- Volume 51, Issue 5, 2001

Four moderately halophilic, exopolysaccharide-producing bacterial strains isolated from soil samples collected from a saltern at Asilah (Morocco) are reported. These four strains were initially considered to belong to the genus Halomonas. Their DNA G+C contents varied between 62.2 and 64.1 mol %. DNA-DNA hybridization revealed a considerable degree of DNA-DNA similarity amongst all four strains (75.5-80.8%). Nevertheless, similarity with the reference strains of phylogenetically close relatives was lower than 40%. 16S rRNA gene sequences were compared with those of other species of Halomonas and other gram-negative bacteria and they were sufficiently distinct phylogenetically from other recognized Halomonas species to warrant their designation as a novel species. The name Halomonas maura sp. nov. is therefore proposed, with strain S-31T (= CECT 5298T= DSM 13445T) as the type strain. The fatty acid composition of strain S-31T revealed the presence of 18:1omega7c, 16:1omega7c/2-OH i15:0 and 16:0 as the major components. Growth rate analysis showed that strain S-31T had specific cationic requirements for Na+ and Mg2+.

The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows: 99.4-100% within the same species, 96% at the subspecies level, and 73-96% (mean 85%) at the interspecies level (and 98% in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 165 rRNA sequence similarities are all above 99%). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79% (mean 75%). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol% G+C) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-G+C (55-67 mol%) bifidobacteria were grouped into one cluster, whereas the low-G+C species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol%) and Bifidobacterium denticolens (55 mol%); a Bifidobacterium species of intermediate G+C content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.

Bacterial strains were isolated from sponge and green algae which were collected on the coast of Japan and Palau. The phylogenetic relationships of these isolates among marine species of the Cytophaga-Flavobacterium-Bacteroides complex were analysed by using their gyrB nucleotide sequences and translated peptide sequences (GyrB) in addition to 16S rDNA sequences. These isolates were closely related to the previously characterized marine Flexibacter species, [Flexibacter] maritimus and [Flexibacter] ovolyticus. These Flexibacter species are distantly related to Flexibacter flexilis, the type species of the genus Flexibacter, and phylogenetically belong to the family Flavobacteriaceae (according to analysis using both 16S rDNA and GyrB sequences). Their phylogenetic, chemotaxonomic and phenotypic characteristics prompted the proposal that these two species should be transferred to the new genus Tenacibaculum, as Tenacibaculum maritimum and Tenacibaculum ovolyticum, respectively. Two additional new species of the genus Tenacibaculum, Tenacibaculum mesophilum gen. nov., sp. nov. (= MBIC 1140T = IFO 16307T) and Tenacibaculum amylolyticum gen. nov., sp. nov. (= MBIC 4355T = IFO 16310T), which were isolated from sponges and macroalgae, are also reported. For taxonomic considerations at the species level, the resolution of gyrB sequences was superior to that of 16S rDNA sequences, and the grouping based on the gyrB phylogram was consistent with DNA-DNA hybridization results.

Three facultatively anaerobic, Gram-positive bacteria, strains Se-3111T, Se-13111 and Se-1311A, were isolated from an anaerobic, dechlorinating bioreactor culture enriched from sediment of the River Saale in Germany. All strains were isolated from the dechlorinating mixed culture through their ability to reduce selenate anaerobically to elemental selenium. All three strains shared identical 16S rDNA sequences and phylogenetic analysis revealed that strain Se-3111T forms a novel taxon within the suborder Micrococcineae of the class Actinobacteria, related most closely to Beutenbergia cavernae. On the basis of genotypic, chemotaxonomic and physiological characteristics, it is proposed that the novel strains Se-3111T, Se-13111 and Se-1311A be classified in a new genus as Salana multivorans gen. nov., sp. nov. The type strain of the novel species is Se-3111T (= DSM 13521T = NRRL B-24118T).

A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan. The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated. Motility was not observed. The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines. The optimum temperature and pH were 25-30 degrees C and 6.7-7.2. Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety. The G+C content of the DNA was 62.2 mol %. Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus. The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp. nov. is proposed for strain MG62T (= JCM 10825T = DSM 13459T). The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.

Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.

An azo-dye-reducing, endospore-forming bacterium isolated from textile industry wastewater has been taxonomically studied. Particularly interesting was the ability of this organism to decolorize the azo dye Remazol Black B by 98% within 24 h. Levels of 16S rRNA similarity between the isolate and Paenibacillus species ranged from 92.1 to 95.0%. The DNA G+C content was 46.8 mol % and anteiso-branched C15:0 was the major fatty acid. Based upon the phenotypic properties and the phylogenetic inference, it is proposed that the bacterium should be designated Paenibacillus azoreducens sp. nov. The type strain of Paenibacillus azoreducens is CM1T (= DSM 13822T = NCIMB 13761T).

Endospore-forming strains were isolated from corn-compost treated with olive-mill wastewater ('alpechin'). The strains were taxonomically studied and proposed as a novel Paenibacillus species. These organisms (strains B.3T, B.7 and B.9) were particularly distinguishable from other aerobic spore-forming species by their ability to grow optimally in 100% (v/v) olive-mill wastewater at 30 degrees C and pH 7.0 and concomitant production of an interesting exopolysaccharide. Chemotaxonomic analysis revealed that MK-7 was the predominant menaquinone, the major fatty acid was anteiso C15:0 and the cell wall contained meso-diaminopimelic acid. The DNA G+C content was 40.7 mol%. Comparative sequence analysis of 16S rDNA with different reference species from the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Alicyclobacillus, Halobacillus, Virgibacillus, Amphibacillus, Coprobacillus and Gracilibacillus indicated that the isolated strains were highly related to the genus Paenibacillus. Strain B.3T formed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus. Strain B.3T was a close relative of Paenibacillus polymyxa, but DNA-DNA relatedness data with this species was very low (relative binding ratio < 16%). Based on the morphological and physiological characteristics, as well as on the phylogenetic position determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these strains should be designated a novel species, for which the name Paenibacillus jamilae sp. nov. is proposed. The type strain is B.3T (= CECT 5266T = DSM 13815T).

Two haloalkaliphilic archaeal strains, X21T and C112T, were isolated from soda lakes in Inner Mongolia Autonomous Region, China. Their morphology, physiology, biochemical features, polar lipid composition and 16S rRNA genes were characterized in order to elucidate their taxonomy. According to these data, strains X21T and C112T belong to the genus Natrialba, although there are clear differences with respect to their physiology and polar lipid composition between the two strains and the type species, Natrialba asiatica. On the basis of low DNA-DNA hybridizations, these two strains should be considered as new species of genus Natrialba. The names Natrialba hulunbeirensis sp. nov. (type strain X21T = AS 1.1986T = JCM 10989T) and Natrialba chahannaoensis sp. nov. (type strain C112T = AS 1.1977T = JCM 10990T) are proposed.

On the basis of its close phylogenetic relationship to Lamprocystis roseopersicina, the phototrophic purple sulfur bacterium originally described as Amoebobacter purpureus and recently transferred to Pfennigia purpurea is reclassified as Lamprocystis purpurea comb. nov. In addition, an emended description of the genus Lamprocystis is given.

Seven isolates from various human body sites displayed general chemotaxonomic and phenotypic characteristics of the genus Brevibacterium. This was corroborated by the 16S rRNA gene sequence analysis of strain CF62T, showing a sequence similarity of 99% to Brevibacterium mcbrellneri. However, DNA-DNA hybridization, a peculiar amino acid content of the cell wall and some phenotypic properties clearly suggested that these strains belong to a new species, for which the name Brevibacterium paucivorans sp. nov. is proposed. The type strain of B. paucivorans is CF62T (= DSM 13657T = LMG 19814T). The DNA G+C content of the type strain is 55.8 mol%.

Strain IPT101T, isolated from the soil of a sugar-cane plantation in Brazil, was analysed in a polyphasic taxonomic study. The strain produces polyhydroxyalkanoates from sucrose and other carbon sources. Morphological, physiological and biochemical data as well as 16S rDNA, whole-cell protein and fatty acid analyses indicated that strain IPT101T represents a new species in the genus Burkholderia. The name Burkholderia sacchari sp. nov. is proposed, with strain IPT101T (= LMG 19450T = CCT 6771T) as the type strain.

