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Analysis of the 16S rRNA gene sequences of species currently assigned to the genus Flexibacter has shown extensive intrageneric phylogenetic heterogeneity. It has been shown in previous studies that the species [Flexibacter] sancti, [Flexibacter] filiformis and [Flexibacter] japonensis were most closely related to Chitinophaga pinensis. In addition, [Cytophaga] arvensicola and species of the genus Terrimonas also clustered into this phylogenetic group. Although the similarities of 16S rRNA gene sequences were low (88.5–96.4 %), there is no evidence for clear phenotypic differences between these organisms that justify assignment to different genera. A proposal is made to transfer these species to the genus Chitinophaga as Chitinophaga sancti comb. nov., Chitinophaga filiformis comb. nov., Chitinophaga japonenis comb. nov. and Chitinophaga arvensicola comb. nov. on the basis of phylogenetic and phenotypic data. Furthermore, a novel species is described within this genus, Chitinophaga skermanii sp. nov., with strain CC-SG1BT (=CCUG 52510T=CIP 109140T) as the type strain.

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers ( Cellvibrio , Chondromyces , Flexibacter , Lysobacter , and Pseudomonas ). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.

We studied the effects of 2 bacterivorous protistan predators on the phenotypic and taxonomic successions of an experimental bacterial community growing in continuous culture with the cryptophyte Cryptomonas phaseolus. Two predators were inoculated to the cultivation system, the mixotrophic flagellate Ochromonas sp., an interception feeder, and the filter-feeding heterotrophic ciliate Cyclidium glaucoma. The system was sampled every second day over a period of 3 mo. The competition between algae and bacteria for limiting phosphorus (20 μg P l - 1 d - 1 ) was reflected in pronounced fluctuations of the ratio of algal versus bacterial biomass. High grazing pressure exerted on bacteria by the flagellate led to a strong decrease of bacterial abundance and biomass, with only few cells left freely dispersed. A succession of various grazing-resistant morphologies was observed, such as microcolonies, large aggregates and filaments. A population affiliated with Aquabacterium sp. formed grazing-resistant co-colonies with a Caulobacter spp., indicating that these 2 bacteria might find a common refuge from protistan grazing within aggregates or biofilms. During the course of the experiment these large colonies were succeeded by grazing-protected morphotypes related to the Flexibacter sancti-Flavobacterium ferrugineum lineage of the Sphingobacteriales. In contrast, the ciliate induced only a slight reduction of bacterial abundance, and bacteria even recovered to higher than original numbers after a few weeks. However, pronounced successions of microbial populations were also observed in the presence of the ciliate.

The taxonomic position of a novel marine bacterium isolated from the green alga Ulva fenestrata collected in the Sea of Japan was established. Cells of the strain studied, designated KMM 6017T, were strictly aerobic, heterotrophic, pink-pigmented, non-motile by gliding, Gram-negative and oxidase-, catalase-, β-galactosidase- and alkaline phosphatase-positive. 16S rRNA gene sequence analysis indicated that the strain occupied a distinct lineage within the phylum ‘Bacteroidetes’ and formed a cluster with [Flexibacter] tractuosus and Reichenbachia agariperforans. The G+C content of the DNA of KMM 6017T was 40·2 mol%. The major respiratory quinone was MK-7. The predominant fatty acids were i15 : 1, i15 : 0 and i17 : 0 3-OH (34·2, 24 and 7·7 %, respectively). On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium was assigned to the genus Roseivirga gen. nov., as Roseivirga ehrenbergii gen. nov., sp. nov. The type strain is KMM 6017T (=KCTC 12282T=LMG 22567T).

Thlaspi goesingense is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing T. goesingense were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction—Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the Holophaga/Acidobacterium division and α-Proteobacteria were found. In addition, high-G+C gram-positive bacteria, Verrucomicrobia , and microbes of the Cytophaga/Flexibacter/Bacteroides division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the Proteobacteria , but also bacteria belonging to the Cytophaga/Flexibacter/Bacteroides division, the Holophaga/Acidobacterium division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related Sphingomonas 16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera Methylobacterium, Rhodococcus , and Okibacterium , whereas the majority of endophytes showed high levels of similarity to Methylobacterium mesophilicum . Additionally, Sphingomonas spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.

The possibility of the absence of cellular triamines in the four flavobacteria, Flammeovirga aprica IAM 14298, 'Flexibacter aurantiacus subsp. Copepodarum' IFO 15978, 'Microscilla arenaria' IFO 15982 and Saprospira grandis NCIMB 1363, phylogenetically loc

