The involvement of α-proteobacteria Phenylobacterium in maintaining the dominance of toxic Microcystis blooms in Lake Taihu, China.

Lake Taihu in China has suffered serious harmful cyanobacterial blooms for decades. The algal blooms threaten the ecological sustainability, drinking water safety, and human health. Although the roles of abiotic factors (such as water temperature and nutrient loading) in promoting Microcystis blooms have been well studied, the importance of biotic factors (e.g. bacterial community) in promoting and meditating Microcystis blooms remains unclear. In this study, we investigated the ecological dynamics of bacterial community, the ratio of toxic Microcystis, as well as microcystin in Lake Taihu. High-throughput 16S rRNA sequencing and principal component analysis (PCA) revealed that the bacteria community compositions (BCCs) clustered into three groups, the partitioning of which corresponded to that of groups according to the toxic profiles (the ratio of toxic Microcystis to total Microcystis, and the microcystin concentrations) of the samples. Further Spearman's correlation network showed that the α-proteobacteria Phenylobacterium strongly positively correlated with the toxic profiles. Subsequent laboratory chemostats experiments demonstrated that three Phenylobacterium strains promoted the dominance of the toxic Microcystis aeruginosa PCC7806 when co-culturing with the non-toxic PCC7806 mcyB- mutant. Taken together, our data suggested that the α-proteobacteria Phenylobacterium may play a vital role in the maintenance of toxic Microcystis dominance in Lake Taihu.

Metabolite-based mutualism enhances hydrogen production in a two-species microbial consortium.

Sustainable hydrogen production from renewable and low-cost substrates is very important to mitigate environmental and energy-related issues. Microbial consortia are promising for diverse bioenergy and environmental applications, yet microbial interactions are not fully understood. Here, we present comprehensive investigation on how two species in an artificial microbial consortium, consisting of Bacillus cereus A1 and Brevundimonas naejangsanensis B1, mutually cooperate to achieve an overall enhancement in hydrogen production and starch utilization. In this consortium, strains A1 and B1 secrete α-amylase and glucoamylase that are functionally complementary in starch hydrolysis. Moreover, strain A1 converts starch into lactate as a carbon source and electron donor, supporting the cell growth and hydrogen generation of strain B1. In return, strain B1 produces formate as an electron shuttle to strain A1 to enhance hydrogen production. The co-culture re-directs the overall metabolic flux, facilitates the cell growth, and up-regulates the key genes of hydrogen production and starch hydrolysis.

Aquidulcibacter paucihalophilus gen. nov., sp. nov., a novel member of family Caulobacteraceae isolated from cyanobacterial aggregates in a eutrophic lake.

A bacterial strain, TH1-2T, was isolated from cyanobacterial aggregates in the eutrophic Lake Taihu, Jiangsu Province, China. Cells were observed to be Gram-negative, slightly curved and rod-shaped. Optimal growth was obtained at pH 7.0 (range 5.5-8.5) and 30 °C (range 20-37 °C) in trypticase soy broth (TSB) without NaCl. Growth was not observed in TSB with 1.0% (w/v) NaCl added. The cells were found to be positive for oxidase and catalase activities. The major fatty acids were identified as 3-hydroxy hexadecanoic acid (C16:0 3-OH), C16:1 ω5c and summed feature 8 (consisting of C18:1 ω6c and cis-11-Octadecenoic acid (C18:1 ω7c). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The 16S rRNA gene sequence of strain TH1-2T was found to be phylogenetically related to those of Glycocaulis abyssi MCS 33T and Brevundimonas naejangsanensis BIO-TAS2-2T (90.5 and 90.0% similarity, respectively). The genomic G+C content of strain TH1-2T was 55.6 mol% based on whole genome calculations. Average nucleotide identities (ANI) and the digital DNA-DNA hybridizations (DDH) for complete genomes ranged from 66.84 to 67.32 and 21.3 to 31.8% between strain TH1-2T and type strains within the family Caulobacteraceae, higher than those between strain TH1-2T and the strains within the family Hyphomonadaceae. The phenotypic, chemotaxonomic and phylogenetic properties, and genome analysis, indicate that strain TH1-2T (=CGMCC 1.12979T = LMG 28362T) represents a novel species in a new genus within the family Caulobacteraceae; thus, the name Aquidulcibacter paucihalophilus gen. nov., sp. nov., is proposed.

Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems.

The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co-ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.

Role of Brevundimonas vesicularis in supporting the growth of Legionella in nutrient-poor environments.

In 1986, we encountered the first case of Legionella micdadei pneumonia in Japan. In the follow-up study to determine the infection route of L. micdadei, we isolated Brevundimonas vesicularis from the shower hose of the patient�s home. This motivated us to explore the symbiosis between B. vesicularis and Legionella in this study. B. vesicularis type strain, B. vesicularis Kobe strain, Legionella pneumophila serogroup 1 type strain, and L. micdadei Kobe strain were used. B. vesicularis was inoculated into 0.01 M phosphate buffer solution containing artificial sand, and varying concentrations of glucose at 0.1%, 0.01%, and 0.001%. Legionella was added to the cultures after ten days of incubation, and Legionella viable counts were monitored over time. After three days of incubation, Legionella counts increased approximately twofold in flasks containing 0.001% glucose, but Legionella counts decreased in both B. vesicularis inoculated and non-inoculated flasks containing higher concentrations of glucose. The counts were significantly higher in flasks inoculated with B. vesicularis than in non-inoculated flasks throughout the experiments. Under the nutrient-poor conditions, the presence of B. vesicularis was found to aid a further increase in Legionella counts. Further research is necessary to understand the symbiotic conditions most supporting the growth of L. micdadei.

New taxa in Alphaproteobacteria: Brevundimonas olei sp. nov., an esterase-producing bacterium.

A polyphasic taxonomic approach was used to characterize a Gram-negative, non-motile bacterium, designated MJ15(T), that was isolated from soil of a GS-Caltex Oil reservoir in Korea. As shown by comparative 16S rRNA gene sequence analysis, strain MJ15(T) belongs to genus Brevundimonas. The 16S rRNA gene sequence similarities ranged from 95.6-99.2% between strain MJ15(T) and validated representatives of the genus Brevundimonas. With respect to Brevundimonas species, strain MJ15(T) exhibited DNA-DNA relatedness values below 40.7%. The G+C content of the genomic DNA was 61.7 mol%. Strain MJ15(T) contained ubiquinone Q-10. The major fatty acids were C(16:0) (27.7%), C(19:0) cyclo ω8c (23.2%), summed feature 8 (containing C(18:1) ω7c/C(18:1) 6c) (28.5%), and major hydroxyl fatty acid was C(12:0) 3OH (3.7%). Based upon its phenotypic and genotypic properties, as well as its phylogenetic distinctiveness, strain MJ15(T) (KCTC 22461(T); JCM 16237(T)) should be classified in the genus Brevundimonas as the type strain of a novel species. The name Brevundimonas olei sp. nov. is proposed for this new species.

Investigation of the microbial degradation of phenazone-type drugs and their metabolites by natural biofilms derived from river water using liquid chromatography/tandem mass spectrometry (LC-MS/MS).

The degradation of the pharmaceuticals phenazone and metamizole, two pyrazolone-derivates in widespread use, using biofilms created by natural organisms from the national park Unteres Odertal, Germany, were investigated. An analytical method based on LC-MS/MS was optimised to determine the substances phenazone and methylaminoantipyrine (MAA), the hydrolysis product of metamizole (also known as dipyrone), as well as their metabolites 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP), acetaminoantipyrine (AAA), formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA). Performance characteristics of the method were evaluated in terms of recovery, standard deviation, coefficient of variation, method detection limits (MDL) and method quantification limits (MQL). Degradation studies of phenazone and MAA were conducted using a laboratory-scale continuous flow biofilm reactor fed with different nutrient media and with variable hydraulic retention times of 24 and 32 h. MAA was degraded rapidly to FAA and AA, while phenazone was not degraded under the prevailing conditions even after 32 h. By operating the bioreactor in batch mode to study the phenazone degradation potential of the biofilm under limiting nutrient conditions, an elimination rate of 85% phenazone was observed, but because of the slow elimination rate and aerobic conditions, the metabolite DP was not detected. In additional batch experiments using bacterial isolates from the natural biofilm to decompose phenazone, some bacterial strains were able to form DP from phenazone in marginal concentrations over the sampling period of eight weeks. Obviously, the microorganisms need a reasonably long time to adapt their metabolisms to enable the removal of phenazone from water samples.

