- Volume 54, Issue 4, 2004

A novel species, Swaminathania salitolerans gen. nov., sp. nov., was isolated from the rhizosphere, roots and stems of salt-tolerant, mangrove-associated wild rice (Porteresia coarctata Tateoka) using nitrogen-free, semi-solid LGI medium at pH 5·5. Strains were Gram-negative, rod-shaped and motile with peritrichous flagella. The strains grew well in the presence of 0·35 % acetic acid, 3 % NaCl and 1 % KNO3, and produced acid from l-arabinose, d-glucose, glycerol, ethanol, d-mannose, d-galactose and sorbitol. They oxidized ethanol and grew well on mannitol and glutamate agar. The fatty acids 18 : 1ω7c/ω9t/ω12t and 19 : 0cyclo ω8c constituted 30·41 and 11·80 % total fatty acids, respectively, whereas 13 : 1 AT 12–13 was found at 0·53 %. DNA G+C content was 57·6–59·9 mol% and the major quinone was Q-10. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains were related to the genera Acidomonas, Asaia, Acetobacter, Gluconacetobacter, Gluconobacter and Kozakia in the Acetobacteraceae. Isolates were able to fix nitrogen and solubilized phosphate in the presence of NaCl. Based on overall analysis of the tests and comparison with the characteristics of members of the Acetobacteraceae, a novel genus and species is proposed for these isolates, Swaminathania salitolerans gen. nov., sp. nov. The type strain is PA51T (=LMG 21291T=MTCC 3852T).

A pink-pigmented, aerobic, facultatively methylotrophic bacterium, strain BJ001T, was isolated from internal poplar tissues (Populus deltoides×nigra DN34) and identified as a member of the genus Methylobacterium. Phylogenetic analyses showed that strain BJ001T is related to Methylobacterium thiocyanatum, Methylobacterium extorquens, Methylobacterium zatmanii and Methylobacterium rhodesianum. However, strain BJ001T differed from these species in its carbon-source utilization pattern, particularly its use of methane as the sole source of carbon and energy, an ability that is shared with only one other member of the genus, Methylobacterium organophilum. In addition, strain BJ001T is the only member of the genus Methylobacterium to be described as an endophyte of poplar trees. On the basis of its physiological, genotypic and ecological properties, the isolate is proposed as a member of a novel species of the genus Methylobacterium, Methylobacterium populi sp. nov. (type strain, BJ001T=ATCC BAA-705T=NCIMB 13946T).

A Gram-negative, motile, non-spore-forming, rod-shaped bacterium, designated strain TF-22T, was isolated from an intertidal sediment in Korea. This organism grew optimally at 30–37 °C and in the presence of 2–5 % (w/v) NaCl. It did not grow without NaCl or in the presence of more than 14 % (w/v) NaCl. Strain TF-22T was characterized chemotaxonomically as having ubiquinone-8 as the predominant respiratory lipoquinone and C16 : 0, C16 : 1 ω7c and/or iso-C15 : 0 2-OH and C18 : 1 ω7c as the major fatty acids. The DNA G+C content of strain TF-22T was 46·0 mol%. Phylogenetic analyses based on 16S rDNA sequences showed that strain TF-22T falls within the γ-subclass of the Proteobacteria and forms a coherent cluster with Alteromonas macleodii and Alteromonas marina. Levels of 16S rDNA similarity between strain TF-22T and the type strains of two Alteromonas species were in the range 98·1–98·6 %. The level of DNA–DNA relatedness between strain TF-22T and the type strains of two Alteromonas species was 15·7–18·5 %. Therefore, on the basis of phenotypic properties, phylogeny and genomic distinctiveness, strain TF-22T should be placed in the genus Alteromonas as a novel species, for which the name Alteromonas litorea sp. nov. is proposed. The type strain is TF-22T (=KCCM 41775T=JCM 12188T).

Novel alkane-degrading strains of bacteria were isolated from soil contaminated with fuel oil from a leaking underground tank in New Jersey, USA. Two phenotypically similar strains (designated AP102 and AP103T) possessed 16S rRNA sequences unique among the majority of known hydrocarbon-degrading bacteria. The 16S rRNA sequences showed a moderate but distant relationship to the genus Nevskia and a substantial similarity to strains that had previously been isolated for growth on phenol (in Japan) and on toluene (in Canada) by other researchers. The hydrocarbon-degrading strains from Japan, Canada and New Jersey showed no resemblance to the typical morphology of Nevskia but did share a striking similarity among themselves in cell morphology, in the unusual appearance of colonies on various solid media and in various physiological properties. A full taxonomic analysis was performed, including DNA–DNA hybridization and nutritional screening with 117 organic compounds as sole sources of carbon and energy. The strains are active in the degradation of important environmental pollutants, and their phenotypic, physiological, metabolic and genomic properties suggest that they are members of a novel taxon in the γ-Proteobacteria, for which the name Hydrocarboniphaga gen. nov. is proposed, with the single species Hydrocarboniphaga effusa sp. nov. The type strain is AP103T (=ATCC BAA-332T=DSM 16095T).

