INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY logo - Volume 52, Issue 5, 2002

A representative selection of coryneform bacteria, isolated from the phyllosphere of grasses and the litter layer after mulching the sward, was characterized by a polyphasic approach to clarify their taxonomic position in the family Microbacteriaceae, with particular reference to potentially plant-pathogenic bacteria. On the basis of 16S rDNA analysis, the isolates can be classified into six genotypes representing the genera Curtobacterium, Clavibacter, Subtercola and a subgroup, which was not affiliated to a known genus. One genotype, belonging to the genus Curtobacterium, had an identical 16S rDNA sequence to reference strains of the Curtobacterium flaccumfaciens pathovars. Another genotype, closely related to the potentially pathogenic Curtobacterium flaccumfaciens, could be distinguished from known species of the genus on the basis of phylogenetic and phenotypic characterization and is consequently proposed as a novel species, Curtobacterium herbarum sp. nov. (type strain P 420/07T DSM 14013T = LMG 19917T). Two genotypes assigned to Clavibacter showed a close relationship to Clavibacter michiganensis subsp. tessellarius, a pathogenic bacterium causing foliar lesions on wheat. A further genotype, which clustered clearly in the genus Subtercola by comparison of 16S rDNA sequences, showed a hitherto undescribed B-type of peptidoglycan containing the diagnostic diamino acids ornithine and 2,4-diaminobutyric acid, in the cell wall; this genotype is proposed as Subtercola pratensis sp. nov. (type strain P 229/10T = DSM 14246T = LMG 21000T). For one genotype, which formed a phylogenetically separate branch in the family of Microbacteriaceae showing chemotaxonomic similarities to the genus Rathayibacter, a novel genus, Plantibacter gen. nov., is proposed; the type species is Plantibacter flavus sp. nov. (type strain P 297/02T = DSM 14012T = LMG 19919T).

A facultatively psychrophilic bacterium, strain 4-22T, was isolated from a cold current off the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan. The isolate was a rod-shaped facultative anaerobe that reduced nitrate to nitrite and hydrolysed starch, DNA and alginic acid, but not chitin or gelatin. The isolate grew at 0 degrees C, but not at 26 degrees C; the optimum growth temperature was 14-16 degrees C. NaCl was required for growth. The DNA G+C content was 43.5 mol%. The whole-cell fatty acids consisted of significant amounts of an unsaturated fatty acid, C16:1, and a saturated fatty acid, C16:0. A polyunsaturated fatty acid, docosahexaenoic acid (C22:6), was also detected (1.6%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 4-22T was closely related to Psychromonas antarctica (95.7% similarity). DNA-DNA hybridization revealed a relatedness of 31% between strain 4-22T and P. antarctica. Based on physiological and biochemical characteristics and the phylogenetic position as determined by 16S rRNA gene analysis and DNA-DNA relatedness, it is concluded that the isolate represents a novel species, for which the name Psychromonas marina sp. nov. is proposed. The type strain is 4-22T (= JCM 10501T = IAM 14899T = NCIMB 13792T).

A strictly anaerobic, gram-positive, sporulating rod (0.5-0.6 x 2.0-4.0 microm), designated strain Lup 21T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating cheese-factory wastewater. Strain Lup 21T was motile by means of peritrichous flagella, had a G+C content of 31.4 mol% and grew optimally at 37 degrees C, pH 7.4, in the absence of NaCl. It is a heterotrophic micro-organism, utilizing proteinaceous compounds (gelatin, peptides, Casamino acids and various single amino acids) but unable to use any of the carbohydrates tested as a carbon and energy source. It reduced thiosulfate and elemental sulfur to sulfide in the presence of Casamino acids as carbon and energy sources. Acetate, butyrate, isobutyrate, isovalerate, CO2 and sulfide were end products from oxidation of gelatin and Casamino acids in the presence of thiosulfate as an electron acceptor. In the absence of thiosulfate, serine, lysine, methionine and histidine were fermented. On the basis of 16S rRNA similarity, strain Lup 21T was related to members of the low-G+C Clostridiales group, Clostridium subterminale DSM 6970T being the closest relative (with a sequence similarity of 99.4%). DNA-DNA hybridization was 56% with this species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the isolate was designated as a novel species of the genus Clostridium, Clostridium thiosulfatireducens sp. nov. The type strain is strain Lup 21T (= DSM 13105T = CIP 106908T).

