Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., Isolated from Soil.

Two gram-negative, catalase-positive, strictly aerobic, and white colony-forming bacteria, strains H242T and B156T, were isolated from soil in South Korea. Cells of strain H242T were oxidase-positive and non-motile short rods, while those of strain B156T were oxidase-negative and long non-motile rods. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. C16:0, cyclo-C17:0, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were identified in both strains as the major cellular fatty acids and polar lipids, respectively. The DNA G+C contents of strains H242T and B156T were 69.4 mol% and 69.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA and 92 concatenated core gene sequences revealed that strains H242T and B156T formed distinct phylogenic lineages from other Ramlibacter type strains. The DNA-DNA hybridization (DDH) value between strains H242T and B156T was 24.6%. Strains H242T and B156T were most closely related to Ramlibacter ginsenosidimutans BXN5-27T and Ramlibacter monticola G-3-2T with 98.4% and 98.6% 16S rRNA gene sequence similarities, respectively. Digital DDH values between strain H242T and R. ginsenosidimutans and between strain B156T and R. monticola were 23.5% and 26.1%, respectively. Phenotypic, chemotaxonomic, and molecular analyses indicated that strains H242T and B156T represent two novel species of the genus Ramlibacter, for which the names Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., respectively, are proposed. The type strains of R. terrae and R. montanisoli are H242T (=KACC 21667 T =JCM 33922T) and B156T (=KACC 21665 T =JCM 33920T), respectively.

Whole-cell biocatalysis using the Acidovorax sp. CHX100 Δ6HX for the production of ω-hydroxycarboxylic acids from cycloalkanes.

Omega hydroxycarboxylic acids (ω-HAs) possess two functional groups, a hydroxyl group and a carboxyl group, and are essential precursors for the production of biodegradable polyester polymers. In this work, an Acidovorax mutant was investigated as a whole-cell biocatalyst for the conversion of cycloalkanes to their respective ω-hydroxycarboxylic acids. This Acidovorax sp. strain CHX100 originated from a wastewater treatment plant and uses cyclohexane as the sole source of carbon and energy with excellent growth rates (0.199 h-1). The metabolic efficiency of Acidovorax CHX100 is based on a highly efficient enzyme cascade used for the mineralization of cyclohexane. A deletion of 6-hydroxyhexanoate dehydrogenase in the native cycloalkane pathway resulted in the Acidovorax sp. strain CHX100 Δ6HX mutant, which accumulated short ω-hydroxycarboxylic acids (C5 to C10) from cycloalkanes. This mutant transformed cyclopentane and cyclohexane (5 mM) to 5-hydroxypentanoic acid and 6-hydroxyhexanoic acid, respectively, with a molar conversion above 98% in 6 h. An elementary environmental and economical assessment based on E-factor and biocatalyst yield suggests the use of inexpensive electron donor and carbon sources, with subsequent efforts to minimize waste generation. Such an early-stage analysis highlights the main bottlenecks that need to be solved in developing a sustainable bioprocess.

Casamino acids slow motility and stimulate surface growth in an extreme oligotroph.

Environmental cues that regulate motility are poorly understood, but specific carbon and nitrogen sources, such as casamino acids (CAA), are known to stimulate motility in model organisms. However, natural environments are commonly more nutrient-limited than laboratory growth media, and the effect of energy-rich CAA on the motility of oligotrophic microorganisms is unknown. In this study, an extreme oligocarbotroph, Variovorax paradoxus YC1, was isolated from weathered shale rock within a disused mine level in North Yorkshire, UK. The addition of 0.1% CAA to minimal media significantly reduced the motility of YC1 after 72 h and inhibited swimming motility resulting in enhanced surface growth. We propose this response to CAA is a physiological adaptation to oligotrophy, facilitating the colonization of nutrient-rich environments.

Association Between Loss of Type IV Pilus Synthesis Ability and Phenotypic Variation in the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

Polaromonas eurypsychrophila sp. nov., isolated from an ice core.

A Gram-stain-negative, aerobic, rod-shaped, beige bacterium, strain B717-2T, was isolated from an ice core drilled from Muztagh Glacier on the Tibetan Plateau, China. According to phylogenetic analyses based on 16S rRNA gene sequences, the novel strain was related most closely to Polaromonas vacuolataand shared 97.7 % similarity with the type strain of this species. It grew optimally at pH 7, at 15 °C and with 2 % (w/v) NaCl. Major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), summed feature 8 (C18 : 1ω7c, C18 : 1ω6c) and C16 : 0. The sole respiratory quinone was Q-8. The DNA G+C content was 63.4 mol %. In DNA-DNA hybridization tests, strain B717-2T shared 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 and 18.4±1.9 % DNA-DNA relatedness with Polaromonas jejuensisJS12-13T, P. vacuolata 34-PT, Polaromonas aquatica CCUG 39402T, Polaromonas glacialisCr4-12T and Polaromonas cryoconitiCr4-35T, respectively. Based on the phenotypic, phylogenetic and genetic characteristics, strain B717-2T represents a novel species of the genus Polaromonas, for which the name Polaromonaseurypsychrophila sp. nov. is proposed. The type strain is B717-2T (=CGMCC 1.15322T=JCM 31171T).