Two cauliform bacteria (CM243T and CM251) isolated by J. Poindexter from the Atlantic Ocean were characterized by 16S rRNA gene sequencing, TaqI restriction fragment length polymorphism and single-strand conformation polymorphism analyses of the internally transcribed 16S–23S rDNA spacer (ITS1) region, analysis of fatty acids from cellular lipids, mass spectrometry of polar lipids and physiological properties. The two strains showed very low diversity of polar lipids with diacyl-sulfoquinovosyl glycerols as the predominant lipids. The two bacterial strains were observed to have nearly identical 16S rRNA gene sequences and could not be differentiated by their ITS1 regions. The isolates differed from species of the genus Maricaulis by their 16S rRNA gene sequences, polar lipids and fatty acid patterns. On the basis of the genotypic analyses and estimations of phylogenetic similarities, physiological and chemotaxonomic characteristics, it is proposed that the isolates represent a new genus and species, for which the name Woodsholea maritima gen. nov., sp. nov. (type strain CM243T=VKM B-1512T=LMG 21817T) is proposed.

A taxonomic study was performed on 26 strains isolated from microbial mats in Antarctic lakes of the Vestfold Hills and the McMurdo Dry Valleys. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the Rhodobacter group of the α-subclass of the Proteobacteria. Sequence similarity values for the strains with their nearest phylogenetic neighbours (Jannaschia, Octadecabacter and Ketogulonicigenium) ranged between 94·0 and 95·8 %. DNA–DNA hybridizations and comparison of repetitive extragenic palindromic DNA–PCR (rep-PCR) fingerprinting patterns revealed that these strains are members of three distinct species. The isolates are Gram-negative, chemoheterotrophic, non-motile rods and their DNA G+C contents range from 59·4 to 66·4 mol%. Whole-cell fatty acid profiles are similar and the primary fatty acid in all the strains is 18 : 1 ω7c (74·1–87·7 % of total). Genotypic results together with phenotypic characteristics allowed the differentiation of these species from related recognized species of the α-Proteobacteria and the strains are assigned to a new genus, Loktanella gen. nov., with three novel species: Loktanella salsilacus sp. nov. (type species), consisting of ten strains with LMG 21507T (=CIP 108322T) as type strain; Loktanella fryxellensis sp. nov., consisting of 12 strains with LMG 22007T (=CIP 108323T) as type strain; and Loktanella vestfoldensis sp. nov., consisting of four strains with LMG 22003T (=CIP 108321T) as type strain.

Some varieties of sugar beet, Beta vulgaris, cultivated in northern Spain have large deformations that resemble the tumours produced by Agrobacterium species. In an attempt to isolate the agent responsible for these deformations, several endophytic slow-growing bacterial strains were isolated, the macroscopic morphology of which resembled that of Bradyrhizobium species. These strains were not able to produce tumours in Nicotiana tabacum plants and, based on phylogenetic analysis of their 16S rRNA, they are closely related to the genus Bradyrhizobium. Phenotypic and molecular characteristics of these strains revealed that they represent a species different from all Bradyrhizobium species previously described. Sequence analysis of the 16S–23S rDNA intergenic spacer region indicated that these novel strains form a homogeneous group, related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense. DNA–DNA hybridization confirmed that these strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium betae sp. nov. is proposed. The type strain is PL7HG1T (=LMG 21987T=CECT 5829T).

