Different types of agricultural land use drive distinct soil bacterial communities.

Biogeographic patterns in soil bacterial communities and their responses to environmental variables are well established, yet little is known about how different types of agricultural land use affect bacterial communities at large spatial scales. We report the variation in bacterial community structures in greenhouse, orchard, paddy, and upland soils collected from 853 sites across the Republic of Korea using 16S rRNA gene pyrosequencing analysis. Bacterial diversities and community structures were significantly differentiated by agricultural land-use types. Paddy soils, which are intentionally flooded for several months during rice cultivation, had the highest bacterial richness and diversity, with low community variation. Soil chemical properties were dependent on agricultural management practices and correlated with variation in bacterial communities in different types of agricultural land use, while the effects of spatial components were little. Firmicutes, Chloroflexi, and Acidobacteria were enriched in greenhouse, paddy, and orchard soils, respectively. Members of these bacterial phyla are indicator taxa that are relatively abundant in specific agricultural land-use types. A relatively large number of taxa were associated with the microbial network of paddy soils with multiple modules, while the microbial network of orchard and upland soils had fewer taxa with close mutual interactions. These results suggest that anthropogenic agricultural management can create soil disturbances that determine bacterial community structures, specific bacterial taxa, and their relationships with soil chemical parameters. These quantitative changes can be used as potential biological indicators for monitoring the impact of agricultural management on the soil environment.

Occurrence of four waterborne viruses at five typical raw water resources in the Republic of Korea during August 2013 to February 2019.

Waterborne diseases have critical public health issues and socioeconomic relevancy worldwide. Various viral pathogens are ordinarily associated with waterborne diseases. Six-year-surveillance (a total of 20 times) of norovirus, hepatitis A virus, group C rotavirus, and enterovirus was conducted at five raw water sampling sites including two lakes (Lakes Soyang and Juam), Hyundo region of Geum River in Daejeon City, and Guui region of Han River in Seoul Metropolitan City and Moolgeum region of Nakdong River in Gimhae City which are located near two water intake plants. In this study, we routinely investigated virus contamination in water samples through reverse transcription polymerase chain reaction (RT-PCR) and integrated cell culture RT-PCR with high sensitivity and specificity. A total 100 samples were tested. Most of the targeted viruses were found in 32% of the samples and at least one of the indicator bacteria was detected in 65% of these occurrences. Among all the detected viruses, enterovirus was the most prevalent with a detection frequency of 12% and 2.71 MPN/10 L on average, while hepatitis A virus was the least prevalent with a detection frequency of 4%. Nearly all of the analyzed viruses (except for group C rotavirus) were present in samples from Han River (the Guui region), Geum River (the Hyundo region), Lake Juam, and Nakdong River (the Moolgeum region), while group C rotavirus was detected in those from the Guui region. During the six-year sampling period, the targeted waterborne viruses in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five representative Korean water environments makes it necessary to establish the chemical and biological analysis systems for waterborne viruses and sophisticated management systems.

A preliminary examination of bacterial, archaeal, and fungal communities inhabiting different rhizocompartments of tomato plants under real-world environments.

Plant microbiota is a key determinant of plant health and productivity. The composition and structure of plant microbiota varies according to plant tissue and compartment, which are specific habitats for microbial colonization. To investigate the structural composition of the microbiome associated with tomato roots under natural systems, we characterized the bacterial, archaeal, and fungal communities of three belowground compartments (rhizosphere, endosphere, and bulk soil) of tomato plants collected from 23 greenhouses in 7 geographic locations of South Korea. The microbial diversity and structure varied by rhizocompartment, with the most distinctive community features found in the endosphere. The bacterial and fungal communities in the bulk soil and rhizosphere were correlated with soil physicochemical properties, such as pH, electrical conductivity, and exchangeable cation levels, while this trend was not evident in the endosphere samples. A small number of core bacterial operational taxonomic units (OTUs) present in all samples from the rhizosphere and endosphere represented more than 60% of the total relative abundance. Among these core microbes, OTUs belonging to the genera Acidovorax, Enterobacter, Pseudomonas, Rhizobium, Streptomyces, and Variovorax, members of which are known to have beneficial effects on plant growth, were more relatively abundant in the endosphere samples. A co-occurrence network analysis indicated that the microbial community in the rhizosphere had a larger and more complex network than those in the bulk soil and endosphere. The analysis also identified keystone taxa that might play important roles in microbe-microbe interactions in the community. Additionally, profiling of predicted gene functions identified many genes associated with membrane transport in the endospheric and rhizospheric communities. Overall, the data presented here provide preliminary insight into bacterial, archaeal, and fungal phylogeny, functionality, and interactions in the rhizocompartments of tomato roots under real-world environments.