Nine strains of anaerobic, non-spore-forming, gram-positive bacilli, isolated from the human oral cavity and provisionally identified as belonging to the genus Eubacterium, were subjected to a comprehensive range of phenotypic and genetic tests. Biochemically, they were found to comprise a homogeneous group, and phylogenetic analysis of their 16S rRNA sequences indicated that they constitute a unique branch within the Clostridium-Bacillus subphylum of the phylum Firmicutes. All of the isolates displayed an unusual colonial morphology after extended incubation. This resembled a contaminated culture in that small, secondary colonies were seen to arise around and from within the primary colony form, and a third, independent, colony type was also seen. However, inspection of the colonies by Gram-staining and scanning electron microscopy together with protein profile analysis and 16S rRNA gene sequence comparison of the two independent colony types revealed that only a single organism was present. A new genus, Shuttleworthia, and the species Shuttleworthia satelles gen. nov., sp. nov., are proposed. The cells are saccharolytic, and acetate, butyrate and lactate are produced as end products of glucose fermentation. Aesculin is hydrolysed and indole is produced. The G+C content of the DNA of the type strain is 51 mol%. The type strain is strain DSM 14600T (= CCUG 45864T = VPI D143K-13T).

In this study, we have sequenced the rpoB gene, encoding the beta subunit of DNA-dependent RNA polymerase, from a selection of gram-positive and gram-negative bacteria. The presence of insertions and deletions (indels) in the beta subunit separate the gram-positive and gram-negative bacteria from each other and support the division of the gram-positive organisms into two clades based on DNA G+C content. Phylogenetic and amino acid content analyses further separate the clostridia from bacilli, leuconostocs, listeriae and relatives, forming an early branch after the common gram-positive ancestor. The occurrence in the beta subunit of Asn-Ala at positions 471-472 in Porphyromonas cangingivalis and Asn at position 372 in Weissella paramesenteroides are postulated to be the cause of the natural rifampicin resistance of these species.

The 16S rDNA sequence similarities between the type strains of Sphingomonas paucimobilis and 32 other Sphingomonas species range from 90.2 to 99.6%. It might be possible to divide the genus into several new genera according to a dendrogram drawn from 16S rDNA sequence similarity. However, the phenotypic and biochemical information needed to define clusters of strains representing distinct genera within this group of organisms was not previously available. Although the cellular lipids of type strains of all 28 Sphingomonas species tested contained glucuronosyl-(1 --> 1)-ceramide together with 2-hydroxymyristic acid, other molecular species of sphingoglycolipids were distributed randomly. Sphingomonas natatoria and Sphingomonas ursincola, bacteriochlorophyll a-containing, gram-negative facultative phototrophs, belong to the cluster of the genus Sphingomonas. Other phototrophic Porphyrobacter and Erythrobacter species in the Sphingomonadaceae were classified into a cluster different from the genus Sphingomonas, as reported previously. None of the physiological and biochemical characteristics considered, including cellular lipids and fatty acid composition, provided evidence for the division of the current genus Sphingomonas. It is therefore concluded that the genus Sphingomonas should remain undivided at this time. The species of three recently proposed genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastobacter ursincola, are junior objective synonyms of species of the genus Sphingomonas.