金沢大学大学院自然科学研究科物質情報解析, Heterocapsa circularisquama, a noxious marine dinoflagellate, has frequently caused red tides and killed cultured bivalves in western Japanese embayments. Observations by electron and epifluorescence microscopy revealed that many bacterial particles were detected inside the H. circularisquama cells. To elucidate the identity and origin of the intracellular bacteria associated with H. circularisquama, bacterial 16S ribosomal RNA genes (16S rDNA) were directly amplified by polymerase chain reaction from 5 clonal cultures of the algal strains that had been isolated from different localities. After cloning, randomly selected clones including the bacterial 16S rDNA fragments were sequenced. The results showed that intracellular bacterial populations consisted of only 2 ribotypes of bacteria, even though the algal strains were established from different localities. One ribotype (bac-G), which was dominant in the intracellular bacterial population, belonged to the gamma-proteobacteria group, and the other (bac-F) clustered with the Flexibacter-Cytophaga-Bacteroides group. Both of these are novel species of endosymbiotic bacteria because of their unique 16S rDNA sequences. Furthermore, the populations of extracellularly associated bacteria were also composed of bac-G and bac-F, indicating that they originated from the intracellular bacteria. Fluorescence in situ hybridization targeting 16S rRNA indicated that bac-G appeared to localize on the algal nuclear surface, while bac-F was distributed in the cytoplasmic space of algal cells. These results strongly suggest that only a few species of specific bacteria reside and share their habitat in the H. circularisquama cells as endosymbionts.

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.

Storage of swine manure is associated with the microbiological production of a variety of odorous chemicals including ammonia, organic acids and alcohols, and sulphides. Although largely the product of microbiological activity, little is known about the microorganisms present in swine manure. In order to gain a better understanding of the types and activities of the microorganisms present, representative strains of microorganisms were isolated from faeces and stored manure slurry, identified, and physiologically characterized. For swine manure slurry samples, total anaerobe colony counts were greatest when a non-selective, habitat simulating medium containing clarified swine manure slurry was used whereas the highest counts for faecal anaerobes were obtained on rumen fluid containing medium. Faecal and slurry samples were also plated onto the appropriate medium containing the antibiotics tetracycline, erythromycin and tylosin (10 micro g ml-1, individually) and the proportional counts of organisms capable of growing in the presence of these antibiotics determined. Randomly selected isolates from the highest dilutions were identified by 16 s rDNA sequence analysis, and selected physiological characteristics were determined. The results of these examinations indicate that the predominant culturable microorganisms from these environments are obligately anaerobic, low mol percentage G + C Gram positive bacteria (Firmicutes) who are members of Clostridial, Eubacterial, and Lactobacillus/Streptococcus phylogenetic groups. Isolates similar to Sporomusa and Flexibacter/Cytophaga/Bacteroides (CFB or Bacteroidetes) groups were also obtained. Although similar overall, faecal and slurry samples differed in bacterial composition. Manure slurry samples were dominated by organisms similar to Clostridium coccoides and Enterococcus species whereas the distribution of species present in faeces appeared much broader. Whereas most of the pure cultures could be assigned to known phylogenetic groupings, few could be identified as known species. Examination of some growth and physiological characteristics of faecal and slurry isolates showed these to be primarily carbohydrate fermenters, although some were able to ferment lactate and amino acids. When the ability of manure and faecal isolates to ferment protein, peptides and amino acids was examined, a relatively small percentage of these were able to do so and most of these fermented carbohydrates in addition to the amino acid sources provided. The predominant amino acid fermenters were most closely related to C. coccoides and C. botulinum, but representatives of the Bacteroides, Staphylococcus, Enterococcus and other phylogenetic groups were also found. The results reported here are compared with those obtained from clone libraries prepared from the same environmental samples.

The present study was undertaken to describe the epiflora of the eggs of an important fishing species collected in a coastal zone. Microflora associated with sardine (Sardina pilchardus) eggs collected in the Ría de Vigo was examined from January to June 2000. The count was carried out in three different ways: a total direct count by epifluorescence, a heterotrophic bacteria count on marine agar (MA) and a total vibrio count on thiosulfate citrate bile sucrose (TCBS). It was observed that the counts of total bacteria by epifluorescence were always higher by 2-3 logarithms than the bacterial counts on MA, and by 3-4 logarithms than the count of vibrios on TCBS. In both cases the differences were statistically significant. Throughout the sampling period only a slight variation was observed in the counts undertaken, and in the measured physicochemical parameters. For the qualitative study, 250 strains isolated from MA and 81 strains recovered on TCBS were identified. Members of the genera Vibrio, Pseudoalteromonas, Pseudomonas and Moraxella were found to dominate on the culturable adherent microflora of sardine eggs and Aeromonas, Tenacibaculum (Flexibacter), Flavobacterium and Cytophaga spp. were present in minor amounts. Vibrio anguillarum and Vibrio fischeri, pathogens of fish larvae, as well as Tenacibaculum ovolyticum, a pathogen of fish eggs, were detected.

5 pages, 5 references., Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. The present study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain). One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first time microbiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCR amplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). The applied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteria with potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint, the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of the sequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related to environmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% to members of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number of clones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteria in Altamira cave.

River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the α subdivision of the division Proteobacteria (α- Proteobacteria ), γ- Proteobacteria , gram-positive bacteria, Cyanobacteria , β- Proteobacteria , δ- Proteobacteria , and the Prosthecobacter group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups Cytophaga , Sphingobacterium , Lewinella , and Cytophaga aurantiaca . CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.

Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.

