- Volume 52, Issue 5, 2002

To re-examine the taxonomic status of Pseudomonas corrugata, 27 strains of this species were studied using a polyphasic approach. Numerical analysis of phenotypic data revealed two phena, A (including the P. corrugata type strain) and B, which could be clearly differentiated by the assimilation of mesotartrate, 2-ketogluconate and histamine. The mean DNA reassociation values with labelled DNA of P. corrugata type strain CFBP 2431T (phenon A) and strain CFBP 5447T (phenon B) were high for strains belonging to the same phenon (96.9 and 98.5%, respectively), whereas the DNA relatedness between the two phena was assessed as being close to 70%, which represents the value that is accepted for the definition of a bacterial species. Phena A and B were also differentiated by means of DNA profiles generated by heteroduplex mobility assay of PCR products of 16S rDNA hypervariable region 2, HaeIII restriction of the amplified internal transcribed spacer, REP- and BOX-PCR profiles, and by PCR with two pairs of specific primers. A comparison of the 16S rRNA sequences of strains CFBP 5447T and CFBP 5458 from phenon B with the available sequences of Pseudomonas species showed that these strains formed a cluster distinct from the P. corrugata type strain. Thus, a new species, Pseudomonas mediterranea, is proposed for strains of phenon B. The type strain is strain CFBP 5447T (= ICMP 14184T); its G+C content is 60.2 mol%.

On the basis of phenotypic and genotypic characteristics and analysis of 16S rRNA sequences, two novel species belonging to the genus Pseudoalteromonas are described. A pale-orange-pigmented strain, KMM 3548T, isolated from a sponge and a non-pigmented strain, KMM 520T, isolated from sea water are marine, gram-negative, aerobic, rod-shaped organisms. One of the strains, KMM 520T, had bipolar flagella. Both strains had the ability to degrade gelatin, DNA and Tween 80 but not chitin or agar. Strain KMM 520T decomposed elastin and grew at NaCl concentrations of 1-8%, while strain KMM 3548T grew at 1-6% NaCl. The temperature range for both strains was 4-30 degrees C. The DNA G+C contents were 46.3 (KMM 520T) and 41.1 mol% (KMM 3548T). The level of DNA relatedness between the two strains was 20%. DNA from strain KMM 520T showed 8-34% genetic relatedness and that of KMM 3548T showed 17-53% relatedness to the DNA of other type strains of the genus Pseudoalteromonas. 16S rRNA analysis indicated a clear affiliation of these novel bacteria with the genus Pseudoalteromonas. The type strains of the novel species are Pseudoalteromonas translucida sp. nov. KMM 520T (= LMG 19696T = ATCC BAA-3157T) and Pseudoalteromonas paragorgicola sp. nov. KMM 3548T (= LMG 19694T = ATCC BAA-322T).

The marine bacterium strain KMM 1376T was isolated from gill tissue of the ascidian Halocynthia aurantium, an inhabitant of the coastal waters of the Sea of Japan. Strain KMM 1376T is an aerobic, gram-negative, non-motile, encapsulated, non-pigmented, slightly halophilic bacterium that is characterized by fimbria-like structures, growth in 0.5-15% NaCl at 7-35 degrees C and absence of acid production from many carbohydrates. The DNA G+C content is 54 mol%. The main fatty acids are C16:0, C16.1omega7c and C18:1omega9c. Comparative 16S rDNA sequence analysis of strain KMM 1376T revealed 92.7-95.3% sequence similarity to members of Halomonas. Based on phenotypic and phylogenetic characteristics, it is proposed that the novel isolate be classified as Halomonas halocynthiae sp. nov., with the type strain KMM 1376T (= DSM 14573T).

On the basis of phenotypic characterization and DNA relatedness, two novel species are proposed, Afipia birgiae sp. nov. (type strain 34632T = CIP 106344T = CCUG 43108T) and Afipia massiliensis sp. nov. (type strain 34633T = CIP 107022T = CCUG 45153T). A new genospecies is described, named Afipia felis genospecies A, closely related to Afipia felis. The complexity encountered in the taxonomy of the Bradyrhizobiaceae group within the alpha-2 subgroup of the Proteobacteria is discussed and the description of these novel species highlights the need for new tools for phylogenetic analysis in the group. The novel species herein described are fastidious bacteria isolated from a hospital water supply in co-culture with amoebae. It is hypothesized that this group of bacteria are a potential cause of nosocomial infections.

