Ribosomes lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae.

Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3' tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU'-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < -13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System.

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Large-Scale Vortices with Dynamic Rotation Emerged from Monolayer Collective Motion of Gliding Flavobacteria.

A collective motion of self-driven particles has been a fascinating subject in physics and biology. Sophisticated macroscopic behavior emerges through a population of thousands or millions of bacterial cells propelling itself by flagellar rotation and chemotactic responses. Here, we found a series of collective motions accompanying successive phase transitions for a nonflagellated rod-shaped soil bacterium, Flavobacterium johnsoniae, which was driven by a surface cell movement known as gliding motility. When we spotted the cells on an agar plate with a low level of nutrients, the bacterial community exhibited vortex patterns that spontaneously appeared as lattice and integrated into a large-scale circular plate. All patterns were exhibited with a monolayer of bacteria, which enabled us to two-dimensionally visualize an individual cell with high resolution within a wide-range pattern. The single cells moved with random orientation, but the cells that were connected with one another showed left-turn-biased trajectories in a starved environment. This feature is possibly due to the collision of cells inducing a nematic alignment of dense cells as self-propelled rods. Subsequently, each vortex oscillated independently and then transformed to the rotating mode as an independent circular plate. Notably, the rotational direction of the circular plate was counterclockwise without exception. The plates developed accompanying rotation with constant angular velocity, suggesting that the mode is an efficient strategy for bacterial survival. IMPORTANCE Self-propelled bacteria propelled by flagellar rotation often display highly organized dynamic patterns at high cell densities. Here, we found a new mode of collective motion in nonflagellated bacteria; vortex patterns spontaneously appeared as lattice and were integrated into a large-scale circular plate, comprising hundreds of thousands of cells, which exhibited unidirectional rotation in a counterclockwise manner and expanded in size on agar. A series of collective motions was driven by gliding motility of the rod-shaped soil bacterium Flavobacterium johnsoniae. In a low-nutrient environment, single cells moved with random orientation, while cells at high density moved together as a unitary cluster. This might be an efficient strategy for cells of this species to find nutrients.

Application of spherical substrate to observe bacterial motility machineries by Quick-Freeze-Replica Electron Microscopy.

3-D Structural information is essential to elucidate the molecular mechanisms of various biological machineries. Quick-Freeze Deep-Etch-Replica Electron Microscopy is a unique technique to give very high-contrast surface profiles of extra- and intra-cellular apparatuses that bear numerous cellular functions. Though the global architecture of those machineries is primarily required to understand their functional features, it is difficult or even impossible to depict side- or highly-oblique views of the same targets by usual goniometry, inasmuch as the objects (e.g. motile microorganisms) are placed on conventional flat substrates. We introduced silica-beads as an alternative substrate to solve such crucial issue. Elongated Flavobacterium and globular Mycoplasmas cells glided regularly along the bead's surface, similarly to those on a flat substrate. Quick-freeze replicas of those cells attached to the beads showed various views; side-, oblique- and frontal-views, enabling us to study not only global but potentially more detailed morphology of complicated architecture. Adhesion of the targets to the convex surface could give surplus merits to visualizing intriguing molecular assemblies within the cells, which is relevant to a variety of motility machinery of microorganisms.

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake.

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose-dependently inhibited by addition of D-glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose-mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N-acetyl-glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose-mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose-negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose-mediated inhibition of F. johnsoniae colony spreading.

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.

Microbial Cells with a Fe3 O4 Doped Hydrogel Extracellular Matrix: Manipulation of Living Cells by Magnetic Stimulus.

This study aims to develop an effective method to control motile microorganisms and enable their manipulation as functional 'live micro/nano robots'. A novel strategy based on Fe3 O4 nanoparticle-doped alginate hydrogel is developed to fashion an artificial extracellular matrix (ECM) for microbial cells (e.g., Saccharomyces cerevisiae and Flavobacterium heparinum). During this strategy, a single layer of alginate hydrogel is coated around the microbial cells doped with Fe3 O4 nanoparticles to form the alg-mag-cells. Transmission electron microscopy shows that Fe3 O4 nanoparticles are uniformly distributed in the hydrogel shell. Together with maintaining the cell activity and metabolism, the hydrogel coated microbial cells demonstrate high magnetic responsiveness in an external magnetic field and are able to form micro-scaled patterns using the magnetic template designed in this study. This strategy provides a building block to fabricate advanced biological models, medical therapeutic products, and non-medical biological systems using different microorganisms.

Comparative Analysis of Cellulophaga algicola and Flavobacterium johnsoniae Gliding Motility.