Effects of Extended Storage of Chlorhexidine Gluconate and Benzalkonium Chloride Solutions on the Viability of Burkholderia cenocepacia.

Chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) formulations are frequently used as antiseptics in healthcare and consumer products. Burkholderia cepacia complex (BCC) contamination of pharmaceutical products could be due to the use of contaminated water in the manufacturing process, over-diluted antiseptic solutions in the product, and the use of outdated products, which in turn reduces the antimicrobial activity of CHX and BZK. To establish a "safe use" period following opening containers of CHX and BZK, we measured the antimicrobial effects of CHX (2-10 µg/ml) and BZK (10-50 µg/ml) at sublethal concentrations on six strains of Burkholderia cenocepacia using chemical and microbiological assays. CHX (2, 4, and 10 µg/ml) and BZK (10, 20, and 50 µg/ml) stored for 42 days at 23°C showed almost the same concentration and toxicity compared with freshly prepared CHX and BZK on B. cenocepacia strains. When 5 µg/ml CHX and 20 µg/ml BZK were spiked to six B. cenocepacia strains with different inoculum sizes (10° -105 CFU/ml), their toxic effects were not changed for 28 days. B. cenocepacia strains in diluted CHX and BZK were detectable at concentrations up to 10² CFU/ml after incubation for 28 days at 23°C. Although abiotic and biotic changes in the toxicity of both antiseptics were not observed, our results indicate that B. cenocepacia strains could remain viable in CHX and BZK for 28 days, which in turn, indicates the importance of control measures to monitor BCC contamination in pharmaceutical products.

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).

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).

Effects of diet type, developmental stage, and gut compartment in the gut bacterial communities of two Cerambycidae species (Coleoptera).

The gut bacterial community of wood-feeding beetles has been examined for its role on plant digestion and biocontrol method development. Monochamus alternatus and Psacothea hilaris, both belonging to the subfamily Lamiinae, are woodfeeding beetles found in eastern Asia and Europe and generally considered as destructive pests for pine and mulberry trees, respectively. However, limited reports exist on the gut bacterial communities in these species. Here, we characterized gut bacterial community compositions in larva and imago of each insect species reared with host tree logs and artificial diets as food sources. High-throughput 454 pyrosequencing of bacterial 16S rRNA gene revealed 225 operational taxonomic units (OTUs) based on a 97% sequences similarity cutoff from 138,279 sequence reads, the majority of which were derived from Proteobacteria (48.2%), Firmicutes (45.5%), and Actinobacteria (5.2%). The OTU network analysis revealed 7 modules with densely connected OTUs in specific gut samples, in which the distributions of Lactococcus-, Kluyvera-, Serratia-, and Enterococcus-related OTUs were distinct between diet types or developmental stages of the host insects. The gut bacterial communities were separated on a detrended correspondence analysis (DCA) plot and by c-means fuzzy clustering analysis, according to diet type. The results from this study suggest that diet was the main determinant for gut bacterial community composition in the two beetles.

Soil pH and electrical conductivity are key edaphic factors shaping bacterial communities of greenhouse soils in Korea.