The vernacular name 'fluorescent Pseudomonas group 97-514' was coined for a group of 43 strains isolated from two French natural mineral waters. All these strains were gram-negative, rod-shaped and motile by means of a single polar flagellum. They produced fluorescent pigment (pyoverdin) on King B medium, catalase and cytochrome oxidase. They were capable of respiratory but not fermentative metabolism. They were not able to accumulate poly-beta-hydroxybutyrate and possessed an arginine dihydrolase system. DNA-DNA relatedness studies (S1 nuclease method) showed that the 43 strains of 'fluorescent Pseudomonas group 97-514' formed a genetically homogeneous group (DNA-DNA relatedness ranged from 70 to 100%). A total of 76 strains representing well-known or partially characterized species of the genus Pseudomonas sensu stricto had 7-56% DNA hybridization with strain CFML 97-514T. The highest DNA binding values were found with Pseudomonas veronii CIP 104663T (52%), Pseudomonas rhodesiae CIP 104664T (56%), Pseudomonas marginalis ATCC 10844T (56%), Pseudomonas gessardii CIP 105469T (53%) and Pseudomonas cedrella CIP 105541T (52%). Their unrelatedness was confirmed by deltaTm values greater than 7 degrees C. On the basis of the results of phenotypic and DNA-DNA hybridization studies, a novel Pseudomonas species, Pseudomonas grimontii sp. nov., is proposed for the 43 strains of 'fluorescent Pseudomonas group 97-514'. The type strain is strain CFML 97-514T (= CIP 106645T = ATCC BAA-140T). The G+C content of the DNA of the type strain was 58 mol%. A comparison of the complete 16S rRNA gene sequence of the type strain CFML 97-514T and the sequence of other strains of the genus Pseudomonas revealed that the novel species fell within the 'Pseudomonas fluorescens intrageneric cluster'. Members of P. grimontii grew at 4 degrees C but not at 41 degrees C. They were able to use D-xylose, alpha-L-rhamnose, alpha-aminobutyrate, meso-erythritol and itaconate as sole sources of carbon and energy and formed levan from sucrose. Strains do not possess lecithinase or Tween esterase activities. The clinical significance of P. grimontii is unknown.

In an attempt to clarify the taxonomy of the Mycobacterium avium complex, the relationship between IS1245 RFLP, growth temperature, 16S rDNA signature sequences and the 16S-23S rDNA internally transcribed spacer (ITS) of 160 M. avium-complex isolates from different sources was investigated. All 70 isolates identified as M. avium by INNO-LiPA MYCOBACTERIA (Innogenetics, Belgium), a DNA probe test that targets the ITS, and by 16S rDNA analysis carried multiple copies of IS1245. Three isolates with multiple copies of IS1245 were identified by 16S rDNA analysis as Mycobacterium intracellulare and by LiPA as M. intracellulare (n = 1) and M. avium-intracellulare complex (n = 2). A dichotomy among the M. avium isolates was found on the basis of a C and a G signature nucleotide at position 228 of the 16S-23S rDNA spacer sequence, and this grouping was largely confirmed on the basis of similarities in IS1245 RFLPs. Strains with the characteristic three-band IS1245 'bird-type', as well as M. avium subsp. silvaticum or 'wood-pigeon' strains, invariably contained the C signature. A third characteristic that separated the M. avium bird-type isolates from M. avium isolates from humans and other mammals was growth-temperature tolerance: in contrast to bird isolates, human/porcine isolates grew at 24 and 45 degrees C. Based on differences in IS1245 RFLP, 16S-23S rDNA ITS and growth temperature, M. avium isolates originating from birds should be considered as a separate, evolutionarily conserved taxon. Because all M. avium isolates from birds are invariably of this type, the designation M. avium subsp. avium should be reserved for these bird-type strains. For clarity in the epidemiology of M. avium-related disease, isolates from humans and pigs with multibanded IS1245 RFLPs merit a separate designation. The designation 'M. avium subsp. hominissuis' is suggested for this group of bacteria.