A polycyclic aromatic hydrocarbon-degrading bacterium isolated from coal tar-contaminated soil in Denmark was characterized by a polyphasic approach. Phylogenetically and chemotaxonomically, it was related to members of the genus Mycobacterium. The isolate contains chemotaxonomic markers that are diagnostic for the genus Mycobacterium; i.e. the meso isomer of 2,6-diaminopimelic acid, arabinose and galactose as diagnostic whole-cell sugars, MK-9(H2) as the principal isoprenoid quinone, a mycolic acid pattern of alpha-mycolates, ketomycolates and wax-ester mycolates, unbranched saturated and unsaturated fatty acids plus a small amount of tuberculostearic acid and a significant amount of a C18:0 secondary alcohol. Based on the unique combination of chemical markers among mycobacteria, it is proposed that the isolate should be assigned to a new species, Mycobacterium frederiksbergense sp. nov. This novel species is phylogenetically closely related to Mycobacterium diernhoferi, Mycobacterium neoaurum and Mycobacterium hodleri. The type strain of M. frederiksbergense is strain FAn9T (= DSM 44346T = NRRL B-24126T).

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

A polyphasic taxonomic study, including 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base ratio determinations, amplified 165 rDNA restriction analysis, whole-cell protein analyses and extensive biochemical characterization, was conducted to clarify the relationships of eight isolates from root nodules of Mimosa species and one isolate from sputum of a cystic fibrosis patient. All nine isolates were classified as a novel Ralstonia species, for which the name Ralstonia taiwanensis sp. nov. is proposed. The type strain is LMG 19424T (= CCUG 44338T). R. taiwanensis effectively nodulated the Mimosa species and is the first beta-proteobacterium that is known to be capable of root nodule formation and nitrogen fixation.

Recent insights have been brought to the taxonomy of the genus Enterococcus by studies applying whole-cell protein analysis and DNA-DNA reassociation experiments, in addition to conventional physiological tests. Using these techniques, a group of 10 strains resembling the physiological group III of enterococcal species was characterized. Five strains were recovered from pigs and five from rats with enteric disorders. On the basis of the results of conventional physiological tests, the most likely identity of these strains was Enterococcus durans or Enterococcus hirae. Analysis of the electrophoretic whole-cell protein profiles showed two distinct clusters of virtually indistinguishable profiles: one composed of strains isolated from pigs, and one composed of strains isolated from rats. These protein profiles were not similar to the profiles of any previously described Enterococcus species. The results of DNA-DNA relatedness experiments were consistent with the results of the protein-profile analysis. The high levels of DNA relatedness found for pig isolates demonstrated that they belong to a new enterococcal species, for which the designation Enterococcus porcinus sp. nov. is proposed (type strain =DS 1390-83T =ATCC 700913T =CCUG 43229T =NCIMB 13634T). Strains isolated from rats were found to comprise another new species, for which the designation Enterococcus ratti sp. nov. is proposed (type strain =DS 2705-87T =ATCC 700914T =CCUG 43228T =NCIMB 13635T). This report provides data on the phenotypic and genotypic characterization of these two new enterococcal species, which may represent diarrhoeagenic pathogens for animals.

DNA-DNA hybridization experiments and comparative 165 rDNA sequence analysis revealed that six isolates from purulent joint and lung infections in calves, from a lung lesion in a sheep, and from a joint infection of a pig represented a novel species belonging to the genus Globicatella. Whole-cell protein electrophoresis and biochemical activity testing revealed that the isolates formed a homogeneous group differing from Globicatella sanguinis, the only species of this genus described to date. These animal isolates were classified as Globicatella sulfidifaciens sp. nov. with LMG 188441 (= CCUG 44365T), isolated from the lung of a calf, as the type strain. A detailed description of its phenotypic characteristics is presented. Hydrogen sulphide production was found to be a very useful diagnostic feature.

PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.