Los microorganismos han sido siempre objeto de estudios como productores de sustancias antibacterianas; sin embargo, también son consideradas productores de sustancias antifúngicas, antivirales, antiparasitarias, citotóxicas e inhibitorias de otras formas de crecimiento celular. Este trabajo describe el aislamiento, el potencial inhibitorio y la caracterización fenotípica de cepas bacterianas nativas asociadas a los moluscos bivalvos Argopecten purpuratus “concha de abanico” y Crassostrea gigas “ostra” en sistemas de cultivo. De un total de 345 cepas de bacterias colectadas se recuperaron 20 cepas marinas con capacidad inhibitoria de amplio espectro de bacterias patógenas de peces, moluscos y crustáceos; y los más sensibles fueron los patógenos Aeromonas sobria P-281, Aeromonas hydrophila ATCC 7966, Vibrio vulnificus ATCC 27562 y Vibrio parahaemolyticus ATCC 17803. La caracterización fenotípica de las cepas con capacidad inhibitoria permitió identificar los siguientes géneros: Vibrio (40%), Aeromonas (15%), Flavobacterium (10%), Pseudomonas (5%), Moraxella (5%), Flexibacter (5%). No fueron identificadas 20%. Los resultados sugieren que las bacterias aisladas podrían ser aprovechadas como agentes probióticos en el control biológico de patógenos de organismos marinos de interés en maricultura.

Electron microscopy and biomolecular methods have been used to describe and identify microbial communities inhabiting the glassy margins of ocean floor basalts. The investigated samples were collected from a neovolcanic ridge and from older, sediment-covered lava flows in the rift valley of the Knipovich Ridge at a water depth around 3500 m and an ambient seawater temperature of −0.7°C. Successive stages from incipient microbial colonisation, to well-developed biofilms occur on fracture surfaces in the glassy margins. Observed microbial morphologies are various filamentous, coccoidal, oval, rod-shaped and stalked forms. Etch marks in the fresh glass, with form and size resembling the attached microbes, are common. Precipitation of alteration products around microbes has developed hollow subspherical and filamentous structures. These precipitates are often enriched in Fe and Mn. The presence of branching and twisted stalks that resemble those of the iron-oxidising Gallionella , indicate that reduced iron may be utilised in an energy metabolic process. Analysis of 16S-rRNA gene sequences from microbes present in the rock samples, show that the bacterial population inhabiting these samples cluster within the γ- and ϵ-Proteobacteria and the Cytophaga/Flexibacter/Bacteroides subdivision of the Bacteria, while the Archaea all belong to the Crenarchaeota kingdom. This microbial population appears to be characteristic for the rock and their closest relatives have previously been reported from cold marine waters in the Arctic and Antarctic, deep-sea sediments and hydrothermal environments.

Microbial mats from hydrogen sulfide-rich waters and cave-wall biofilms were investigated from Cesspool Cave, Virginia, to determine community composition and potential geomicrobiological functioning of acid-producing bacteria. Rates of microbial mat chemoautotrophic productivity were estimated using [ 14 C]-bicarbonate incorporations and microbial heterotrophy was determined using [ 14 C]-leucine incubations. Chemoautotrophic fixation was measured at 30.4 - 12.0 ng C mg dry wt -1 h -1 , whereas heterotrophic productivity was significantly less at 0.17 - 0.02 ng C mg dry wt -1 h -1 . The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that the mats are not a high quality food source for higher trophic levels. Ribosomal RNA-based methods were used to examine bacterial diversity in the microbial mats, revealing the presence of at least five strains of bacteria. The identity of some of the strains could be resolved to the genus Thiothrix and the Flexibacter - Cytophaga - Bacteriodes...

The composition and chlorophenol-degrading potential of groundwater bacterial community in a permanently cold, oxygen-deficient chlorophenol contaminated aquifer at Kärkölä, Finland was studied with the aim of evaluating in situ bioremediation potential. The groundwater contained from 10(4) to 10(7) microscopically counted cells/ml and up to 10(5) CFU/ml heterotrophic bacteria cultivable at 8 and 20 degrees C. Of the 102 pure cultures, of which 86% Gram-negative, from the plume area (10,000 microg of chlorophenols/l), 57% degraded 2, 3, 4, 6-tetrachlorophenol (TeCP), the main component of the wood preservative which was the source of contamination: 17% also degraded pentachlorophenol (PCP). The degraders were scattered among 16 different clusters of Gram-negatives mainly proteobacteria and members of Cytophaga/Flexibacter/Bacteroides phylum judged by the composition of whole-cell fatty acids. Only one Gram-positive degrading cluster was found containing seven actinobacteria closest to Nocardioides. Of the 88 pure cultures isolated from outside the plume (10 microg of chlorophenols/l) 67% were Gram-negative. Seven percent of the isolates degraded 2, 3. 4, 6-TeCP and/or PCP. Five of the Gram-positive isolates from outside the plume were Mycobacterium/Rhodococcus-related actinobacteria and O-methylated 2, 3, 4, 6-TeCP and PCP. The results show that chlorophenol degrading bacterial flora had been enriched as a result of contamination of the aquifer. This suggests significant in situ bioremediation potential of the site.

