Flavobacterium inviolabile sp. nov. isolated from stream water.

A Gram-stain-negative, rod-shaped, aerobic and non-motile bacterium, designated P2-65T, was isolated from Moonsan stream water in the Republic of Korea. The temperature, NaCl concentration and pH ranges for growth of strain P2-65T were 10-37 °C, 0.0-3.0% (w/v) and 6.5-8.5 with optimum growth at 25-30 °C, 0.0-1.0% and 7.0-7.5, respectively. Comparison of 16S rRNA gene sequence showed that strain P2-65T was closely related to Flavobacterium cauense (95.4%) and Flavobacterium cheniae (95.3%). The major fatty acids were iso-C15:0, iso C17:0 3-OH, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0) and iso-C15:0 3-OH. The predominant respiratory quinone was menaquinone-6 (MK-6). The major polar lipids detected in the strain were phosphatidylethanolamine, one aminophospholipid, one unidentified aminolipid and one unidentified polar lipid. The G + C content of the genomic DNA was 39.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for strain P2-65T with closely related Flavobacterium species were below 74.8% and 20%, respectively. Based on polyphasic features, strain P2-65T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium inviolabile sp. nov. is proposed. The type strain is P2-65T (= KCTC 62055T = NBRC 112953T).

Sphingobacterium praediipecoris sp. nov. isolated from effluent of a dairy manure treatment plant.

A novel Gram-reaction-negative, rod-shaped, non-motile bacterium, designated as strain G2-10T was isolated from effluent of a dairy manure treatment plant. Growth occurred at 20-40 °C (optimum at 25-30 °C), pH 7.0-8.0 (optimum at pH 7.0). The range of NaCl concentration for growth was between 0% and 3% (w/v) (optimum 0-1%, w/v). Comparison of 16S rRNA gene sequence indicated that strain G2-10T was moderately related to the type strains of Sphingobacterium nematocida M-SX103T and Sphingobacterium suaedae T47T with a pair-wise sequence similarity of 94.3% and 94.0%, respectively. The major fatty acid constituents of strain G2-10T were identified as iso-C15:0 (37.6%), summed feature 3 (consisting of C16:1ω7c and/or C16:1ω6c, 29.6%) and iso-C17:0 3-OH (15.2%). Phosphatidylethanolamine was the major polar lipids of strain G2-10T. Sphingophospholipids were present. The isoprenoid quinone was composed of only MK-7. The DNA G + C content of strain G2-10T was found to be 42.5 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain G2-10T represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium praediipecoris is proposed. The type strain is G2-10T (= KCTC 52880T = NBRC 112848T).

Flavobacterium amnigenum sp. nov. Isolated from a River.

A yellowish, flexirubin-pigment-producing strain I3-3T was isolated from river water in Iksan, the Republic of Korea. The strain was gram-negative, aerobic, non-motile, showed catalase and oxidase activities, and could grow at a temperature range of 10-35°C, pH 5.0-10 and 0-2.0% (w/v) of NaCl. The major fatty acids were iso-C15:0, iso-C17:0 3-OH and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c). The isolate contained phosphatidylethanolamine, one aminolipid, and two unidentified lipids as the major polar lipids. Menaquinone-6 (MK6) was the major respiratory quinone. The G+C content of the genomic DNA of strain I3-3T was 35.6%. Comparison of the 16S rRNA gene sequence with the sequences of the closely related type strains showed highest sequence similarity of 96.95% and 96.93% to Flavobacterium nitrogenifigens NXU-44T and Flavobacterium compostarboris 15C3T, respectively. Based on phenotypic and phylogenetic distinctiveness, strain I3-3T is considered as a member of novel species within the genus Flavobacterium, for which Flavobacterium amnigenum sp. nov. is proposed. The type strain is I3-3T (=KCTC 52884T =NBRC 112871T).

Soonwooa purpurea sp. nov., isolated from a fresh water river.