Streptococcus ferus was originally isolated from rats and, although it is often considered a member of the 'mutans group' streptococci on the basis of phenotypic criteria, its phylogenetic position has not been investigated. Here, the relationship of this organism to other streptococcal groups has been determined by DNA-DNA hybridization and by partial sequencing of 16S rDNA and the sodA gene. S. ferus was found to be distantly related to all currently described streptococci, with no strong evidence in support of its inclusion within any of the currently recognized streptococcal groups.

The existence of tremendous genetic diversity within Campylobacter species has been well documented. To analyse the population structure of Campylobacter and determine whether or not a clonal population structure could be detected, genetic diversity was assessed within the genus Campylobacter by multilocus enzyme electrophoresis of 156 isolates representing 11 species and subspecies from disparate sources. Analyses of electrophoretic mobility of 11 enzymes revealed 109 electrophoretic types (ETs) and 118 ETs when nulls were counted as an allele. Cluster analysis placed most ETs into groups that correlated with species. With nulls counted as alleles, 19 ETs were identified among 33 isolates of Campylobacter lari, 31 ETs among 34 isolates of Campylobacter coli and 43 ETs among 59 isolates of Campylobacter jejuni subsp. jejuni. Nine C. jejuni subsp. jejuni isolates, confirmed as this species by DNA-DNA hybridization, were hippuricase-negative. Reported linkage analyses were done with nulls ignored. Scores for mean genetic diversity (H) were high for the total population (mean H = 0.802). Allelic mismatch-frequency distributions and allelic tracing pointed to possible genetic exchange between subpopulations. C. lari appears to be a panmictic species. Some pairs of species shared multiple alleles of certain loci, possibly indicating genetic exchange between species. Of the species tested, C. jejuni appeared to be the most active in sharing alleles. However, there was evidence of variable involvement in recombination by the different loci. Linkage analysis of loci in C. jejuni and C. coli revealed a clonal framework, with some loci tightly linked to each other. The loci appeared to occur in linkage groups or islands. Campylobacter may have a clonal framework with other portions of the genome involved in frequent recombination. Population genetic structure among Campylobacter is inconclusive and it remains to be seen if pathogenic types can be identified.

Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan. The cells of strain P2F9704aT were non-motile, irregular cocci 0.9-1.4 microm in diameter. They stained gram-negative. The cells catabolized formate and H2+CO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates. The optimal growth parameters for strain P2F9704aT were pH 6.7, 37 degrees C and 0.5% NaCl. Acetate was required for cell growth even though it was not a substrate for methanogenesis. The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT. However, tungsten extended the growth ranges relating to temperature, pH and salt. The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 99.1 and 98.5% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species. On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis. The type strain is P2F9704aT (= OCM 671T = CCRC 16182T = DSM 14663T).

Several extremely halophilic coccoid archaeal strains were isolated from pieces of dry rock salt that were obtained three days after blasting operations in an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225-280 million years ago). On the basis of their polar-lipid composition, 16S rRNA gene sequences, cell shape and growth characteristics, the isolates were assigned to the genus Halococcus. The DNA-DNA reassociation values of one isolate, strain H4T, were 35 and 38% with Halococcus salifodinae and Halococcus saccharolyticus, respectively, and 65.8-67.8% with Halococcus morrhuae. The polar lipids of strain H4T were C20-C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate. Whole-cell protein patterns, menaquinone content, enzyme composition, arrangements of cells, usage of carbon and energy sources, and antibiotic susceptibility were sufficiently different between strain H4T and H. morrhuae to warrant designation of strain H4T as a new species within the genus Halococcus. It is proposed that the isolate be named Halococcus dombrowskii, and the type strain is H4T (= DSM 14522T = NCIMB 13803T = ATCC BAA-364T).