Distinct profiles of extracellular proteolytic enzyme activity were observed in the water column of the North Atlantic, with maximum potential proteolytic activity occurring in the top 35 m. The proteolytic enzyme V max values varied significantly and decreased from 1.46 nM min −1 in surface waters to 0.365 nM min −1 at 100 m. In contrast, K m values increased with depth from about 70 to 360 μM. Cell-associated enzymes accounted for the majority of the observed proteolytic activity. Dissolved enzymes comprised only 30–40% of the total extracellular enzyme activity and exhibited a low substrate affinity ( K m =∼1000 μM). These observations indicate clear stratification of bacterial associated extracellular enzyme activity, with the maximum activity in surface waters. This is consistent with some environmental changes in the water column, especially algal biomass and nitrate concentration. Bacterial mediated nitrogen remineralization in surface waters was approximately three times the total nitrogen demand of phytoplankton and bacteria. We determined bacterial population diversity using 16S rRNA sequence analysis and found evidence for stratification, with a higher representation of the Cytophaga/Flexibacter/Bacteriodes group at 5 m compared to 100 m. No similar stratification was observed among the α -proteobacterial SAR11 cluster, which were especially prevalent in the PRIME eddy. However, sequences phylogenetically related to another marine cluster, SAR122, were only observed at 100 m. We suggest that stratification of proteolytic activity within the water column may be explained at least in part, by differences in the composition of the bacterial community.

The aim of this work was to isolate, identify and type carbofuran-degrading bacteria from two geographically distant soils. Restriction Fragment Length Polymorphism (RFLP) patterns of the 16S rRNA gene and partial 16S rRNA sequence analysis were used to classify the 23 isolates obtained. Nine of them showed high similarity to Pseudomonas strains, seven showed similarity to the Flexibacter/Cytophaga/Bacteroides group and the remainder showed similarity to other bacterial genera. Isolates within the same group were sub-typed by comparing partial 16S rRNA sequences and SDS-PAGE analysis of their total protein profiles. Many of the UK isolates showed similarity to the Pseudomonas genera, while most of the Greek isolates showed similarity to the Flexibacter/Cytophaga/Bacteroides group. Only two Chrysobacterium strains isolated from both the UK and Greek soils were identical.

Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T); (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albensis M384(T); (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.

Rust-coloured shells of the gastropod Hydrobia ulvae collected in the Wadden Sea near Texel and in the Jade Busen were analysed under the scanning electron microscope. Most of the shells were found to be covered with a microbial community encrusted with an iron-rich mineral containing traces of Mn, Mg, Ca and Si (EDAX analysis). The community formed a biofilm including two morphotypes of diatoms identified as Cocconeis placentula and Achnanthes lemmermanni, two morphotypes of slender filamentous bacteria resembling Leucothrix and Flexibacter, aggregates of coccoid cells and large trichomes resembling members of the cyanobacterial orders Pleurocapsales and Stigonematales, respectively. The most frequent microorganisms of the biofilm were diatoms and filamentous bacteria.

Genetic variability among strains of Flavobacterium columnare (formerly Flexibacter columnaris) was characterized using restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, 16S rDNA sequencing, and DNA-DNA hybridization. A total of 23 Fl. columnare strains isolated from diseased fishes in various areas were divided into three distinct genomic groups. The main group, genomovar 1 consisted of 20 strains, including the type strain. Two strains were assigned to genomovar 2, and one to genomovar 3. However, as we found their phenotypic characteristics to be identical, there is no evidence to classify strains of these genomovars as belonging to new species or sub-species.

The ability of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of common carp, Cyprinus carpio L., was evaluated using a gill perfusion model. A comparison between a high and a low virulence strain of F. columnare was made and evaluated in comparison to results obtained previously with an in vivo model. The ion composition of the water of the organ bath in which the gills were suspended was varied and the influence on adhesion processes assessed. Experiments were carried out to examine the influence of water quality (i.e. nitrite and organic matter) and temperature on the capacity of the bacteria to adhere. It was found that the high virulence strain adhered more readily than the low virulence strain, as was found during the in vivo experiments. Moreover, it was observed that adhesion of the high virulence strain was enhanced by a number of factors. These were immersion of the gill in bivalent, ion-rich water, the presence of nitrite or organic matter, and high temperatures.

The nearly complete small subunit 16S ribosomal RNA (rRNA) gene sequence amplified by polymerase chain reaction was determined for Flavobacterium psychrophilum (formerly Flexibacter psychrophilus) by using automated nucleotide sequencing. The sequence was found to be 1,465 base pairs (bp) in length, a size consistent with previously determined sequences for 14 other bacterial species from various taxa, including the yellow-pigmented bacteria. Sequence signatures confirmed that this organism was a member of the bacterial division Bacteroides–Flavobacterium. Parsimonious and additive phylogenetic trees were constructed with homology, and pairwise evolutionary distances were used to estimate phylogenetic relationships. Data show that F. psychrophilum, F. columnare, and Flexibacter maritimus are closely related, have a common descent, and represent a distinct group within the division Bacteroides–Flavobacterium. This group also included other organisms from the genera Flavobacterium and Cytophaga. Fu...

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare (Flexibacter columnaris) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum (Flexibacter psychrophila), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter mari...