Taxonomic studies were performed on a phenotypically homogeneous group of 13 mycobacteria isolated from clinical, veterinary and stream-water samples. The methods applied included chromatographic analyses of bacterial lipids, biochemical tests and sequencing of the 16S rDNA and the internal transcribed spacer 1 (ITS1) region. Positive results in urease, Tween 80 hydrolysis and pyrazinamidase tests and a negative result in a semi-quantitative catalase test, combined with the ability to grow at 42 degrees C, distinguished this group among the yellow-pigmented, slowly growing mycobacteria. Unique fatty acid and mycolic acid profiles in chromatographic analyses and the results of gene sequencing indicated that the novel isolates represent a previously undescribed species, for which the name Mycobacterium palustre sp. nov. is proposed. The fatty acid profile obtained by GLC was characterized by the presence of several methyl-branched fatty acid markers. The most prominent markers were 2-methyleicosanoic, tetracosanoic and hexacosanoic acids. According to 16S rDNA sequencing, M. palustre is phylogenetically closest to Mycobacterium kubicae, a recently described species. M. palustre gives a false-positive result in a hybridization test with the AccuProbe Mycobacterium avium complex. One of the strains was isolated from a lymph-node biopsy from a child with cervical lymphadenitis. Thus, M. palustre should be listed among potential inducers of paediatric lymphadenitis. The veterinary isolates originated from the lymph nodes of slaughter pigs. The majority of the strains were recovered from natural waters, which highlights the role of the environment as a source of potentially pathogenic mycobacteria. The type strain of M. palustre is strain E846T (= DSM 44572T = ATCC BAA-377T).

Two strains of obligately piezophilic bacteria were isolated from sediment collected from the deepest cold-seep environment with chemosynthesis-based animal communities within the Japan Trench, at a depth of 7434 m. The isolated strains, JT7301 and JT7304T, were closely affiliated with members of the genus Psychromonas on the basis of 16S rDNA sequence analysis. Hybridization values for DNA-DNA relatedness between these strains and the Psychromonas antarctica reference strain were significantly lower than that accepted as the phylogenetic definition of a species. The optimal temperature and pressure for growth of the isolates were 10 degrees C and 50 MPa and they produced both eicosapentaenoic acid (C20:5omega3) and docosahexaenoic acid (C22:6) in the membrane layer. Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Psychromonas species. The name Psychromonas kaikoae sp. nov. (type strain JT7304T = JCM 11054T = ATCC BAA-363T) is proposed. This is the first proposed obligately piezophilic species of the genus Psychromonas.

Several strains isolated from Antarctic winter sea water, sea-ice algal assemblages and quartz stone subliths were found to belong to a novel 16S rDNA sequence cluster within the family Flavobacteriaceae (Cytophaga-Flavobacterium-Bacteroides division). The strains were gram-negative, non-motile, psychrotolerant, strictly aerobic, chemoheterotrophic rod-shaped cells that contained orange or yellow carotenoid pigments and required yeast extract when grown in defined mineral-salts media. The requirement for sodium ions varied between strains. Results of DNA-DNA hybridization analysis were used to divide the strains into four distinct genospecies, which were differentiated by physiological and nutritional characteristics. The DNA G+C content of the strains was 33-39 mol%. The fatty acid profiles of representative strains were very similar, with major constituents including i15:1omega10c, a15:1omega10c, i15:0, a15:0, i16:1omega6c, i17:1omega5c and 3-OH i16:0. The novel genus Aequorivita gen. nov., which has widespread distribution in the Antarctic region, is proposed. The genus comprises four species: the type species Aequorivita antarctica sp. nov. (type strain SW49T = ACAM 640T = DSM 14231T), Aequorivita lipolytica sp. nov. (type strain Y10-2T = ACAM 641T = DSM 14236T), Aequorivita crocea sp. nov. (type strain Y12-2T = ACAM 642T = DSM 14239T) and Aequorivita sublithincola sp. nov. (type strain 9-3T= ACAM 643T = DSM 14238T).

In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches' broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 1.4-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches' broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, 'Candidatus Phytoplasma castaneae'.

Thirty-four Acetobacter strains, representing Acetobacter aceti, Acetobacter pasteurianus, Acetobacter pomorum, Acetobacter peroxydans, Acetobacter lovaniensis, Acetobacter estunensis, Acetobacter orleanensis, Acetobacter indonesiensis and Acetobacter tropicalis, were subjected to a polyphasic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis and phenotypic characterization. Two novel species are proposed, Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. The type strains of these species are respectively LMG 1625T (= DSM 14362T = NCIB 8894T = ATCC 23765T) and LMG 1746T (= DSM 14337T).