A distinct actinomycete isolated from soil was subjected to polyphasic taxonomic analysis. It is demonstrated by comparative 16S rDNA gene sequencing that the organism, designated strain AS 4.0578T, represents a novel species of the genus Lentzea. The phylogenetic results also showed that it formed a monophyletic lineage distinct from the available members of the genera Lentzea, Lechevalieria and Saccharothrix. The organism was distinguished from all the validly described type strains of the genus Lentzea by a combination of phenotypic features and DNA-DNA hybridization. It is proposed, therefore, that strain AS 4.0578T (= JCM 11373T) be classified in the genus Lentzea as Lentzea flaviverrucosa sp. nov. In addition, it is proposed that Saccharothrix aerocolonigenes subsp. staurosporea NRRL 11184T be transferred to Lentzea albida on the basis of phylogenetic analysis, DNA-DNA homology, nucleotide signatures and phenotypic properties.

The taxonomic relationships between Phyllobacterium myrsinacearum LMG 2t2T, Phyllobacterium rubiacearum LMG 1t1T and two Phyllobacterium strains representing a collection of isolates from sugar-beet roots were investigated by means of fatty acid analysis and DNA-DNA hybridization. The strains showed very similar fatty acid compositions and more than 96% DNA reassociation to each other. On the basis of these results and available data from the literature, it is proposed that P. rubiacearum be classified as a junior subjective synonym of P. myrsinacearum, and that the sugar-beet isolates be included in the latter species. An emended description of P. myrsinacearum is given.

Comparative 16S rDNA studies of Nocardia type and reference strains revealed that strain DSM 43406T, identified as Nocardia vaccinii, was wrongly classified. The strain was aerobic, gram-positive and produced scarce, white, branched aerial mycelium and a beige-red substrate mycelium. The reverse side of the colonies was yellow-orange. It showed chemotaxonomic markers that were consistent with its classification in the genus Nocardia. The mycolic acids had chain lengths from 50 to 58 carbon atoms. The 16S rDNA sequence showed the highest similarity to Nocardia nova (97.7%) and N. vaccinii (97.6%), but the strain could be clearly separated from these species and other members of the N. vaccinii cluster by significant differences in biochemical test results and unique fatty acid and mycolic acid patterns. These data led to the conclusion that the isolate represents a novel species within the genus Nocardia, for which the name Nocardia pseudovaccinii sp. nov. is proposed. The type strain is strain AR 368,38366-20T (= DSM 43406T = NRRL B-24154T).

Haloarcula mukohataei, previously isolated from soils of salt flats in Argentina, was originally placed in the genus Haloarcula on the basis of 16S rRNA gene sequence comparison. However, its morphology and polar lipid composition differs from that of the other Haloarcula species. In addition, its phylogenetic distance from other Haloarcula species is rather large, and the 16S rDNA sequence does not contain many of the signature bases characteristic of the genus. Transfer of the species to a new genus was therefore recommended by the International Committee on Systematic Bacteriology Subcommittee on the taxonomy of Halobacteriaceae. A full description of the isolate, proposed as a member of a new genus, Halomicrobium, as Halomicrobium mukohataei comb. nov., is presented. The type strain is strain arg-2T (= JCM 9738T = DSM 12286T = ATCC 700874T = NCIMB 13541T).

A stretch of nucleotides consisting of a conserved region and an adjacent upstream variable region in the 16S rDNA of members of domain Bacteria was identified as a suitable target site for developing a real-time PCR adjacent hybridization assay. A single universal fluorogenic cyanin 5-labelled probe, CY5 1046+, targeting the conserved region, and two FITC-labelled probes, Calo and Fervi, targeting the variable region were designed and synthesized for the identification and differentiation of the thermophilic anaerobes Caloramator and Fervidobacterium. The simultaneous hybridization of probes CY5 1046+ and Fervi to the 16S rDNA target sites of Fervidobacterium species during PCR amplification resulted in an increase in fluorescence emission that was monitored continuously in a LightCycler. The differences in the temperature of disassociation of the hybridization probes (Tm) due to compositional variation in the nucleotide bases of the probe and target sequences enabled Fervidobacterium islandicum DSM 5733T to be differentiated from Fervidobacterium gondwanense ACM 5017T and Fervidobacterium nodosum ATCC 35602T. The simultaneous hybridization of probes CY5 1046+ and Calo to the 16S rDNA target sequence of Caloramator indicus ACM 3982T during PCR amplification also resulted in an increase in fluorescence emission. However, as the target sequence of C. indicus ACM 3982T is identical to those of the other three Caloramator species, Caloramator fervidus ATCC 43204T (formerly Clostridium fervidus), Caloramator proteoclasticus DSM 10124T and Caloramator coolhaasii DSM 12679T, further species discrimination on the basis of Tm will not be possible, making probe Calo a useful genus-specific probe. The devised method was subsequently used to detect and identify Fervidobacterium and Caloramator isolates from our previously uncharacterized culture collection and from enrichment cultures. The assay is cheap and flexible, as only a battery of inexpensive FITC probes for differentiating other members of domain Bacteria needs to be synthesized and DNA templates prepared by a simple lyse-boil method, in addition to purified DNA, can also be used.