UNLABELLED: Gliding motility is common in members of the phylum Bacteroidetes, including Flavobacterium johnsoniae and Cellulophaga algicola. F. johnsoniae gliding has been extensively studied and involves rapid movement of the cell surface adhesin SprB. Genetic analysis of C. algicola allowed a comparative analysis of gliding. Sixty-three HimarEm1-induced mutants that formed nonspreading colonies were characterized. Each had an insertion in an ortholog of an F. johnsoniae motility gene, highlighting similarities between the motility systems. Differences were also observed. C. algicola lacks orthologs of the F. johnsoniae motility genes gldA, gldF, and gldG that are thought to encode the components of an ATP-binding cassette (ABC) transporter. In addition, mutations in any of 12 F. johnsoniae gld genes result in complete loss of motility, whereas all C. algicola gld mutants retained slight residual motility. This may indicate that C. algicola has multiple motility systems, that the motility proteins exhibit partial redundancy of function, or that essential components of the motility machinery of both C. algicola and F. johnsoniae remain to be discovered. IMPORTANCE: The development of genetic tools for C. algicola and comparative analysis of F. johnsoniae and C. algicola motility mutants identified similarities and differences between their gliding motility machineries. Gliding motility is common in the phylum Bacteroidetes Proteins that are important for gliding in both C. algicola and F. johnsoniae are potential core components of the Bacteroidetes gliding motility machinery.

Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

Association of colony morphotypes with virulence, growth and resistance against protozoan predation in the fish pathogen Flavobacterium columnare.

Many opportunistic pathogens can alternate between inside- and outside-host environments during their life cycle. The opportunistic fish pathogen Flavobacterium columnare is an inhabitant of the natural microbial community and causes significant yearly losses in aquaculture worldwide. The bacterium grows in varying colony morphotypes that are associated with either virulence (rhizoid type) or resistance to starvation and phages (rough type). Rough type strains can arise spontaneously or can be induced by phage infection. To identify the determinants of morphotype fitness, we measured virulence, growth parameters, biofilm-forming ability and resistance to amoeba and ciliate predation of both morphotypes in thirteen F. columnare strains. The (phage-sensitive) rhizoid type had a fitness advantage over the rough type in virulence, growth rate and maximum population size. Phage-induced rough type was found to be significantly weakest in resisting both ciliate and amoeba predation, and produced more biofilm in the presence of amoebae, whereas the spontaneous rough types did not differ from rhizoid in biofilm production. In co-culture experiment, the ciliate population sizes were higher when co-cultured with rough type than with rhizoid type. Our results thus suggest that the resistance to phages and starvation of the F. columnare rough type may have strong a trade-off, as the performance of the ancestral rhizoid type is better under environmental conditions.

Solvent immersion imprint lithography.

We present Solvent Immersion Imprint Lithography (SIIL), a technique for polymer functionalization and microsystem prototyping. SIIL is based on polymer immersion in commonly available solvents. This was experimentally and computationally analyzed, uniquely enabling two practical aspects. The first is imprinting and bonding deep features that span the 1 to 100 µm range, which are unattainable with existing solvent-based methods. The second is a functionalization scheme characterized by a well-controlled, 3D distribution of chemical moieties. SIIL is validated by developing microfluidics with embedded 3D oxygen sensors and microbioreactors for quantitative metabolic studies of a thermophile anaerobe microbial culture. Polystyrene (PS) was employed in the aforementioned applications; however all soluble polymers - including inorganic ones - can be employed with SIIL under no instrumentation requirements and typical processing times of less than two minutes.

Columnaris disease in fish: a review with emphasis on bacterium-host interactions.

Flavobacterium columnare (F. columnare) is the causative agent of columnaris disease. This bacterium affects both cultured and wild freshwater fish including many susceptible commercially important fish species. F. columnare infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely remains unclear, compromising the further development of efficient curative and preventive measures to combat this disease. Besides elaborating on the agent and the disease it causes, this review aims to summarize these pathogenesis data emphasizing the areas meriting further investigation.

Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes.

BACKGROUND: The ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated. RESULTS: Starvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge experiments shown that starved cells were avirulent for a fish host model. CONCLUSIONS: Specific morphological and ultrastructural changes allowed F. columnare cells to remain viable under adverse conditions. Those changes were reversed by the addition of nutrients. This bacterium can survive in water without nutrients for extended periods of time although long-term starvation appears to decrease cell fitness and resulted in loss of virulence.

Generation of biotechnology-derived Flavobacterium columnare ghosts by PhiX174 gene E-mediated inactivation and the potential as vaccine candidates against infection in grass carp.

Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene.

AIMS: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. METHODS AND RESULTS: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. CONCLUSIONS: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. SIGNIFICANCE AND IMPACT OF THE STUDY: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare.