Bacteria dispersal by hitchhiking on zooplankton.

Microorganisms and zooplankton are both important components of aquatic food webs. Although both inhabit the same environment, they are often regarded as separate functional units that are indirectly connected through nutrient cycling and trophic cascade. However, research on pathogenic and nonpathogenic bacteria has shown that direct association with zooplankton has significant influences on the bacteria's physiology and ecology. We used stratified migration columns to study vertical dispersal of hitchhiking bacteria through migrating zooplankton across a density gradient that was otherwise impenetrable for bacteria in both upward and downward directions (conveyor-belt hypothesis). The strength of our experiments is to permit quantitative estimation of transport and release of associated bacteria: vertical migration of Daphnia magna yielded an average dispersal rate of 1.3 x 10(5) x cells x Daphnia(-1) x migration cycle(-1) for the lake bacterium Brevundimonas sp. Bidirectional vertical dispersal by migrating D. magna was also shown for two other bacterial species, albeit at lower rates. The prediction that diurnally migrating zooplankton acquire different attached bacterial communities from hypolimnion and epilimnion between day and night was subsequently confirmed in our field study. In mesotrophic Lake Nehmitz, D. hyalina showed pronounced diel vertical migration along with significant diurnal changes in attached bacterial community composition. These results confirm that hitchhiking on migrating animals can be an important mechanism for rapidly relocating microorganisms, including pathogens, allowing them to access otherwise inaccessible resources.

Brevundimonas basaltis sp. nov., isolated from black sand.

A Gram-negative, aerobic, rod-shaped, motile Brevundimonas-like bacterial strain, J22(T), was isolated from black sand collected from Soesoggak, Jeju Island, Korea. Growth of strain J22(T) was observed in R2A medium at temperatures between 10 and 42 degrees C (optimum 30 degrees C), between pH 6.5 and 10.5 (optimum pH 7.5) and at a NaCl concentration between 0 and 4 % (w/v) (optimum 0-1 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain J22(T) belonged to the genus Brevundimonas, with high sequence similarities of >97 % to the sequence of the type strains Brevundimonas alba CB88(T), Brevundimonas lenta DS-18(T), Brevundimonas variabilis CB17(T), Mycoplana bullata TK0051(T), Brevundimonas kwangchunensis KSL-102(T), Brevundimonas intermedia CB63(T), Brevundimonas subvibrioides CB81(T) and Brevundimonas bacteroides CB7(T). Strain J22(T) exhibited DNA-DNA relatedness values of less than 22.2 % with the phylogenetically related species of the genus Brevundimonas. The DNA G+C content of strain J22(T) was 66.3 mol%. The predominant cellular fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(16 : 1)omega9c; C(12 : 0) 3-OH was present, which chemotaxonomically characterizes the members of the genus Brevundimonas. Phylogenetic, genomic and biochemical characteristics served to differentiate this isolate from recognized members of the genus Brevundimonas. Strain J22(T) (=KCTC 22177(T)=JCM 15911(T)) should be classified as a novel species in the genus Brevundimonas, for which the name Brevundimonas basaltis sp. nov. is proposed.

Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from fresh water and human blood. As determined by analyses of 16S rRNA gene sequences, the prosthecate strain FWC 38T was affiliated to the alphaproteobacterial genus Caulobacter, with Caulobacter henricii (96.8 %) and Caulobacter fusiformis (96.8 %) as its closest relatives. The non-prosthecate strain LMG 11050T and the prosthecate strain FWC 21T both belonged to the genus Phenylobacterium with Phenylobacterium koreense (96.9 %) and Phenylobacterium immobile (96.3 %) as the closest relatives. This affiliation was supported by chemotaxonomic data (polar lipids and cellular fatty acids). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of the novel strains from all hitherto recognized species of the genera Caulobacter and Phenylobacterium. The strains therefore represent novel species, for which the names Caulobacter mirabilis sp. nov. (type strain FWC 38T=LMG 24261T=CCUG 55073T), Phenylobacterium conjunctum (type strain FWC 21T=LMG 24262T=CCUG 55074T), the first described prosthecate Phenylobacterium species, and Phenylobacterium haematophilum sp. nov. (type strain LMG 11050T=CCUG 26751T) are proposed. Marker nucleotides within the 16S rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.