A high-G+C gram-positive, motile, non-spore-forming coccus capable of accumulating significant amounts of polyhydroxyalkanoate (PHA) was isolated from an inefficient biological phosphorus removal activated sludge reactor. The cell wall of strain Lpha5T was characterized by the presence of meso-diaminopimelic acid, menaquinone MK-8(H4) and a complex fatty-acid pattern consisting of C16:0 and at least five other major straight-chain saturated and unsaturated fatty acids. Strain Lpha5T also had a high G+C content (71.3 mol%). The nearest phylogenetic relative of strain Lpha5T, based on 16S rDNA sequence analysis, was the high-G+C gram-positive bacterium Dermatophilus congolensis (similarity value of 94%), of the family Dermatophilaceae, class Actinobacteria. As strain Lpha5T was distinct from D. congolensis in its morphological, phenotypical (i.e. its PHA-accumulating ability and fatty-acid profile) and genetic traits (phylogeny and G+C content), it is proposed that strain Lpha5T be designated as the type species of a novel genus within the Dermatophilaceae. The name Kineosphaera limosa is proposed for strain Lpha5T (= JCM 11399T = DSM 14548T).

A set of taxonomically unique, blue-coloured bacterial isolates are described on the basis of physiological and biochemical characterization, fatty acid profiling and analyses of 16S rDNA sequences. The flagellated, non-fermentative strains were isolated in 1986, 1987 and 1998 from different layers of the water column of the central Baltic Sea. According to 16S rDNA sequences, all strains are very closely related to each other and to strains from several other marine environments, including the deep sea. Thus, the described species seems to be widespread in marine habitats. According to DNA-DNA hybridization, the strains described can be considered to belong to the same species. The bacteria grew at temperatures from 4 to 30 degrees C, with an optimum around 20-25 degrees C. Growth was observed at salinities from 0 to 30, with an optimum between 10 and 30 and no growth at high salinities. The dominant fatty acids were 16:1omega7c, 16:0 and 18:1omega7c. The G+C content of the DNA ranged from 47.8 to 48.9 mol%. Phylogenetic analyses of the 16S rDNA sequences revealed a clear affiliation with members of the gamma-Proteobacteria. The closest relationship was seen with Alishewanella fetalis but, in terms of physiology, colour and fatty acids, the bacteria described are rather distant from A. fetalis. To honour the marine microbiologist Gerhard Rheinheimer, the name Rheinheimera baltica gen. nov., sp. nov., is suggested for the Baltic isolates, with the type strain OSBAC1T (= DSM 14885T = LMG 21511T).

A novel, extremely thermophilic, chemolithoautotrophic bacterium was isolated from the submarine hydrothermal system off the beach of Lihir Island, Papua New Guinea. Cells of the organism were curved rods of about 1.5-3 microm in length and 0.5-0.8 microm in width. The bacterium grew within the temperature range 50-80 degrees C (optimum around 75 degrees C) and was an obligate anaerobe. Molecular hydrogen was used as the sole electron donor by the bacterium, and nitrate or elemental sulfur served as electron acceptors, producing ammonium or H2S, respectively. Complex organic substrates stimulated growth of the bacterium, but they could not be used as the sole energy source. Based on 16S-rDNA-based phylogenetic analyses and on physiological, biochemical and structural characteristics, the novel organism was found to represent a novel genus for which the name Thermovibrio is proposed. This novel genus, together with Desulfurobacterium thermolithotrophum, may represent a new order within the phylum Aquificae. Since cell pellets of the novel bacterium had an intense red colour, the name Thermovibrio ruber is proposed for the novel organism. The type strain of Thermovibrio ruber gen. nov., sp. nov. is ED11/3LLKT (= DSM 14644T = JCM 11468T).