Prey-Specific Growth Responses of Freshwater Flagellate Communities Induced by Morphologically Distinct Bacteria from the Genus Limnohabitans.

Because their large growth potential is counterbalanced with grazing by heterotrophic nanoflagellates (HNF), bacteria of the genus Limnohabitans, which are common in many freshwater habitats, represent a valuable model for examining bacterial carbon flow to the grazer food chain. We conducted experiments with natural HNF communities taken from two distinct habitats, the meso-eutrophic Rímov Reservoir and the oligo-mesotrophic Lake Cep (South Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal bloom and a late May clear water phase, were each fed 3 Limnohabitans strains of differing cell sizes. Water samples were prefiltered (5 µm) to release natural HNF communities from zooplankton control and then amended with the Limnohabitans strains L. planktonicus II-D5 (medium sized, rod shaped), Limnohabitans sp. strain T6-5 (thin, long, curved rod), and Limnohabitans sp. strain 2KL-3 (large solenoid). Using temporal sampling and prey treatment, we determined HNF growth parameters such as doubling time, growth efficiency, and length of lag phase prior starting to exponential growth. All three Limnohabitans strains supported HNF growth but in significant prey-, site-, and season-dependent fashions. For instance, addition of the moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat protected against grazing by smaller HNF, resulting in slower HNF growth and longer lag phases. These trends were particularly pronounced during the late May clear-water phase, which was dominated by smaller HNF cells. This may indicate a longer "adaptation time" for the flagellate communities toward the large prey size offered.

Aerobic mineralization of nitroguanidine by Variovorax strain VC1 isolated from soil.

Nitroguanidine (NQ) is an energetic material that is used as a key ingredient of triple-base propellants and is currently being considered as a TNT replacement in explosive formulations. NQ was efficiently degraded in aerobic microcosms when a carbon source was added. NQ persisted in unamended microcosms or under anaerobic conditions. An aerobic NQ-degrading bacterium, Variovorax strain VC1, was isolated from soil microcosms containing NQ as the sole nitrogen source. NQ degradation was inhibited in the presence of a more favorable source of nitrogen. Resting cells of VC1 degraded NQ effectively (54 µmol h(-1) g(-1) protein) giving NH(3) (50.0%), nitrous oxide (N(2)O) (48.5%) and CO(2) (100%). Disappearance of NQ was accompanied by the formation of a key intermediate product that we identified as nitrourea by comparison with a reference material. Nitrourea is unstable in water and suffered both biotic and abiotic decomposition to eventually give NH(3), N(2)O, and CO(2). However, we were unable to detect urea. Based on products distribution and reaction stoichiometry, we suggested that degradation of NQ, O(2)NN═C(NH(2))(2), might involve initial enzymatic hydroxylation of the imine, -C═N- bond, leading first to the formation of the unstable α-hydroxynitroamine intermediate, O(2)NNHC(OH)(NH(2))(2), whose decomposition in water should lead to the formation of NH(3), N(2)O, and CO(2). NQ biodegradation was induced by nitroguanidine itself, L-arginine, and creatinine, all being iminic compounds containing a guanidine group. This first description of NQ mineralization by a bacterial isolate demonstrates the potential for efficient microbial remediation of NQ in soil.

The cyst-dividing bacterium Ramlibacter tataouinensis TTB310 genome reveals a well-stocked toolbox for adaptation to a desert environment.

Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical "cyst-like" cells ("cyst-cyst" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.

Caldimonas hydrothermale sp. nov., a novel thermophilic bacterium isolated from Roman hot bath in south Tunisia.