The relationship of Photorhabdus isolates that were cultured from human clinical specimens in Australia to Photorhabdus asymbiotica isolates from human clinical specimens in the USA and to species of the genus Photorhabdus that are associated symbiotically with entomopathogenic nematodes was evaluated. A polyphasic approach that involved DNA–DNA hybridization, phylogenetic analyses of 16S rRNA and gyrB gene sequences and phenotypic characterization was adopted. These investigations showed that gyrB gene sequence data correlated well with DNA–DNA hybridization and phenotypic data, but that 16S rRNA gene sequence data were not suitable for defining species within the genus Photorhabdus. Australian clinical isolates proved to be related most closely to clinical isolates from the USA, but the two groups were distinct. A novel subspecies, Photorhabdus asymbiotica subsp. australis subsp. nov. (type strain, 9802892T=CIP 108025T=ACM 5210T), is proposed, with the concomitant creation of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. Analysis of gyrB sequences, coupled with previously published data on DNA–DNA hybridization and PCR-RFLP analysis of the 16S rRNA gene, indicated that there are more than the three subspecies of Photorhabdus luminescens that have been described and confirmed the validity of the previously proposed subdivision of Photorhabdus temperata. Although a non-luminescent, symbiotic isolate clustered consistently with P. asymbiotica in gyrB phylogenetic analyses, DNA–DNA hybridization indicated that this isolate does not belong to the species P. asymbiotica and that there is a clear distinction between symbiotic and clinical species of Photorhabdus.

Three Halomonas strains, FP34, FP35T and FP36, which were isolated from soil samples taken from Fuente de Piedra, a saline wetland in the province of Málaga in southern Spain, are described. Phylogenetic analyses based on 16S rRNA gene sequences show that the three isolates belong to the genus Halomonas in the γ-Proteobacteria and form an independent genetic line. Phenotypically, they share the characteristics of Halomonas and differ from the most closely related species, Halomonas campisalis, in the following features: they are strictly aerobic and, because of their production of exopolysaccharides, form cream-coloured, mucoid colonies; they produce phosphatase and grow within narrow pH and temperature ranges; and they are susceptible to kanamycin and streptomycin. Their G+C content varies between 60·0 and 61·4 mol%. The name Halomonas anticariensis sp. nov. is proposed for these isolates. Strain FP35T (=LMG 22089T=CECT 5854T) is the type strain. The bacterium grows best in 7·5 % (w/v) NaCl and does not require magnesium or potassium salts for growth, although they do stimulate growth somewhat when present. Its major fatty acids are 18 : 1ω7c, 16 : 0, 16 : 1ω7c, 15 : 0 iso 2-OH, 12 : 0 3-OH, 12 : 0, 10 : 0 and 19 : 0 cyclo ω8c. Its predominant respiratory lipoquinone is ubiquinone with nine isoprene units (Q-9).

Five strains with strong chitinolytic activity were isolated from a soil sample collected from southern Taiwan. The strains shared more than 92 % DNA–DNA similarity, indicating membership of the same genospecies. This close relationship was supported by high similarities in fatty acid composition and biochemical characteristics. A 16S rRNA gene sequence analysis indicated that the isolates were members of the class ‘Betaproteobacteria’, in which they formed an individual subline of descent that was distantly related (<94 % similarity) to lineages defined by Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of the phylogenetic and phenotypic distinctness of these novel chitin-degrading organisms, a new genus, Chitinibacter, is proposed, with Chitinibacter tainanensis (type strain, S1T=BCRC 17254T=DSM 15459T) as the type species.

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97·5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel ‘Candidatus’ species under the names ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Gram-positive, spore-forming rods were isolated from blood cultures of three different patients. Based on phylogenetic analyses, these strains were placed within the Paenibacillus cluster and specific phenotypic characteristics for each strain were described. Levels of 16S rRNA gene sequence similarity between existing Paenibacillus species and the three novel strains 2301065T, 2301032T and 2301083T were 87·6–94·4, 88·5–95·4 and 87·5–96·0 %, respectively, and anteiso-branched C15 : 0 was the major fatty acid. On the basis of phenotypic data and phylogenetic inference, it is proposed that these strains should be designated Paenibacillus massiliensis sp. nov., Paenibacillus sanguinis sp. nov. and Paenibacillus timonensis sp. nov. The type strains are respectively strain 2301065T (=CIP 107939T=CCUG 48215T), strain 2301083T (=CIP 107938T=CCUG 48214T) and strain 2301032T (=CIP 108005T=CCUG 48216T).

Nine unidentified Gram-positive, lipophilic corynebacteria were isolated from clinical and food samples and subjected to a polyphasic taxonomic analysis. The bacteria were distinguished from Corynebacterium species with validly published names by biochemical tests, fatty acid content and whole-cell protein analysis. Comparative 16S rRNA gene sequence analysis demonstrated unambiguously that the nine strains were related phylogenetically to the species ‘Corynebacterium tuberculostearicum’ and represented a distinct subline within the genus Corynebacterium. On the basis of both phenotypic and phylogenetic evidence, the formal description of Corynebacterium tuberculostearicum sp. nov. is proposed. The type strain of C. tuberculostearicum is Medalle XT (=LDC-20T=CIP 107291T=CCUG 45418T=ATCC 35529T).