Morphological analysis and sequencing of the 165 rRNA gene, the spacer region of the ribosomal operon (ITS1) and the rbcLX (RubisCO) region was performed on 26 Anabaena strains and 14 Aphanizomenon strains isolated from several lakes in Denmark, Finland and France. Based on their morphology, Anabaena strains differed from strains of Aphanizomenon: the vegetative cells, heterocysts and akinetes were significantly wider in Anabaena than in Aphanizomenon. Phylogenetic trees based on the 16S rDNA, ITS1 and rbcLX regions showed that the planktic Anabaena strains were not distinguishable from Aphanizomenon strains. The results of the clustering of Anabaena and Aphanizomenon strains based on 16S rDNA sequences showed that these two genera are not monophyletic. Sequence analysis of the 16S rDNA, ITS1-S and rbcLX regions of the planktic Anabaena strains showed that this genus is heterogeneous. In all methods, Anabaena strains that produced different toxic compounds (e.g. anatoxin-a, microcystin and an unknown neurotoxin) were clustered separately from each other but were grouped either with non-toxic Anabaena and/or Aphanizomenon strains. Our data suggest that the planktic Anabaena and Aphanizomenon isolates belong to the same genus, regardless of their morphological differences. Thus, a taxonomic revision of the two genera is required.

A facultatively anaerobic, non-spore-forming, psychrophilic, gram-positive, non-aciduric but alkaliphilic, rod-shaped bacterium (MPL-11T) was found to be responsible for green discoloration of refrigerated vacuum-packaged bologna upon opening of the package. Although Aerococcus viridans, which had been implicated earlier in causing the same problem, was also found, this is the first report of discoloration caused by an organism shown to be a species of Carnobacterium. Bacterial discoloration was caused by H2O2 production upon exposure of the meat to air. Strain MPL-11T is catalase- and oxidase-negative. It is not motile and does not reduce nitrate to nitrite or produce ammonia from arginine. It does not grow in acetate-containing broth or agar (Rogosa) or produce H2S. The peptidoglycan is of the meso-diaminopimelic acid type and it produces predominantly L(+)-lactic acid from glucose. It grows from at least 2 to 30 degrees C over a pH range from 5.5 to 9.1. Ribotyping suggested that strain MPL-11T could be a species of either Lactobacillus or Carnobacterium, but analysis using DNA sequences from the 16S rRNA gene showed conclusively that the organism belonged to the genus Carnobacterium. Since acid is not produced from amygdalin, inulin, mannitol, methyl alpha-D-glucoside or D-xylose, the organism differs from the seven described species of Carnobacterium. In addition, strain MPL-11T is the first member of the genus found that does not produce acid from ribose. It is capable of acid production/growth on galactose, glucose, fructose, mannose, N-acetylglucosamine, aesculin, cellobiose, maltose, lactose, sucrose, trehalose and tagatose. Although extremely salt tolerant, it does not grow in > or = 4% NaCl. On the basis of phenotypic and genotypic data, it is concluded that this isolate represents a separate, novel species. Accordingly, the name Carnobacterium viridans sp. nov. is proposed. The type strain is strain MPL-11T (= ATCC BAA-336T = DSM 14451T).

Fifteen ballistoconidium-forming yeasts, isolated from the leaves of plants in Hungary, showed morphological, physiological and biochemical characteristics similar to those of Udeniomyces pyricola. The identical sequences of internal transcribed spacer regions for selected strains (HY-16T, HY-29, HY-111 and HY-186) indicated that they should be classified as one species. Although a representative strain, HY-16T, showed a closer relationship to Itersonilia perplexans than to known Udeniomyces species in phylogenetic trees constructed using 18S rDNA and the D1/D2 region of the 26S rDNA sequence, this species was placed in the genus Udeniomyces on the basis of its morphological and chemotaxonomic characteristics. Udeniomycespannonicus sp. nov. (type strain HY-16T = JCM 11145T = NCAIM Y 01556T = CBS 9123T) is proposed.