A Taxonomic Study of Flavobacteria and Related Gram Negative Yellow Pigmented Rods

One hundred and seventy-nine yellow pigmented Gram negative rods, including 170 new isolates obtained from a variety of habitats, were investigated and the results analysed by computer. Nine phena, embracing 146 strains, were distinguished. Five of these comprised 88 of the 109 non-motile, aflagellate strains (i.e. 'atypical cytophagas') and they should be assigned to a re-defined genus Flavobacterium . A separate phenon containing 41 of the gliding isolates was delineated which should be ascribed provisionally to the genus Flexibacter . Only low G + C contents have been recorded for representative strains from these six phena but one strain (which would have been ascribed to Flavobacterium ) had a high G + C content; such organisms (both peritrichous and non-motile) should comprise a separate genus Empedobacter (Brisou). The remaining three phena respectively comprised strains of Erwinia herbicola , pseudomonads and unclassified aflagellate isolates; their taxonomic position is discussed.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and IB comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and 1B comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

Ecology and taxonomy of chitinoclasticCytophaga and related chitin-degrading bacteria isolated from an estuary

A total of 103 strains of estuarine, Chitinoclastic bacteria isolated from water, and sediment samples collected from the upper Chesapeake Bay, including 17 freshwater and 11 seawater isolates, were subjected to numerical taxonomy analysis. The isolates included 44 yellow-orange pigmented strains classified asCytophaga-like bacteria (CLB) of theCytophagaceae. Salt requirement of the strains ranged from tolerance to 1% NaCl to an absolute requirement for NaCl, with 1% NaCl satisfying this requirement. The largest phenon consisted of facultatively anaerobic, oligo-nitrophilic, and flexirubin pigment-producing freshwater and estuarine isolates, and included reference strains of bothCytophaga johnsonae Stanier andCytophaga aquatilis Strohl and Tait. Other phena, containing a smaller number of strains, comprised marine and estuarine isolates which did not produce flexirubin pigments, and required organic nitrogen for growth and for production of chitinolytic enzymes. Salt-requiring, flexirubin pigment-producing, chitin-degrading strains were, on occasion, isolated from estuarine samples and represented phena found in estuaries. Most of theCytophaga isolates, as well as chitin-degrading species not of the genusCytophaga that were isolated from Chesapeake Bay, clustered in phena representing previously described species of aerobic, zymogenic, chitinoclastic bacteria. When the frequency of occurrence of features related to environmental parameters, viz., pH, salinity, temperature range of growth, and growth on media lacking organic nitrogen, was calculated, ecological groupings of strains in the 2 major phena of CLB could be distinguished among the estuarine, chitin-degrading bacteria.     

Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni

AIMS: To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. METHODS AND RESULTS: Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. CONCLUSIONS: Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. Significance and IMPACT OF THE STUDY: This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.     

Numerical taxonomic study of thermophilic Bacillus isolated from three geographically separated deep-sea hydrothermal vents

Abstract: A numerical taxonomic study has been carried out with 80 strains, newly isolated, from three geographically separated deep-sea hydrothermal vents (Mid-Atlantic Ridge, Guaymas Basin and Lau Basin) and eleven thermophilic reference strains representing 11 Bacillus species. The deep-sea isolates were all halotolerant spore-forming rods and grew aerobically above 65°C. Results from unweighted average linkage cluster analysis of a similarity matrix derived from the simple matching coefficient, showed formation of nine major phena, which were defined at the 83% similarity level or above. Seven phena were composed exclusively of strains isolated from the same site (4 from Mid-Atlantic Ridge, 1 from Guaymas Basin and 2 from Lau Basin). The majority of the Lau Basin isolates clustered with 6 of the reference strains in one phenon, while isolates from Mid-Atlantic Ridge and Guaymas Basin were found separated from this phenon at the 69% similarity level. The other reference strains showed less than 69% similarity with the deep-sea isolates.     

Isolation and characterization of moderately halophilic bacteria from fully cured salted cod (bachalao)

A total of 128 Strains of moderately halophilic bacteria were isolated from wet, dry and desalted bachalao (salted codfish) as well as from fresh cod and curing salt. The viable count of these bacteria in fully cured wet and dry bachalao ranged from 10³ to 10⁷ per g. All strains were characterized with 40 phenotypic tests and clustered using the S _SM coefficient and UPGMA linking analysis. The strains clustered into five phena at 75% similarity, with 77 strains in phenon A and 37 in phenon E. Two main colony types, smooth and rough, were observed and correspond to phena A, B and C on one hand and phenon E on the other. These two types seem to represent the dominating bacterial flora in fully cured, wet and dry bachalao, respectively. Representative strains of the smooth colony type were characterized further and found to grow well in 0.1–4.5 mol I⁻¹ NaCl and at 15–37°C. They have not, at present, been assigned to any known bacterial species.     