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac-Col(®) . The strain of F. columnare used to generate the avirulent rifampicin-resistant mutant used in Aquavac-Col(®) belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin-resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin-resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.

Diversity of marine gliding bacteria in Thailand and their cytotoxicity

Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90 percent sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica, Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.

Niabella aurantiaca gen. nov., sp. nov., isolated from a greenhouse soil in Korea.

An orange-coloured bacterial strain, designated R2A15-11(T), was isolated from greenhouse soil. The strain was found to be strictly aerobic, Gram-negative, non-spore-forming and non-flagellated. The cells were short rods (0.7-0.9x1.0-1.5 mum) and produced flexirubin. Growth of the strain was observed at 10-35 degrees C, pH 5.0-8.0 and 0-3 % (w/v) NaCl. The predominant isoprenoid quinone was MK-7. The major fatty acids were iso-C(15 : 0), iso-C(15 : 1) G, iso-C(17 : 0) 3-OH and summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). The genomic DNA G+C content was 45.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence of strain R2A15-11(T) revealed a clear affiliation with the phylum Bacteroidetes, and the highest levels of sequence similarity were found with respect to Terrimonas ferruginea ATCC 13524(T) (91.5 %), Terrimonas lutea DY(T) (90.2 %), Niastella yeongjuensis GR20-13(T) (89.9 %) and Niastella koreensis GR20-10(T) (89.7 %). On the basis of the polyphasic evidence from this study, strain R2A15-11(T) represents a novel genus and species, for which the name Niabella aurantiaca gen. nov., sp. nov. is proposed. The type strain of Niabella aurantiaca is R2A15-11(T) (=KACC 11698(T)=DSM 17617(T)).

Flavobacterium defluvii sp. nov., isolated from activated sludge.

A Gram-negative bacterium, designated strain EMB117T, was isolated from a municipal wastewater treatment plant and characterized by polyphasic taxonomy. The cells were non-spore-forming rods that showed gliding motility. Optimal growth occurred at 25-30 degrees C and pH 7.0-8.0. Strain EMB117T contained phosphatidylethanolamine as the predominant polar lipid, and the major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH, iso-C(15 : 0) 3-OH and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH). The G+C content of the genomic DNA was 33.5 mol% and the major isoprenoid quinone was MK-6. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain EMB117T belonged to the genus Flavobacterium and was most closely related to Flavobacterium johnsoniae DSM 425(T) (97.8 % sequence similarity). The DNA-DNA relatedness between strain EMB117T and F. johnsoniae ATCC 17061T was about 18 %. On the basis of the phenotypic, chemotaxonomic and molecular data, strain EMB117T represents a novel species within the genus Flavobacterium, for which the name Flavobacterium defluvii sp. nov. is proposed. The type strain is EMB117T (=KCTC 12612T=DSM 17963T).

Flavobacterium aquidurense sp. nov. and Flavobacterium hercynium sp. nov., from a hard-water creek.

Ten new Flavobacterium-like strains were isolated from freshwater of the hard-water creek Westerhöfer Bach, northern Germany. These strains formed two phylogenetic groups: strains WB 1.1-56T, WB 1.1-04, WB 1.1-14, WB 1.1-57 and WB 1.1-63; and strains WB 4.2-33T, WB 4.1-86, WB 4.2-34, WB 4.2-32 and WB 4.2-78. Cells were Gram-negative, yellow-pigmented, chemoheterotrophic rods. Their major fatty acid profiles were similar, consisting of iso-C(15 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 0) 3-OH and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH). DNA G+C contents for strains WB 1.1-56T and WB 4.2-33T were 33.5 and 37.5 mol%, respectively. Phylogenetic analysis based on almost complete 16S rRNA gene sequences indicated that strain WB 1.1-56T was phylogenetically most closely related to Flavobacterium frigidimaris KUC-1T, and that strain WB 4.2-33T was related most closely to F. frigidimaris KUC-1T and Flavobacterium saccharophilum DSM 1811T. Levels of 16S rRNA gene sequence similarity between strains WB 1.1-56T and WB 4.2-33T and the type strains of recognized members of the genus Flavobacterium were below 98 %. DNA-DNA hybridization experiments confirmed the separate genomic status of strains WB 1.1-56T and WB 4.2-33T. Strains WB 1.1-56T and WB 4.2-33T and their respective relatives differed from phylogenetically related Flavobacterium species based on several phenotypic characteristics. On the basis of their phenotypic and phylogenetic distinctiveness, the two groups of strains are considered to represent two novel species, for which the names Flavobacterium aquidurense sp. nov. (type strain WB 1.1-56T=DSM 18293T=CIP 109242T) and Flavobacterium hercynium sp. nov. (type strain WB 4.2-33T=DSM 18292T=CIP 109241T) are proposed.