A pale-brown-coloured, rod-shaped, non-motile, and Gram-reaction-negative bacterium, strain I54T, was isolated from a water sample of a freshwater river in Iksan, Republic of Korea. The phylogenetic affiliation based on 16S rRNA gene sequence analysis showed that strain I54T belonged to the genus Soonwooa of the family Flavobacteriaceae with a sequence similarity of 97.5 % to Soonwooa buanensis HM0024T. The major fatty acids (>5 %) of strain I54T were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C17 : 0 3-OH. Phosphatidylethanolamine, one aminolipid and two unknown lipids were the major polar lipids. The G+C content of the genomic DNA was 34.2 (±0.3)mol% and MK6 was the sole respiratory quinone. On the basis of its molecular and phenotypic characteristics, strain I54T represents a novel species in the genus Soonwooa, for which the name Soonwooapurpurea sp. nov. is proposed. The type strain is I54T (=KCTC 52722T=JCM 31880T).

Chitinimonas naiadis sp. nov., Isolated from a Freshwater River.

A rod shaped, aerobic, Gram-stain-negative, and motile bacterium, strain AR2T, was isolated from a water sample of Yeongsan river, Republic of Korea. Strain AR2T clustered closely with the members of the genus Chitinimonas and showed the highest 16S rRNA gene sequence similarity with Chitinimonas prasina LY03T (96.4%), Chitinimonas viridis HMD2169T (96.4%), Chitinimonas taiwanensis cfT (96.2%), and Chitinimonas koreensis R2A43-10T (94.2%). The predominant fatty acids of strain AR2T were identified to be summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), C16:0, and C10:03-OH. Diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were found to be the major polar lipids. The genomic DNA G+C content was 60.4 mol%. Based on the polyphasic characterization, the isolated strain AR2T is described as a representative of a novel species in the genus Chitinimonas, for which the name Chitinimonas naiadis sp. nov. (type strain =AR2T =KCTC 42755T =JCM 31504T) is proposed.

Reclassification of Aeromonas sharmana to a new genus as Pseudaeromonas sharmana gen. nov., comb. nov., and description of Pseudaeromonas pectinilytica sp. nov. isolated from a freshwater stream.

A Gram-reaction-negative, rod-shaped, facultatively anaerobic, motile bacterium, designated strain AR1T, was isolated from a freshwater stream in Jeonju, South Korea. Strain AR1T showed highest 16S rRNA gene sequence similarity (96.83 %) and also formed a separate clade with Aeromonas sharmana GPTSA-6T in the phylogenetic tree reconstructed among the members of the family Aeromonadaceae. Major cellular fatty acids are summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16: 0. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol are the predominant polar lipids. The genomic DNA G+C content was found to be 54.7 mol%. However, earlier studies on 16S rRNA gene, gyrB, rpoD and universal target region of cpn60 sequences of the members of the genus Aeromonas recommended the transfer of Aeromonas sharmana to a new genus. Hence, based on the comparative polyphasic data obtained during the present study and also on the previous recommendations, it is proposed that Aeromonas sharmana be transferred to a novel genus as Pseudaeromonas sharmana gen. nov., comb. nov. with strain GPTSA-6T (=DSM 17445T=MTCC 7090T=CIP 109378T=CCUG 54939T) as the type strain of the type species of the genus. Also, it is proposed that strain AR1T be designated as a representative of a novel species of this new genus, namely Pseudaeromonas pectinilytica sp. nov. The type strain is AR1T (=KCTC 42754T=JCM 31503T).

Paenibacillus gelatinilyticus sp. nov. a psychrotolerant bacterium isolated from a reclaimed soil and amended description of Paenibacillus shenyangensis.

A rod shaped, Gram-stain positive, non-motile, facultative anaerobic and gelatin hydrolysing bacterium, strain PG1(T), was isolated from reclaimed land soil in Kyehwa-do, Republic of Korea. Strain PG1(T) showed highest 16S rRNA gene sequence similarity (97.4 and 96.5%, respectively) to Paenibacillus shenyangensis A9(T) and Paenibacillus hunanensis FeL05(T), and clustered closely with the members of the family Paenibacillaceae. DNA-DNA hybridization studies revealed a genomic relatedness of 47 ± 9% with P. shenyangensis A9(T). The predominant fatty acids of strain PG1(T) were identified to be anteiso-C(15:0) (46.7%) and C(16:0) (22.7%). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid were found to be the major polar lipids. The genomic DNA G+C content was found to be 47.7 mol%. This polyphasic characterisation of the newly isolated strain PG1(T) justifies its description as representative of a novel species in the genus Paenibacillus, for which the name Paenibacillus gelatinilyticus sp. nov., (type strain = PG1(T) = KCTC 33642(T) = JCM 30624(T)) is proposed.