A polyphasic approach was used to characterize a bacterium, HAN-85(T), isolated from thermal water in natural thermal spring at Tozeur, an oasis in southwest Tunisia. The novel isolate was thermophilic, strictly aerobic and amylolytic bacterium, which stained Gram negative. Cells were short rods motile by means of a single polar flagellum. Their optimum temperature and pH required for growth were 55 degrees C and pH 7, respectively. Comparative 16S rRNA gene sequence analyses showed that strain HAN-85(T) belonged to the genus Caldimonas, with highest sequence similarity to the type strains Caldimonas manganoxidans and Caldimonas taiwanensis. DNA-DNA hybridization measurements revealed low DNA relatedness (35.2-44.5%) between the novel isolate and its closest relative, C. manganoxidans. The major cellular fatty acid components were 16:0, 17:0 cyclo and summed feature 3. The DNA G+C content was 68.3 mol%. Taken together, the results of DNA-DNA hybridization, fatty acids profile, physiological tests and biochemical analyses have allowed the genotypic and phenotypic differentiation of the isolate from currently recognized Caldimonas species. Therefore, we suggest that this isolate is a novel species within the genus Caldimonas and propose that it should be named Caldimonas hydrothermale sp. nov. The type strain is HAN-85(T) (=DSM 18497(T) =LMG 23755(T)).

Biological nitrification-denitrification with alternating oxic and anoxic operations using aerobic granules.

Efficient nitrification and denitrification of wastewater containing 1,700 mgl(-1) of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH (4) (+) -N m(-3) day(-1), a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH (4) (+) -N g(-1) volatile suspended solids(-1) h(-1) at 6.4 kg NH (4) (+) -N m(-3) day(-1) loading and 41.3 +/- 2.0 at 10.2 kg NH (4) (+) -N m(-3) day(-1) loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification-denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.

Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures.

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

Proposal of Giesbergeria voronezhensis gen. nov., sp. nov. and G. kuznetsovii sp. nov. and reclassification of [Aquaspirillum] anulus, [A.] sinuosum and [A.] giesbergeri as Giesbergeria anulus comb. nov., G. sinuosa comb. nov. and G. giesbergeri comb. nov., and [Aquaspirillum] metamorphum and [A.] psychrophilum as Simplicispira metamorpha gen. nov., comb. nov. and S. psychrophila comb. nov.

Five Gram-negative, motile, spiral-shaped strains were isolated from a sulfide spring (D-412T), active sludge of wastewater (D-419T, D-420, D-424) and industrial wastewater (D-416). Comparative 16S rRNA gene sequence analysis showed that the isolates belong to the family Comamonadaceae, within the class Betaproteobacteria, but fall into a distinct cluster. On the basis of phenotypic, chemotaxonomic and phylogenetic data, a new genus, Giesbergeria gen. nov., is proposed, including five species. The type species of the genus is Giesbergeria voronezhensis sp. nov. (type strain D-419T = DSM 12825T = CIP 107340T = VKM B-2350T) and other novel members of the genus are Giesbergeria kuznetsovii sp. nov. (type strain D-412T = DSM 12827T = VKM B-2352T), Giesbergeria giesbergeri comb. nov. (basonym Aquaspirillum giesbergeri), Giesbergeria sinuosa comb. nov. (basonym Aquaspirillum sinuosum) and Giesbergeria anulus comb. nov. (basonym Aquaspirillum anulus). Using the same criteria, isolate D-416 (= DSM 12826) was identified as a strain of [Aquaspirillum] metamorphum. Strain D-416, the type strain of [A.] metamorphum and the type strain of [Aquaspirillum] psychrophilum form a distinct cluster within the family Comamonadaceae (97-97.2% 16S rRNA gene sequence similarity) and share phenotypic and chemotaxonomic properties. Therefore, it is proposed that these strains are reclassified as members of a new genus, Simplicispira gen. nov., as Simplicispira metamorpha comb. nov. (the type species) and Simplicispira psychrophila comb. nov., respectively.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

A gas vesiculate planktonic strain of the purple non-sulfur bacterium Rhodoferax antarcticus isolated from Lake Fryxell, Dry Valleys, Antarctica.

A moderately psychrophilic purple non-sulfur bacterium, Rhodoferax antarcticus strain Fryx1, is described. Strain Fryx1 was isolated from the water column under the ice of the permanently frozen Lake Fryxell, Antarctica. Cells of Fryx1 are long thin rods and contain gas vesicles, the first report of such structures in purple non-sulfur bacteria. Gas vesicles are clustered at 2-4 sites per cell. Surprisingly, the 16S rRNA gene sequence of strain Fryx1 is nearly identical to that of Rfx. antarcticus strain AB, a short, vibrio-shaped phototroph isolated from an Antarctic microbial mat. Although showing physiological parallels, strains AB and Fryx1 differ distinctly in their morphology and absorption spectra. DNA-DNA hybridization shows that the genomes of strains AB and Fryx1 are highly related, yet distinct. We conclude that although strains AB and Fryx1 may indeed be the same species, their ecologies are quite different. Unlike strain AB, strain Fryx1 has adapted to a planktonic existence in the nearly freezing water column of Lake Fryxell.