cells (GB8) were large coccobacilli (2-4 µm in size) with the ability to take up acetate under anaerobic conditions, but unable to accumulate polyphosphate under the subsequent aerobic conditions, consistent with in situ phenotypes of GB. FISH analyses on several sludge samples showed that members of GB8 were commonly detected as the majority of GB in lab- and full-scale EBPR processes. In conclusion, this study showed that members of GB8 could be a subgroup of GB with an important role in EBPR deterioration. Designs of FISH probes which hybridize with broader GB subgroups at different hierarchical levels will contribute to studies of the distributions and ecophysiologies of GB in lab- or full-scale EBPR plants.

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation.

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36-39 kDa, and their structures were deduced to be dimeric. The K(m) values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.

Metagenomic analysis of kimchi, a traditional Korean fermented food.

Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

Henriciella litoralis sp. nov., isolated from a tidal flat, transfer of Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. and emended description of the genus Henriciella.

A Gram-staining-negative, strictly aerobic bacterium, designated strain SD10(T), was isolated from a tidal flat of the Yellow Sea, South Korea. Cells were non-spore-forming rods that showed catalase- and oxidase-positive reactions. Growth of strain SD10(T) was observed at 15-40 °C (optimum, 25-30 °C), at pH 6.0-9.0 (optimum, pH 6.5-8.5) and in the presence of 1-10 % (w/v) NaCl. Strain SD10(T) contained ubiquinone-10 (Q-10) as a major isoprenoid quinone and C(18 : 1)ω7c (39.3 %), C(16 : 0) (20.2 %), C(17 : 0) (8.9 %) and C(17 : 1)ω6c (8.1 %) as major fatty acids. The cellular polar lipids were identified as phosphatidylglycerol, monoglycosyldiglyceride, glucuronopyranosyldiglyceride and two unidentified glycolipids. The G+C content of the genomic DNA was 55.2 mol%. Based on 16S rRNA gene sequence similarities, the strain was most closely related to Henriciella marina Iso4(T) and Maribaculum marinum P38(T), with similarities of 97.8 and 97.0 %, respectively. The DNA-DNA relatedness between strain SD10(T) and H. marina Iso4(T) was 12.0±3.2 %. A phylogenetic analysis based on 16S rRNA gene sequences showed that M. marinum P38(T) and H. marina Iso4(T) formed a monophyletic cluster and that their 16S rRNA gene sequence similarity was 98.1 %. DNA-DNA hybridization between H. marina Iso4(T) and M. marinum LMG 24711(T) was 22.9±2.7 %, indicating that the two strains belong to separate species. On the basis of chemotaxonomic data and molecular properties, we propose that strain SD10(T) represents a novel species of the genus Henriciella, for which the name Henriciella litoralis sp. nov. is proposed. The type strain is SD10(T) ( = KACC 13700(T)  = DSM 22014(T)). In addition, we propose to transfer Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. (type strain P38(T)  = CCTCC AB 208227(T)  = LMG 24711(T)  = MCCC 1A01086(T)), and we present an emended description of the genus Henriciella.

Litorimicrobium taeanense gen. nov., sp. nov., isolated from a sandy beach.

A Gram-reaction-negative, strictly aerobic, non-motile bacterium, designated strain G4(T), was isolated from a sandy beach of Taean in South Korea. Cells were ovoid rods and were catalase- and oxidase-positive. Growth of strain G4(T) was determined at 15-35 °C (optimum 25-30 °C) and pH 6-8 (optimum pH 6.5-7.5). Strain G4(T) contained Q-10 as the predominant isoprenoid quinone and C(18 : 1)ω7c (59.0 %), C(18 : 1)ω7c 11-methyl (11.3 %) and C(12 : 1) 3-OH (9.8 %) as the major fatty acids. The major cellular polar lipids were identified as phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified amino lipid, an unidentified phospholipid and an unidentified lipid. The DNA G+C content was 62.4 mol%. Phylogenetic and comparative analysis based on 16S rRNA gene sequences indicated that strain G4(T) fell within the family Rhodobacteraceae of Alphaproteobacteria and was most closely related to members of the genera Marinovum, Leisingera and Phaeobacter with 95.5-96.4 % sequence similarities. On the basis of phenotypic, chemotaxonomic and molecular properties, strain G4(T) represents a novel species of a novel genus within the family Rhodobacteraceae, for which the name Litorimicrobium taeanense gen. nov., sp. nov. is proposed. The type strain is G4(T) ( = KACC 13706(T)  = DSM 22007(T)).