Recently, the single reactor system for high activity ammonia removal over nitrite (SHARON) process was developed for the removal of ammonia from wastewater with high ammonia concentrations. In contrast to normal systems, this nitrifying reactor system is operated at relatively high temperatures (35°C) without sludge retention. Classical methods to describe the microbial community present in the reactor failed and, therefore, the microorganisms responsible for ammonia removal in this single reactor system were investigated using several complementary molecular biological techniques. The results obtained via these molecular methods were in good agreement with each other and demonstrated successful monitoring of microbial diversity. Denaturing gradient gel electrophoresis of 16S rRNA PCR products proved to be an effective technique to estimate rapidly the presence of at least four different types of bacteria in the SHARON reactor. In addition, analysis of a 16S rRNA gene library revealed that there was one dominant (69%) clone which was highly similar (98.8%) to Nitrosomonas eutropha. Of the other clones, 14% could be assigned to new members of the Cytophaga/Flexibacter group. These data were qualitatively and quantitatively confirmed by two independent microscopic methods. The presence of about 70% ammonia oxidizing bacteria was demonstrated using a fluorescent oligonucleotide probe (NEU) targeted against the 16S rRNA of the Nitrosomonas cluster. Electron microscopic pictures showed the typical morphology of ammonia oxidizers in the majority of the cells from the SHARON reactor.

A bacteriological survey in three fish farms with intensive culture of gilt-head sea bream (Sparus aurata L.) was conducted in southwestern Spain (provinces of Cadiz, Malaga, and Huelva) from 1990 to 1996. All the disease problems occurring during this period in the S. aurata were investigated, focusing on the isolation, characterization, antimicrobial resistance and pathogenicity of the bacterial isolates affecting the fish. The main pathogenic microorganisms isolated were Vibrio (67.8%), Pseudomonas (13.5%), Photobacterium damsela subsp. piscicida (6.7%), Cytophaga/Flexibacter-like bacteria (4.8%), Aeromonas (0.5%), and Gram positive bacteria (6.7%). Although the highest percentages of isolates corresponded to Vibrio and Pseudomonas spp., the strains of P. damsela subsp. piscicida and Cytophaga/Flexibacter-like bacteria caused epizootics with highest degree of mortalities. From the present study, on the basis of the pathogenicity test, we suggest the consideration of V. anguillarum, V. alginolyticus, V. harveyi and V. splendidus as primary pathogenic species for gilt-head sea bream.

Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.

Since 1993, significant mortality outbreaks of Penaeus stylirostris usually during winter, have been reported from grow out ponds in New Caledonia. During the mortality peaks in 1994 and 1995, moribund and apparently healthy shrimps were collected from all New Caledonian farms for bacteriological and histological studies in order to describe the `Syndrome 93' disease. Hemolymph was collected aseptically and spread on to Marine agar 2216E and TCBS agar (Difco). Hemolymph from apparently healthy shrimps was generally sterile. Each contaminated hemolymph contained various bacteria, most of the time Vibrionaceae, but also Pseudomonas spp., Flavobacterium spp., Acinetobacter spp., Cytophaga spp., Moraxella spp., Flexibacter spp., Alteromonas spp. and Bacillus spp. In contrast, moribund shrimps were always infected with Vibrionaceae and sometimes a combination of Vibrionaceae and Pseudomonas sp. Preliminary identification studies based on morphological, physiological and biochemical characteristics including testing for the utilization of 100 sources of carbon, show that one particular group of Vibrio sp. was present and predominant in shrimps collected throughout farms in New Caledonia. Molecular chemistry techniques like ribotypic patterns and DNA–DNA hybridization were applied, as a complement to conventional identification methods, to 18 selected strains from moribund shrimps randomly selected from nine mortality peaks. They showed the presence of two different groups of Vibrio sp., provisionally called `Noumea 1' and `Noumea 2' for the purpose of this study. The Vibrio belonging to the first group have been identified as Vibrio penaeicida. Identification of the second group is now in progress, and it is already clear that these bacteria belong to a different species. Toxin production capacity and experimental pathogenicity of these species are currently being studied. Some identified V. penaeicida strains demonstrated a very high virulence. The etiological role of these bacteria is also being investigated, particularly through the search for a suitable experimental model.

The structures of topostins, a novel type of mammalian DNA topoisomerase I inhibitors isolated from the bacterium Flexibacter topostinus sp. nov., have remained unknown. Reexamination of the production of topostins resulted in the isolation of original topostins B (B553 (1) and B567 (2)) and new related compounds named topostins D (D640 (3) and D654 (4)). Their structures, including the absolute stereochemistry, were determined by spectroscopic analyses and chemical degradations. These substances are lipid-containing amino acids and peptides.

Sixteen heparinase-producing isolates, related to Sphingobacterium heparinum, were grouped into three major clusters by SDS-PAGE and DNA-rRNA hybridizations. Based on a polyphasic approach, it was shown that isolates of two of these clusters and S. heparinum species belong to a new genus for which the name Pedobacter is proposed. The genus consists of Pedobacter heparinus comb. nov. (formerly Sphingobacterium heparinum), which is the type species, Pedobacter piscium comb. nov. (formerly Sphingobacterium piscium), Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. and four as-yet-unnamed DNA hybridization groups. All the previously named taxa can be discriminated by phenotypic features, but have strong overall similarities with representatives of the genus Sphingobacterium and the misclassified species [Flexibacter] canadensis. All these organisms constitute a separate rRNA branch in rRNA superfamily V for which the family Sphingobacteriaceae fam. nov. is proposed.