Soil microorganisms play an essential role in soil ecosystem processes such as organic matter decomposition, nutrient cycling, and plant nutrient availability. The land use for greenhouse cultivation has been increasing continuously, which involves an intensive input of agricultural materials to enhance productivity; however, relatively little is known about bacterial communities in greenhouse soils. To assess the effects of environmental factors on the soil bacterial diversity and community composition, a total of 187 greenhouse soil samples collected across Korea were subjected to bacterial 16S rRNA gene pyrosequencing analysis. A total of 11,865 operational taxonomic units at a 97% similarity cutoff level were detected from 847,560 sequences. Among nine soil factors evaluated; pH, electrical conductivity (EC), exchangeable cations (Ca2+, Mg2+, Na+, and K+), available P2O5, organic matter, and NO3-N, soil pH was most strongly correlated with bacterial richness (polynomial regression, pH: R2 = 0.1683, P < 0.001) and diversity (pH: R2 = 0.1765, P < 0.001). Community dissimilarities (Bray-Curtis distance) were positively correlated with Euclidean distance for pH and EC (Mantel test, pH: r = 0.2672, P < 0.001; EC: r = 0.1473, P < 0.001). Among dominant phyla (> 1%), the relative abundances of Proteobacteria, Gemmatimonadetes, Acidobacteria, Bacteroidetes, Chloroflexi, and Planctomycetes were also more strongly correlated with pH and EC values, compared with other soil cation contents, such as Ca2+, Mg2+, Na+, and K+. Our results suggest that, despite the heterogeneity of various environmental variables, the bacterial communities of the intensively cultivated greenhouse soils were particularly influenced by soil pH and EC. These findings therefore shed light on the soil microbial ecology of greenhouse cultivation, which should be helpful for devising effective management strategies to enhance soil microbial diversity and improving crop productivity.

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches.

The microbiome in the rhizosphere-the region surrounding plant roots-plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner's 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread. IMPORTANCE: Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

Dynamics of bacterial communities in rice field soils as affected by different long-term fertilization practices.

Fertilization and the response of the soil microbial community to the process significantly affect crop yield and the environment. In this study, the seasonal variation in the bacterial communities in rice field soil subjected to different fertilization treatments for more than 50 years was investigated using 16S rRNA sequencing. The simultaneous application of inorganic fertilizers and rice straw compost (CAPK) maintained the species richness of the bacterial communities at levels higher than that in the case of non-fertilization (NF) and application of inorganic fertilizers only (APK) in the initial period of rice growth. The seasonal variation in the bacterial community structure in the NF and APK plots showed cyclic behavior, suggesting that the effect of season was important; however, no such trend was observed in the CAPK plot. In the CAPK plot, the relative abundances of putative copiotrophs such as Bacteroidetes, Firmicutes, and Proteobacteria were higher and those of putative oligotrophs such as Acidobacteria and Plactomycetes were lower than those in the other plots. The relative abundances of organotrophs with respiratory metabolism, such as Actinobacteria, were lower and those of chemoautotrophs that oxidize reduced iron and sulfur compounds were higher in the CAPK plot, suggesting greater carbon storage in this plot. Increased methane emission and nitrogen deficiency, which were inferred from the higher abundances of Methylocystis and Bradyrhizobium in the CAPK plot, may be a negative effect of rice straw application; thus, a solution for these should be considered to increase the use of renewable resources in agricultural lands.

Siphonobacter intestinalis sp. nov., a bacterium isolated from the feces of Pseudorhynchus japonicus.

Strain 63MJ-2T was isolated from the feces of broad-winged katydid (Pseudorhynchus japonicus) collected in Korea. The 16S rRNA gene sequence of this strain showed the highest sequence similarity with that of Siphonobacter aquaeclarae P2T (96.1%) and had low similarities (below 86.3%) with those of other members of family 'Flexibacteraceae'. The strain 63MJ-2T is a strictly aerobic, Gram-stain-negative, non-motile, rod-shaped bacterium. The strain grew at 4-35°C (optimum, 25-30°C), pH of 5.0-9.0 (optimum, 6.0-7.0), and 0-2.0% (optimum, 1.0-2.0) (w/v) NaCl. The DNA G+C content of strain 63MJ-2T was 43.5 mol%. The major fatty acids were C16:1 ω5c (42.5%), iso-C17:0 3-OH (18.7%), and summed feature 3 (iso-C15:0 2-OH and/or C16:1 ω7c, 18.0%). The major menaquinone was MK-7 and polar lipids were phosphatidylethanolamine, six unknown aminolipids, and five unknown lipids. Based on the evidence from our polyphasic taxonomic study, we conclude that strain 63MJ-2T should be classified as a novel species of the genus Siphonobacter, and propose the name Siphonobacter intestinalis sp. nov. The type strain is 63MJ-2T (=KACC 18663T =NBRC 111883T).

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.

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)).

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)).

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)).

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."

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)).