Numerical Taxonomy of <Emphasis Type="Italic">Vibrionaceae</Emphasis> Isolated from Cultured Amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S_SM and S_J coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S_SM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish. Received: 27 March 2002 / Accepted: 24 May 2002     

A Taxonomic Study to Establish the Relationship between Exopolysaccharide-Producing Bacterial Strains Living in Diverse Hypersaline Habitats

This study was undertaken to identify exopolysaccharide-producing bacteria gathered from 18 hypersaline habitats. Phenotypic studies performed with 134 isolates revealed the majority of them to be Gram-negative rods with respiratory metabolism, belonging to the genus Halomonas. A numerical analysis of the 114 phenotypic data showed that at an 80% similarity level most of the strains (121) could be grouped into six phenotypic groups. Phenon A included 25 new isolates and the reference strain of Halomonas eurihalina, and phenon B was formed by 77 new isolates and Halomonas maura. Phenon C was also related to H. maura although to a lesser extent than strains in group B. Three phena (D, E, and F) could not be grouped with any of the reference strains and may represent new taxa; their G + C contents and DNA-DNA hybridization data corroborated this hypothesis. Results of this work proved that the most abundant halophilic species EPS producer in hypersaline habitats was H. maura, followed by H. eurihalina.     

A detailed phenotypic characterisation of the type strains of Halomonas species

We have made a detailed phenotypic characterisation of the type strains of 21 species within the genus Halomonas and have also studied any possible intraspecific variation of strains within H. eurihalina, H. halophila, H. maura and H. salina. We used 234 morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests. Nutritional assays were carried out using both classical and miniaturized (BIOLOG system) identification methods. Two different numerical analyses were made using the TAXAN program; the first included the differential data from all the tests carried out whilst the second used only the 57 tests with the highest diagnostic scores (> or = 0.5). The results of both analyses were quite similar and demonstrated the phenotypic heterogeneity of the Halomonas species in question. At a 62% similarity level the type species were grouped into three phena, the main difference between them being the capacity of those included within phenon A (H. aquamarina, H. meridiana, H. cupida, H. pantelleriensis and H. halmophila) to produce acids from sugars. The species grouped in phenon C (H. campisalis, H. desiderata and H. subglasciescola) used fewer organic substrates than the others. The remaining strains were included in phenon B. H. marisflavi was clearly distinct and thus was not included in any of the three phena. High phenotypic similarity (more than 88%) was found between Halomonas campisalis and Halomonas desiderata. The results of our work should allow researchers to minimise the tests required to arrive at a reliable phenotypic characterisation of Halomonas isolates and to select those of most use to differentiate Halomonas species from each other.     

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments. Received: February 25, 2001 / Accepted: July 27, 2001     

Numerical Analysis of Astragalus cicer Microsymbionts

Thirty-seven rhizobium strains, isolated from root nodules of Astragalus cicer (L.) (cicer milkvetch) deriving from different geographic regions, were compared with the representative strains of the known rhizobial species and genera by numerical analysis of phenotypic characteristics. Our results indicated that Astragalus cicer rhizobia were related to the bacteria of Mesorhizobium species and formed two major phena. One phenon, localized on Mesorhizobium loti branch, contained strains from Poland. Another cluster, placed in the vicinity of M. tianshanense, M. mediterraneum, M. ciceri, and M. huakuii, comprised cicer milkvetch nodule isolates from Canada, Ukraine, and one strain from Poland. The relationship of Astragalus cicer microsymbionts to bacteria of the Mesorhizobium species was also supported by phage typing. Received: 10 February 2000 / Accepted: 8 March 2000     

<Emphasis Type="Italic">Psychroflexus lacisalsi</Emphasis> sp. nov., a moderate halophilic bacterium isolated from a hypersaline lake (Hunazoko-Ike) in Antarctica

A novel Gram-negative, aerobic, moderate halophilic, and psychrotolerant bacterium, designated as strain H7^T, was isolated from a hypersaline lake located in Skarvsnes, Antarctica. Cells were filaments with varying lengths. Coccoid bodies developed in old cultures. Growth occurred with 0.5–15% (w/v) NaCl (optimum, 5.8–7.0%), at pH 6.0–10.0 (optimum, pH 7.0–8.0), and at 10–28°C (optimum, 25°C). The strain had a G+C content of 34.9 mol%, which is within the range of 32–36 mol% reported for the genus Psychroflexus. Chemotaxonomic data (major respiratory quinone: MK-6; major fatty acids: aC_15:0, iC_16:0 3-OH, and aC_{15: 1} A) supported the classification of strain H7^T within the genus Psychroflexus. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain H7^T should be assigned to the genus Psychroflexus and has a homology with Psychroflexus salinarum (98.2%), P. sediminis (96.1%), P. torquis (95.2%), P. tropicus (95.8%), and P. gondwanense (92.2%). Strain H7 is not identified as P. salinarum because that DNA-DNA hybridization data were 8.5% between strain H7^T and P. salinarum. The combination of phylogenetic analysis, DNA-DNA hybridization data, phenotypic characteristics, and chemotaxonomic differences supported the view that strain H7^T represents a novel species of the genus Psychroflexus. The name Psychroflexus lacisalsi is proposed, and the type strain is H7^T (=JCM 16231^T =KACC 14089^T).     