Brevundimonas lenta sp. nov., isolated from soil.

A Gram-negative, rod-shaped, Brevundimonas-like bacterial strain, DS-18(T), was isolated from soil in Dokdo, Korea, and its exact taxonomic position was investigated by using a polyphasic approach. Strain DS-18(T) grew optimally at pH 6.5-7.0 and 25 degrees C without NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-18(T) belonged to the genus Brevundimonas. Strain DS-18(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and C(16 : 0) as the major fatty acids. The DNA G+C content was 68.7 mol%. Strain DS-18(T) exhibited levels of 16S rRNA gene sequence similarity of 96.3-98.7 % to the type strains of Brevundimonas species and Mycoplana bullata. Mean DNA-DNA relatedness values between strain DS-18(T) and the type strains of phylogenetically related Brevundimonas species and M. bullata were in the range 15-32 %. Strain DS-18(T) differed from Brevundimonas species and M. bullata in several phenotypic characteristics. On the basis of phenotypic, phylogenetic and genetic data, strain DS-18(T) represents a novel species of the genus Brevundimonas, for which the name Brevundimonas lenta sp. nov. is proposed. The type strain is DS-18(T) (=KCTC 12871(T) =JCM 14602(T)).

Phenylobacterium koreense sp. nov., isolated from South Korea.

A Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, Slu-01(T), was isolated from activated sludge from a wastewater treatment plant in Daejeon, South Korea. After 3 days on R2A medium, strain Slu-01(T) developed colourless colonies (0.7-1.2 mm). The non-motile rods (0.7-1.0 x 1.0-2.0 microm) were slightly curved and occurred singly and in pairs. No filamentous cells were found. On the basis of 16S rRNA gene sequence similarity, strain Slu-01(T) was shown to belong to the family Caulobacteraceae and was most closely related to Phenylobacterium immobile (96.6%) and Phenylobacterium lituiforme (96.5%). The genomic DNA G+C content of strain Slu-01(T) was 68.1 mol%, within the range of 67.3-68.4 mol% for the genus Phenylobacterium. Chemotaxonomic data (major ubiquinone, Q-10; major fatty acids C(18:1)omega7c, C(16:0), C(15:0) and C(17:0)) and 16S rRNA gene sequence analysis supported the affiliation of strain Slu-01(T) to the genus Phenylobacterium. However, DNA-DNA hybridization data and phenotypic properties showed that strain Slu-01(T) could be distinguished from the two other Phenylobacterium species with validly published names. Thus, it is suggested that strain Slu-01(T) represents a novel species of the genus Phenylobacterium, for which the name Phenylobacterium koreense is proposed. The type strain is Slu-01(T) (=KCTC 12206(T)=IAM 15119(T)).

Phenylobacterium falsum sp. nov., an Alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium.

A Gram-negative bacterium designated AC-49T was isolated from an alkaline groundwater with a pH 11.4. This organism formed rod-shaped cells, was strictly aerobic, catalase and oxidase positive, with an optimum growth temperature of 35 degrees C and an optimum pH value of 8.0. Strain AC-49T assimilated primarily amino acids and some Krebs cycle metabolites, did not use sugars for growth. The organism did not grow on L-phenylalanine or antipyrin. The G+ C content of DNA was 66.9 mol%. The phylogenetic analyses based on the 16S rRNA sequencing showed that the closest relatives of strain AC-49T were Phenylobacterium lituiforme and Phenylobacterium immobile, indicating that the organism is a member of the order Caulobacterales of the Alphaproteobacteria. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-49T, represents a novel species of the genus Phenylobacterium for which we propose the name Phenylobacterium falsum sp. nov.

Asticcacaulis taihuensis sp. nov., a novel stalked bacterium isolated from Taihu Lake, China.