The taxonomic position of two butane-utilizing bacteria was studied using a polyphasic approach. Biochemical and physiological characteristics indicated these to be members of the genus Pseudomonas, showing more similarity to Pseudomonas mendocina than to any other species. The major fatty acids found in these two strains also pointed to their similarity to P. mendocina. On the other hand, DNA-DNA hybridization studies with seven related Pseudomonas species belonging to the gamma-Proteobacteria and the deltaTm values of reassociated molecules clearly showed that these two strains do not belong to any of the seven species tested. The 16S rRNA gene was sequenced and compared with the sequences available in the GenBank database. Phylogenetic analysis using the region covering positions 31-1488 (Escherichia coli numbering) confirmed these observations and placed these two strains as members of the authentic Pseudomonas, but not in any existing species of the genus. On the basis of biochemical characteristics, fatty acid profiles, DNA-DNA reassociation and deltaTm values, as well as 16S rRNA gene sequence analyses, these two isolates were shown to belong to one species but to have characteristics distinct from those of validly described species of Pseudomonas (sensu stricto). These strains, therefore, should be recognized as a novel species, for which the name Pseudomonas indica sp. nov. is proposed. The type strain is strain IMT37T (= MTCC 3713T = DSM 14015T).

Members of Bisgaard taxon 11 have been isolated from horses. These bacteria are of importance in the veterinary clinic and also to the medical profession, since they may be isolated from infected wounds of humans bitten by horses. Six strains from different continents were identified as taxon 11, with 16S rRNA similarities between 98.0 and 99.7%. A single isolate that represented the so-called (+)L-arabinose-positive Actinobacillus equuli isolated from a diseased foal showed 99.9% 16S rRNA similarity to the type strain of A. equuli. DNA-DNA hybridizations showed that (+)L-arabinose-positive strains of A. equuli represent A. equuli sensu stricto. DNA-DNA hybridizations also showed that A. equuli and Bisgaard taxon 11 represent two genotypes. These genotypes differ with respect to disease pattern and epidemiology. For these reasons, two subspecies of A. equuli are proposed, Actinobacillus equuli subsp. equuli subsp. nov. (type strain NCTC 8529T = ATCC 19392T) and Actinobacillus equuli subsp. haemolyticus subsp. nov. (type strain F 154T = CCUG 19799T = NCTC 13195T).

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 16S rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204T), Planktothrix rubescens (type strain CCAP 1459/22T), Planktothrix pseudagardhii sp. nov. (type strain T1-8-4T), Planktothrix mougeotii (type strain TR1-5T), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207T), Tychonema bourrellyi (type strain CCAP 1459/11BT) and Limnothrix redekei (type strain NIVA CYA 277/1T).

Two bacterial strains that are able to grow specifically on the sheath of a sheathed filamentous bacterium, Sphaerotilus natans, were isolated from soil samples. The sheath-degrading organisms, designated strains TB(T) and TK, are facultatively anaerobic and form endospores. The Gram reaction was negative at all stages of cultivation. The optimum growth temperature and pH were 30 degrees C and pH 7. The DNA G+C content was 54.0-55.8 mol%. MK-7 was the predominant menaquinone and anteiso-C15:0 was the major fatty acid. Phylogenetic analysis based on the 16S rDNA sequences revealed that the isolates were closely related to Paenibacillus chondroitinus, Paenibacillus alginolyticus, Paenibacillus koreensis, Paenibacillus validus, Paenibacillus larvae subsp. larvae and P. larvae subsp. pulvifaciens. The sequences were found to contain consensus sequences characteristic of all Paenibacillus species. The isolates were able to lyse and utilize the purified sheath of S. natans as the sole carbon and energy source. Acid was not produced from common carbon sources, allowing easy distinction from other members of Paenibacillus. It is concluded that the two strains represent a novel Paenibacillus species, for which the name Paenibacillus koleovorans sp. nov. is proposed. The type strain is strain TB(T) (= JCM 11186T = IAM 14926T = KCTC 13912T).