Screening of actinomycetes and fungi for genes involved in beta-lactam production can be carried out using a technique involving both PCR with conserved primers to the isopenicillin N synthase gene and a Southern dot blotting. This technique was successful with nine out of nine known beta-lactam producing Actinomycetes tested and three out of six known beta-lactam producing Fungi tested. It proved unable to detect the isopenicillin N synthase gene in the two Flexibacter beta lactam producing eubacteria strains tested. It also identified the presence of potential beta-lactam production genes in two known none beta-lactam producing actinomycetes and one out of fourteen novel newly isolated actinomycetes. This technique has potential for use in commercial screens for novel beta-lactam producers.

Many cases of fin or tail rot, skin ulcer, and jaw erosion were reported over the years in cultured marine fish. The aetiological role of various bacteria belonging to the Flavobacterium-Cytophaga group was frequently suspected, but only 3 bacterial species pathogenic for marine fish were fully described until now.Flexibacter maritimus was first identified in Japan in the late seventies from several fish species reared in sea water. Subsequently, the disease was recognized in very different geographic areas such as France, Scotland, Spain, Malta, Tasmania and California. The pathogenicity of the bacterium was demonstrated experimentally, phenotypic and genomic characteristics as well as virulence mechanisms were investigated, and treatment methods were proposed. Significant losses due to Flexibacter maritimus may occur locally.In Norway, Flexibacter ovolyticus was isolated from the adherent bacterial epiflora of Atlantic halibut eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae.Chryseobacterium scophthalmum (first described under the epithet Flavobacterium scophthalmum) was isolated 10 years ago in Scotland from turbot suffering from gill hyperplasia and haemorrhagic septicaemia. A large collection of strains was studied, and investigations of the disease included experimental infection, histopathology and immunization trials.Until now, Flexibacter ovolyticus and Chryseobacterium scophthalmum have not been recognized in fish species or regions other than those from which they were initially isolated.

Previously, time courses of the colony formation process of soil bacteria on agar plates were simulated by colony-forming curves (CFCs). Bacteria were isolated from the colonies that appeared during an incubation period corresponding to each component curve of the CFC, respectively, and they were placed into groups I, II, III or IV, which were based on the order of colonization, so that their physiological characteristics could be studied (Gorlach, K. et al. (1994) J. Gen. Appl. Microbiol. 40, 507–517). We report here on the results of phylogenetic analysis of the isolates from each CFC group. A wide variety of eubacteria were included in the collection. Strains closely related to Arthrobacter were those from CFC groups I or II, groups of faster growers, and strains in Proteobacteria α-subdivision were those from groups III or IV, which grew more slowly. In contrast, some phylogenetic groups, such as Firmicutes low G+C, Cytophaga/Flexibacter/Bacteroides and Proteobacteria β-divisions exhibited heterogeneity regarding CFC grouping. As for their requirement for media, isolates belonging to the Cytophaga/Flexibacter/Bacteroides division, the Proteobacteria α-subdivision, β-subdivision and the Firmicutes high G+C division, except for Arthrobacter-related strains, were oligotrophic bacteria. In contrast, those belonging to the Firmicutes low G+C division, the Proteobacteria γ-subdivision and the A. globiformis-related group in the Firmicutes high G+C division were not oligotrophic.

Gliding bacteria of the genera Cytophaga and Flexibacter contain unusual sulfonolipids, called capnoids, in their outer membrane. The parent compound capnine has been identified as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Structural studies on N-acylaminosulfonates purified from a cellulolytic cytophaga, Cytophaga hutchinsonii ATCC 33406, revealed a structural variant of the aminosulfonate core in addition to a unique 2-hydroxy fatty acyl group, i17:0(2-OH), in amide linkage with the core. The source of the structural variation in the aminosulfonate core of Cytophaga hutchinsonii arose from the addition of two carbons to the acyl chain and the insertion of two trans-double bonds. Thus, the aminosulfonate core purified from Cytophaga hutchinsonii was identified as 2-amino-3-hydroxy-tras, trans-17-methylocta-4,7-diene-1-sulfonic acid and assigned the trivial name capnosine. A second cellulolytic cytophaga, CyP2, also showed this structural variation, which was absent from the noncellulolytic isolate CyS1. Further structural studies on sulfonolipids isolated from other cellulolytic and noncellulolytic isolates will determine the taxonomic significance of this structural variant of capnine.Key words: N-acylcapnine, N-acylaminosulfonates, capnosine, Cytophaga, cellulolytic cytophagas.