A chemotaxonomic study of some moderately halophilic Gram-positive isolates

A chemotaxonomic study was carried out on some moderately halophilic Gram-positive motile cocci, previously isolated from the Salar de Atacama (Chile) and grouped in two phenons (A and B) by numerical taxonomic analysis. Strains included in phenon A had a DNA base composition ranging between 42.0 and 44.0 mol%, while that of phenon B ranged from 48.0 to 48.8 mol%. The results of DNA-DNA hybridization studies on representative strains from phenons A and B, indicated that the strains assigned to phenon A comprise a genomically homogeneous group, with a high degree of homology (80%) to the type strain of Marinococcus albus. Similarly, phenon B constituted a homogeneous group and the representative strain studied showed a DNA-DNA homology of 70% with the type strain of Marinococcus halophilus. Representative strains studied from each phenon had meso-diaminopimelic acid in the cell wall and menaquinone systems with seven isoprene units (MK-7) as a major component. All these results, together with those previously reported, indicate that strains included in phenons A and B constitute additional strains of the species Marinococcus albus and Marinococcus halophilus , respectively.     

Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.     

Numerical Taxonomy of Aerobic,Gram-negative Bacteria associated with Oysters and Surrounding Seawater of the Mediterranean Coast

A numerical taxonomic study was performed on 245 strains of heterotrophic, aerobic, marine bacteria, plus 26 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterised by 93 morphological, biochemical, physiological and nutritional tests. Clustering yielded 46 phena at 0.60 S level (S_J coefficient). Some could be identified as species of Alteromonas, Shewanella, Deleya, Flavobacterium, Oceanospirillum, Pseudomonas and marine Agrobacterium-like organisms, others were unidentified groups. Several phena seem to correspond to as yet undescribed taxa.     

Hymenobacter negativus sp. nov., bacteria isolated from mountain soil collected in South Korea

Two novel Gram-negative bacterial strains BT442^T and BT584 were isolated from dry soil collected in mountains Busan and Guri, Korea during wintertime. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT442^T and BT584 both belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia). Strain BT442^T was closely related to Hymenobacter soli PB17^T (98.0% 16S rRNA gene similarity) and Hymenobacter terrae POA9^T (97.6%). No other recognized bacterial species showed more than 97% 16S rRNA gene sequence similarity to strains BT442^T. The genome size of strain BT442^T was 5,143,362 bp. Bacterial growth was observed at 10–30 °C (optimum 25 °C), pH 6.0–8.0 (optimum pH 6.0) in R2A agar and in the presence up to 1% NaCl. The major cellular fatty acids of strains BT442^T and BT584 were iso-C_15:0, anteiso-C_15:0 and summed feature 3 (C_16:1 ω6c / C_16:1 ω7c). In addition, their predominant respiratory quinone was MK-7. The major polar lipids of strains BT442^T and BT584 were identified to be phosphatidylethanolamine, aminophospholipid, and aminolipid. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, strains BT442^T and BT584 are novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter negativus. The strain type of Hymenobacter negativus is BT442^T (=?KCTC 72902^T?=?NBRC XXXX^T).

     

Yeasts and Yeast-Like Fungi Associated with Tree Bark: Diversity and Identification of Yeasts Producing Extracellular Endoxylanases

A total of 239 yeast strains was isolated from 52 tree bark samples of the Medaram and Srisailam forest areas of Andhra Pradesh, India. Based on analysis of D1/D2 domain sequence of 26S rRNA gene, 114 strains were identified as ascomycetous; 107 strains were identified as basidiomycetous yeasts; and 18 strains were identified as yeast-like fungi. Among the ascomycetous yeasts, 51% were identified as members of the genus Pichia, and the remaining 49% included species belonging to the genera Clavispora, Debaryomyces, Kluyveromyces, Hanseniaspora, Issatchenkia, Lodderomyces, Kodamaea, Metschnikowia, and Torulaspora. The predominant genera in the basidiomycetous yeasts were Cryptococcus (48.6%), Rhodotorula (29%), and Rhodosporidium (12.1%). The yeast-like fungi were represented by Aureobasidium pullulans (6.7%) and Lecythophora hoffmanii (0.8%). Of the 239 yeast strains tested for Xylanase, only five strains of Aureobasidium sp. produced xylanase on xylan-agar medium. Matrix-assisted laser desorption ionization-time of flight analysis and N-terminal amino-acid sequence of the xylanase of isolate YS67 showed high similarity with endo-1-4-β-xylanase (EC 3.2.1.8) of Aureobasidium pullulans var. melanigenum. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera,California

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198^T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0–2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198^T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA–DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C_15:0, iso-C_17:0 and anteiso-C_15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by β-galactosidase and β-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198^T (= CGMCC 1.13590^T = KCTC XXX).

     

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage within the γ-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.