Metabolic versatility of toluene-degrading, iron-reducing bacteria in tidal flat sediment, characterized by stable isotope probing-based metagenomic analysis.

DNA stable isotope probing and metagenomic sequencing were used to assess the metabolic potential of iron-reducing bacteria involved in anaerobic aromatic hydrocarbon degradation in oil spill-affected tidal flats. In a microcosm experiment, (13) C-toluene was degraded with the simultaneous reduction of Fe(III)-NTA, which was also verified by quasi-stoichiometric (13) C-CO2 release. The metabolic potential of the dominant member affiliated with the genus Desulfuromonas in the heavy DNA fraction was inferred using assembled scaffolds (designated TF genome, 4.40 Mbp with 58.8 GC mol%), which were obtained by Illumina sequencing. The gene clusters with peripheral pathways for toluene and benzoate conversion possessed the features of strict and facultative anaerobes. In addition to the class II-type benzoyl-CoA reductase (Bam) of strict anaerobes, the class I-type (Bcr) of facultative anaerobes was encoded. Genes related to the utilization of various anaerobic electron acceptors, including iron, nitrate (to ammonia), sulfur and fumarate, were identified. Furthermore, genes encoding terminal oxidases (caa3 , cbb3 and bd) and a diverse array of genes for oxidative stress responses were detected in the TF genome. This metabolic versatility may be an adaptation to the fluctuating availability of electron acceptors and donors in tidal flats.

Biodegradation of BTEX mixture by Pseudomonas putida YNS1 isolated from oil-contaminated soil.

The presence of mixed contaminants, such as BTEX (benzene, toluene, ethylbenzene and xylene isomers) can affect the biodegradation, fate and environmental impacts of each compound. To understand the influence of interactions among BTEX compounds on their biodegradation, four bacteria were isolated from oil-contaminated soil and assayed for BTEX biodegradation in vitro. The isolate exhibiting maximum biodegradation was identified as Pseudomonas putida based on the 16S rDNA sequence. The biodegradation of the BTEX compounds was greatly influenced by pH, temperature, and salinity. Substrate mixture studies (binary, tertiary and quaternary) revealed that the presence of toluene increased the biodegradation rate of benzene, ethylbenzene, and xylene.

Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction.

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 µM. PMA at a concentration of 50 µM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.

Giardia duodenalis: improved detection of viable cysts by reverse transcription-PCR of heat shock-inducible hsp70 gene.

Giardia duodenalis is a waterborne protozoan parasite that causes the diarrhoeal disease, giardiasis. Its durable and thick cell wall allows the parasite to exhibit resistance to environmental stresses. Because G. duodenalis exists in a water system at low levels, it is necessary to develop a sensitive method to detect its viability in aquatic environments. In the present study, specific primers for the heat shock protein (hsp) 70 gene were designed on the basis of G. duodenalis genome sequence and bioinformatic analysis. Viable G. duodenalis cysts were successfully distinguished by reverse transcription-PCR (RT-PCR) analysis using these primers. The amplicon of hsp70 was obtained from one cyst of G. duodenalis/100 microl, and this detection sensitivity significantly increased by 10(3)-fold when the cysts were given heat shock treatment. These findings prove that viable G. duodenalis cysts were successfully detected with a high degree of sensitivity by RT-PCR analysis targeting the hsp70 gene of G. duodenalis, thereby suggesting its practical potential for detecting viable G. duodenalis in environmental samples.

Trisindoline synthesis and anticancer activity.