Psychrophilic, yellow-pigmented, seawater-requiring bacteria isolated from the pycnocline of meromictic Burton Lake and from sea ice cores obtained in the Vestfold Hills (68 degrees S, 78 degrees E) in eastern Antarctica were characterized. Phenotypic analysis showed that the strains isolated formed two distinct taxa. The first taxon included nonmotile, nutritionally fastidious strains that were isolated from the pycnocline of Burton Lake. The cells of these strains were morphologically variant, ranging from vibrioid to ring shaped to coiled and filamentous; in addition, the strains were unable to metabolise carbohydrates or polysaccharides and had DNA G + C contents of 27 to 29 mol%. The strains of the second taxon, which were isolated from sea ice cores and from ice aigal biomass, were saccharolytic, exhibited rapid gliding motility, were rodlike to filamentous, and had DNA G + C contents of 36 to 38 mol%. A 16S ribosomal DNA (rDNA) sequence analysis revealed that the two Antarctic taxa formed related but distinct lineages within the [Flexibacter] maritimus rRNA branch of the family Flavobacteriacrae. The levels of 16S rDNA sequence similarity between the taxa were 90.5 to 91.3%, while the levels of similarity to other members of the [F.] maritimus rRNA branch were 85 to 90%. The whole-cell lipid profiles of the Antarctic strains were mainly comprised of branched and unbranched monounsaturated C15 to C17 fatty acids. The presence of significant levels of the lipids a 15:1 omega 10c and a17:1 omega 7c appeared to be useful biomarkers for the new Antarctic taxa and for differentiating these organisms from other members of the family Flavobacteriaceae. On the basis of polyphasic taxonomic data we propose that the new taxa are novel bacterial species designated Psychroserpens burtonensis gen. nov., sp. nov. (type strain, ACAM 188) and Gelidibacter algens gen. nov., sp. nov. (type strain, ACAM 536).

Phylogenetically, Cytophaga aprica and Cytophaga diffluens occupy independent positions in the flavobacter-bacteroides phylum. Both of these organisms are gram-negative rods that are motile by gliding, chemoorganotrophic, and aerobic, degrade several kinds of biomacromolecules, and inhabit marine environments. Their major isoprenoid quinone is menaquinone 7. The G+C content of the DNA of C. aprica is 35 to 37 mol%, and the G+C content of the DNA of C. diffluens is 40 to 42 mol%. In addition to constituting an independent phylogenetic lineage, each species has a distinctive cellular polyamine constitution. C. aprica is characterized by possessing cadaverine as its major polyamine, and C. diffluens is characterized by possessing spermidine, in contrast to most species of the genera Cytophaga, Flavobacterium, and Flexibacter and related organisms, which possess homospermidine. Transfer of C. aprica to the genus Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. and transfer of C. diffluens to the genus Persicobacter gen. nov. as Persicobacter diffluens comb. nov. are proposed.

Soil contamination with heavy metals is a widespread problem, especially prominent on grounds lying in the vicinity of mines, smelters, and other industrial facilities. Many such areas are located in Southern Poland; they are polluted mainly with Pb, Zn, Cd, or Cu, and locally also with Cr. As for now, little is known about most bacterial species thriving in such soils and even less about a core bacterial community—a set of taxa common to polluted soils. Therefore, we wanted to answer the question if such a set could be found in samples differing physicochemically and phytosociologically. To answer the question, we analyzed bacterial communities in three soil samples contaminated with Pb and Zn and two contaminated with Cr and lower levels of Pb and Zn. The communities were assessed with 16S rRNA gene fragments pyrosequencing. It was found that the samples differed significantly and Zn decreased both diversity and species richness at species and family levels, while plant species richness did not correlate with bacterial diversity. In spite of the differences between the samples, they shared many operational taxonomic units (OTUs) and it was possible to delineate the core microbiome of our sample set. The core set of OTUs comprised members of such taxa as Sphingomonas, Candidatus Solibacter, or Flexibacter showing that particular genera might be shared among sites ~40 km distant. Electronic supplementary material The online version of this article (doi:10.1007/s00248-013-0344-7) contains supplementary material, which is available to authorized users.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO 3 − ) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria ( Flexibacter , Geobacter , Rhodoferax , and Shewanella ) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO 3 , but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus , Thaurea , and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Línea Investigación: Línea 6. Biorremediación de suelos contaminados This work aimed to assess the effectiveness of different in situ bioremediation treatments (bioaugmentation, biostimulation, bioaugmentation and biostimulation, and natural attenuation) on creosote polluted soil. Toxicity, microbial respiration, creosote degradation and the evolution of bacterial communities were analyzed. Results showed that creosote decreased significantly in all treatments, and no significant differences were found between treatments. However, some specific polycyclic aromatic hydrocarbons (PAH) were degraded to a greater extent by biostimulation. The dominance of low temperatures (8.9ºC average) slowed down microbial creosote and PAH uptake and, despite significantly creosote degradation (>60%) at the end of the experiment, toxicity remained constant and high throughout the biodegradation process. DGGE results revealed that biostimulation showed the highest microbial biodiversity, although at the end of the biodegradation process, community composition in all treatments was different from that of the control assay (unpolluted soil). The active uncultured bacteria belonged to the genera Pseudomonas, Sphingomonas, Flexibacter, Pantoea and Balneimonas, the latter two of which have not been previously described as PAH degraders. The majority of the species identified during the creosote biodegradation belonged to Pseudomonas genus, which has been widely studied in bioremediation processes. Results confirmed that some bacteria have an intrinsic capacity to degrade the creosote without previous exposure. Tecnología Química y Ambiental