A moderately thermophilic and alkaliphilic bacillus, which had been reported and designated BLx ( Haruta et al., 2002 ), was isolated from a semi-continuous decomposing system of kitchen refuse. Cells of strain BLxT were strictly aerobic, rod-shaped, motile and spore forming. The optimum temperature and pH for growth were approximately 50 °C and pH 8–9. Strain BLxT was able to grow at NaCl concentrations from 0·5 to 7·5 %, with optimum growth at 0·5 % NaCl. The predominant menaquinone was MK-7, and the major fatty acid was iso-C15 : 0. Phylogenetic analysis showed that strain BLxT was positioned in an independent lineage within the cluster that includes the genera Virgibacillus and Lentibacillus in Bacillus rRNA group 1. Strain BLxT exhibited 16S rDNA similarity of 92·8–94·8 % to Virgibacillus species and 92·3 % to Lentibacillus salicampi. Phenotypic, chemotaxonomic and phylogenetic analyses supported the classification of strain BLxT in a novel genus and species. Cerasibacillus quisquiliarum gen. nov., sp. nov. is proposed on the basis of phenotypic, chemotaxonomic and phylogenetic data. The type strain is BLxT (DSM 15825T=IAM15044T=KCTC 3815T).

Seven strains of aerobic, endospore-forming bacteria were found in soil taken from an active fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago, Antarctica, and four strains were from soil of an inactive fumarole at the foot of the hill. Amplified rDNA restriction analysis, 16S rDNA sequence comparisons, SDS-PAGE and routine phenotypic tests support the proposal of two novel species of Paenibacillus, Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., the type strains of which are LMG 18439T (=CIP 108109T) and LMG 18419T (=CIP 108110T), respectively. A further strain, isolated from a gelatin-production process, showed more than 99 % 16S rDNA sequence similarity to the proposed P. cookii type strain and, although the gelatin isolate was atypical when compared with the fumarole isolates by repeated element primed-PCR, SDS-PAGE and phenotypic analyses, it was shown by DNA–DNA reassociation studies to belong to the same species. Strains of P. cookii produce spreading growth with motile microcolonies. Both species produce swollen sporangia that are typical for the genus, they both show 97·6 % 16S rDNA sequence similarity to Paenibacillus azoreducens, they have 51·5–51·6 mol% G+C in their DNA and their major fatty acid is anteiso-C15 : 0; however, fatty acids C16 : 0 and anteiso-C17 : 0 represent, respectively, 18 and 10 % of the total in P. cineris, but 11 and 20 % in P. cookii.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

The isolation and identification of a novel, slow-growing, scotochromogenic, mycobacterial species is reported. A strain, designated MUP 1182T, was isolated from a cervical lymph node of a 3-year-old child. MUP 1182T is alcohol- and acid-fast, with a lipid pattern that is consistent with those of species that belong to the genus Mycobacterium. It grows slowly at 25–37 °C, but does not grow at 42 °C. The isolate was revealed to be biochemically distinct from previously described mycobacterial species: it has urease and Tween hydrolysis activities and lacks nitrate reductase, 3-day arylsulfatase and β-glucosidase activities. Comparative 16S rDNA sequencing showed that isolate MUP 1182T represents a novel, slow-growing species that is related closely to Mycobacterium lentiflavum and Mycobacterium simiae. On the basis of these findings, the name Mycobacterium parmense sp. nov. is proposed, with MUP 1182T (=CIP 107385T=DSM 44553T) as the type strain.

The taxonomic standing of Bifidobacterium lactis and Bifidobacterium animalis was investigated using a polyphasic approach. Sixteen representatives of both taxa were found to be phenotypically similar and shared more than 70 % DNA–DNA relatedness (76–100 %), which reinforces the conclusions of previous studies in which B. lactis and B. animalis were considered to be one single species. However, the results of protein profiling, BOX-PCR fingerprinting, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), and atpD and groEL gene sequence analysis demonstrate that representatives of B. animalis and B. lactis constitute two clearly separated subgroups; this subdivision was also phenotypically supported based on the ability to grow in milk. Given the fact that B. lactis Meile et al. 1997 has to be considered as a junior synonym of B. animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974 , our data indicate that the latter species should be split into two new subspecies, i.e. Bifidobacterium animalis subsp. animalis subsp. nov. (type strain R101-8T=LMG 10508T=ATCC 25527T=DSM 20104T=JCM 1190T) and Bifidobacterium animalis subsp. lactis subsp. nov. (type strain UR1T=LMG 18314T=DSM 10140T=JCM 10602T).