Expression of a Rhodococcus-derived oxygenase gene in Escherichia coli yielded indigo metabolites with cytotoxic activity against cancer cells. Bioactivity-guided fractionation of these indigo metabolites led to the isolation of trisindoline as the agent responsible for the observed in vitro cytotoxic activity against cancer cells. While the cytotoxicity of etoposide, a common anticancer drug, was dramatically decreased in multidrug-resistant (MDR) cancer cells compared with treatment of parental cells, trisindoline was found to have similar cytotoxicity effects on both parental and MDR cell lines. In addition, the cytotoxic effects of trisindoline were resistant to P-glycoprotein overexpression, one of the most common mechanisms of drug resistance in cancer cells, supporting its use to kill MDR cancer cells.

A new classification system for bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases.

BACKGROUND: Rieske non-heme iron aromatic ring-hydroxylating oxygenases (RHOs) are multi-component enzyme systems that are remarkably diverse in bacteria isolated from diverse habitats. Since the first classification in 1990, there has been a need to devise a new classification scheme for these enzymes because many RHOs have been discovered, which do not belong to any group in the previous classification. Here, we present a scheme for classification of RHOs reflecting new sequence information and interactions between RHO enzyme components. RESULT: We have analyzed a total of 130 RHO enzymes in which 25 well-characterized RHO enzymes were used as standards to test our hypothesis for the proposed classification system. From the sequence analysis of electron transport chain (ETC) components of the standard RHOs, we extracted classification keys that reflect not only the phylogenetic affiliation within each component but also relationship among components. Oxygenase components of standard RHOs were phylogenetically classified into 10 groups with the classification keys derived from ETC components. This phylogenetic classification scheme was converted to a new systematic classification consisting of 5 distinct types. The new classification system was statistically examined to justify its stability. Type I represents two-component RHO systems that consist of an oxygenase and an FNRC-type reductase. Type II contains other two-component RHO systems that consist of an oxygenase and an FNRN-type reductase. Type III represents a group of three-component RHO systems that consist of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNRN-type reductase. Type IV represents another three-component systems that consist of oxygenase, [2Fe-2S]-type ferredoxin and GR-type reductase. Type V represents another different three-component systems that consist of an oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase. CONCLUSION: The new classification system provides the following features. First, the new classification system analyzes RHO enzymes as a whole. RwithSecond, the new classification system is not static but responds dynamically to the growing pool of RHO enzymes. Third, our classification can be applied reliably to the classification of incomplete RHOs. Fourth, the classification has direct applicability to experimental work. Fifth, the system provides new insights into the evolution of RHO systems based on enzyme interaction.

4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 is mediated by a tripartite ATP-independent periplasmic transporter.

The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

Characterization of two small cryptic plasmids from Pseudomonas sp. strain S-47.

Two small cryptic plasmids, p47L and p47S, identified in Pseudomonas sp. S-47 were characterized by determination of DNA sequences and physical and functional maps. They are 3084 and 1782 bp in length, respectively, with GC contents of 63.55 and 65.21%. The detection of single-strand DNAs of both plasmids indicates that they replicate by a rolling-circle mechanism. The deduced polypeptide encoded by the rep gene of p47L is homologous with Rep proteins of plasmids belonging to the pIJ101/pJV1 family, which are known to replicate by the rolling-circle mechanism. Despite containing a homologous signature with Rep proteins of rolling-circle replicating (RCR) plasmids in the pT181 family, the Rep of p47S lacks significant homology with Rep proteins of this family and is missing a region similar to the family's replication origin (dso). Based on the rep sequence comparisons, p47L falls into a previously defined plasmid family whereas p47S defines a new family of RCR plasmid.

Chloroplast-type ferredoxin involved in reactivation of catechol 2,3-dioxygenase from Pseudomonas sp. S 47.

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XylTE products catalyze the dioxygenation of the aromatics. The xylT of the strain S-47 is located just upstream of the xylE gene. XylT is a typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2 (TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XylT for the reactivation of the catechol 2,3-dioxygenase (XylE) that is oxidized with hydrogen peroxide. The pRES1 that was treated with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 minutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XylT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.