Eleven isolates of Flexibacter columnaris from channel catfish Ictalurus punctatus, blue catfish I. furcatus, largemouth bass Micropterus salmoides, and fathead minnow Pimephales promelas exhibiting clinical signs of columnaris disease were studied. All isolates were confirmed as F. columnaris based on their colonial and cellular morphology and on biochemical and physiological characteristics. Although the isolates were from different fish species and different tissues, most of their biochemical characteristics were uniform and were similar to the reference strain of F. columnaris (American Type Culture Collection 49512). Slight variations occurred in growth at 15°C, with 0.5% NaCl, and at pH 6 and pH 10. Three phenotypic variations in colony morphology were observed among the isolates, but these were not related to fish species or to tissues of origin.

The Bacteroides-Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree contains a diverse group of bacterial species. Techniques for the genetic manipulation of Bacteroides spp. are well developed (A. A. Salyers, N. B. Shoemaker, and E. P. Guthrie, Crit. Rev. Microbiol. 14:49-71, 1987). Recently we developed techniques to genetically manipulate the gliding bacterium Cytophaga johnsonae (M. J. McBride and M. J. Kempf, J. Bacteriol. 178:583-590, 1996). We now demonstrate that some of these techniques allow genetic manipulation of a number of environmentally or medically significant bacteria in this group. The Bacteroides transposon Tn4351 was introduced into Cytophaga hutchinsonii, Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadensis, Flexibacter sp. strain FS1, and Sporocytophaga myxococcoides by conjugation. Tn4351 integrated itself into the host chromosomes and conferred erythromycin resistance. We isolated several auxotrophic mutants of Flavobacterium meningosepticum following Tn4351 mutagenesis. The C. johnsonae-Escherichia coli shuttle vector pCP11 functioned in C. succinicans but not in the other bacteria. pLYL03 did not replicate in any of these bacteria and should function as a convenient suicide vector. The identification of a system of gene transfer, a selectable marker, a suicide vector, and a transposon that functions in these diverse bacteria allows genetic manipulations to be performed.

This strain 758 of a new member of Flexibacter species isolated from a soil is a Gram-negative, nonflagellated, gliding, long rod or filamentous. The GC contents of the deoxyribonucleic acid of this strain is 49.8 mol %GC. The isolate can be distinguished from other Flexibacter species on the basis of physiological and biochemical properties and DNA relatedness data. Therefore, we propose a new species, Flexibacter japonensis, for this strain. The strain 758 is deposited to the Japan Collection of Microorganisms as JCM 9735.

A 16S rRNA sequence analysis revealed that the genera Cytophaga, Flavobacterium, and Flexibacter are all polyphyletic and should be redefined and reorganized. Cytophaga hutchinsonii, the type species of the genus Cytophaga, belongs to a lineage that also contains Cytophaga aurantiaca. The genus Cytophaga is emended so that it contains only these two species, which decompose distinctly crystalline cellulose. Cytophaga salmonicolor and Cytophaga agarovorans form a lineage which is intermediate between other aerobic species and anaerobic bacteroides. Phenotypically, these organisms are characterized by being facultative anaerobes, inhabiting marine environments, and containing menaquinone-7 and spermidine. We propose that C. salmonicolor and C. agarovorans should be transferred to the genus Marinilabilia gen. nov. as Marinilabilia salmonicolor comb. nov. and Marinilabilia agarovorans comb. nov., respectively.

A severe necrotic myositis characterized by large intramuscular bullae was identified in 100–500 g rainbow trout, Oncorhynchus mykiss (Walbaum), maintained in freshwater netpens. Mortality increased as the temperature decreased, with mortality rates peaking at 10% per month. Flexibacter psychrophilus was recovered from the muscle lesions and kidneys of affected fish. The gross and histological description of the disease, and the phenotypic characteristics of the causative agent are described. The necrotic myositis isolate was utilized to experimentally reproduce the disease.

Isolates of Flexibacter psychrophila were obtained from chinook salmon Oncorhynchus tshawytscha and coho salmon O. kisutch that had previously sustained epizootics of coldwater disease. The pathogen was readily isolated from kidney and mucus of convalescent fish. The organisms were relatively inert in most standard microbiological media but were structurally and serologically homogenous by examination of whole cell protein lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In contrast to the homogeneity observed in phenotypic and serologic assays, the isolates studied elaborated varied ribotypes. All isolates produced a single rDNA spacer amplification product of about 240 base pairs.

The susceptibility of freshwater and marine fish to infection by Flexibacter columnaris and Flexibacter maritimus was assessed in laboratory based experiments. Flexibactor columnaris produced a more severe disease in barramundi compared to goldfish. Both species suffered greater mortality at 25 °C than at 20 °C. Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) had similar levels of susceptibility to F. maritimus infection in seawater with approximate LC 50 values of 2.3 × 10 5 and 1.6 × 10 6 CFU mL −1 respectively. However, at salinity of 15 %. greenback flounder ( Rhombosolea tapirina ) were more resistant to F. maritimus than Atlantic salmon.