A novel stalked bacterium, designated strain T3-B7(T), was isolated from sediment of Taihu Lake, Jiangsu Province, China, and its taxonomy was studied by using a polyphasic approach. Cell morphology, physiological and biochemical properties, and polar lipids indicated that strain T3-B7(T) represented a member of the genus Asticcacaulis. Based on 16S rRNA gene sequence similarity analysis, strain T3-B7(T) was found to be phylogenetically related to Asticcacaulis biprosthecium DSM 4723(T) (98.5 %) and Asticcacaulis excentricus DSM 4724(T) (95.0 %), but could be differentiated from these two species on the basis of the number and position of prosthecae, assimilation of sugars, nitrate reduction and tolerance to NaCl. Levels of DNA-DNA relatedness of strain T3-B7(T) to A. biprosthecium DSM 4723(T) and A. excentricus DSM 4724(T) were 37.1 and 18.0 %, respectively. The G + C content of strain T3-B7(T) was 59 mol% (T(m)). It is concluded that strain T3-B7(T) represents a novel species of the genus Asticcacaulis, for which the name of Asticcacaulis taihuensis sp. nov. is proposed. The type strain is T3-B7(T) (=AS 1.3431(T) = JCM 12463(T)).

Phenylobacterium lituiforme sp. nov., a moderately thermophilic bacterium from a subsurface aquifer, and emended description of the genus Phenylobacterium.

A facultative anaerobic bacterium, strain FaiI3(T), was isolated from samples collected from the free-flowing waters of a bore well (Fairlea Bore, registration number 3768) which taps into the Australian Great Artesian Basin subsurface thermal aquifer. Strain FaiI3(T) developed yellow to pale-yellow colonies (0.5-1.5 mm) after 48 h. The non-spore forming rods (0.5x1-3 microm) were slightly curved, occurred singly and as pairs and were motile with a single polar flagellum. Cells tended to form clumps in liquid medium and rosettes were commonly observed. The cells stained Gram-negative and electron micrographs of thin sections revealed a multi-layered complex Gram-negative cell wall structure. Strain FaiI3(T) grew optimally at 40-41 degrees C, with growth observed at 45 degrees C but not at 50 degrees C. The pH growth range was between pH 6 and 9 and optimal growth occurred between pH 6 and 6.5. Strain FaiI3(T) grew best with yeast extract as the sole carbon and energy source. Peptone, yeast extract, acetate, xylose, sucrose, glucose, glycerol, succinate, butyrate, lactate, fumarate, citrate, L-phenylalanine, cellobiose and gelatin supported growth but maltose, fructose, glycine, ethanol, benzoate and oxalate did not. Tyrosine was produced from L-phenylalanine. Strain FaiI3(T) was catalase-positive and oxidase-negative and did not hydrolyse starch. Growth was inhibited by neomycin, tetracycline, streptomycin, chloramphenicol, ampicillin, vancomycin and spectinomycin. The G+C content was determined to be 66.5+/-0.5 mol%. On the basis of the 16S rRNA gene sequence analysis, strain FaiI3(T) was assigned as a novel species of the genus Phenylobacterium, Phenylobacterium lituiforme sp. nov. in the order Caulobacterales, subclass alpha-Proteobacteria, class Proteobacteria. The type strain is FaiI3(T) (=ATCC BAA-294(T)=DSM 14363(T)).

Ecological significance of microdiversity: identical 16S rRNA gene sequences can be found in bacteria with highly divergent genomes and ecophysiologies.

A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the B. alba strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.

Woodsholea maritima gen. nov., sp. nov., a marine bacterium with a low diversity of polar lipids.

Two cauliform bacteria (CM243T and CM251) isolated by J. Poindexter from the Atlantic Ocean were characterized by 16S rRNA gene sequencing, TaqI restriction fragment length polymorphism and single-strand conformation polymorphism analyses of the internally transcribed 16S-23S rDNA spacer (ITS1) region, analysis of fatty acids from cellular lipids, mass spectrometry of polar lipids and physiological properties. The two strains showed very low diversity of polar lipids with diacyl-sulfoquinovosyl glycerols as the predominant lipids. The two bacterial strains were observed to have nearly identical 16S rRNA gene sequences and could not be differentiated by their ITS1 regions. The isolates differed from species of the genus Maricaulis by their 16S rRNA gene sequences, polar lipids and fatty acid patterns. On the basis of the genotypic analyses and estimations of phylogenetic similarities, physiological and chemotaxonomic characteristics, it is proposed that the isolates represent a new genus and species, for which the name Woodsholea maritima gen. nov., sp. nov. (type strain CM243T=VKM B-1512T=LMG 21817T) is proposed.