Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov

Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).     

Taxonomic study of bacteria isolated from natural mineral waters: proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov

The taxonomic position of 23 strains isolated from mineral waters and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster IX, subclusters XIIIa and XIIIc of VERHILLE, S., ELOMARI, M., COROLER, L., IZARD, D., LECLERC, H. (Syst. Appl. Microbiol, 20, 137-149, 1997) has been genotypically further studied in the present work. On the basis of hybridization results, these strains were gathered into two new genomic groups for which we propose the names of Pseudomonas jessenii sp. nov. (Type strain CIP 105274) and Pseudomonas mandelii sp. nov. (Type strain CIP 105273). Deoxyribonucleic acid relatedness levels showed homologies ranging from 78 to 100% for Pseudomonas jessenii and from 77 to 100% for Pseudomonas mandelii. Furthermore, hybrization rates with 66 representative well characterized species or only partially characterized species of the genus Pseudomonas were below 53%, with delta Tm values of 7 degrees C and more. The mol% G + C content ranged from 57 to 58. The two new species presented basic morphological characteristics common to all pseudomonads. Various phenotypic features, such as denitrification, growth at 4 degrees C or 41 degrees C, trigonelline assimilation, alpha-L-glutamyl-L-histidine arylarmidase activity, growth on benzoate and meso-tartrate were found to differentiate Pseudomonas jessenii from Pseudomonas mandelii and from other Pseudomonas species. Pseudomonas jessenii encompassed a total of 9 strains from both phenotypic groups IX and XIIIa. Pseudomonas mandelii clustered a total of 13 strains from both phenotypic groups IX and XIIIc. Their clinical significance is unknown. The 16S rDNA of each type strain was sequenced and compared with the known sequences of the representative strains of the genus Pseudomonas. A phylogenetic tree was constructed to determine the intrageneric relationships within the genus Pseudomonas.     

Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster

The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria. Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed. It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A. lipoferum, A. doebereinerae, A. largimobile, A. brasilense and A. halopraeferens, for the Azospirilli subcluster II including A. amazonense, A. irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster. The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels. The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set. In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A. brasilense on maize roots were demonstrated using confocal laser scanning microscopy.     

The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov.

Large numbers of strains selectively isolated from soil, water and deteriorating vulcanised natural rubber pipe rings were provisionally assigned to the genus Nocardia. Twenty-eight representative isolates were found to have chemical and morphological properties typical of nocardiae. These organisms formed a monophyletic clade in the 16S rDNA tree together with Nocardia salmonicida. Three of the strains, isolates S1, W30 and R89, were distinguished from one another and from representatives of the validly described species of Nocardia using genotypic and phenotypic data. These organisms were considered to merit species status and were named Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. respectively. Additional comparative studies are needed to resolve the finer taxonomic relationships of the remaining isolates assigned to the Nocardia salmonicida clade and to further unravel the extent of nocardial diversity in artificial and natural ecosystems.     

Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov

Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

The phylogenetic relationship among Ectothiorhodospiraceae: a reevaluation of their taxonomy on the basis of 16S rDNA analyses

Sequences of the 16S rRNA gene were determined from all type strains of the recognized Ectothiorhodospira species and from a number of additional strains. For the first time, these data resolve the phylogenetic relationships of the Ectothiorhodospiraceae in detail, confirm the established species, and improve the classification of strains of uncertain affiliation. Two major groups that are recognized as separate genera were clearly established. The extremely halophilic species were removed from the genus Ectothiorhodospira and reassigned to the new genus Halorhodospira gen. nov., to recognize that the most halophilic eubacteria are species of this genus. These species are Halorhodospira halophila comb. nov., Halorhodospira halochloris comb. nov., and Halorhodospira abdelmalekii comb. nov. Among the slightly halophilic Ectothiorhodospira species, the classification of strains belonging to Ectothiorhodospira mobilis and Ectothiorhodospira shaposhnikovii was improved. Several strains that were tentatively identified as Ectothiorhodospira mobilis form a separate cluster on the basis of their 16S rDNA sequences and are recognized as two new species: Ectothiorhodospira haloalkaliphila sp. nov., which includes the most alkaliphilic strains originating from strongly alkaline soda lakes, and Ectothiorhodospira marina, describing isolates from the marine environment. Received: 12 October 1995 / Accepted: 1 December 1995     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Acidovorax-like symbionts in the nephridia of earthworms

Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms). These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling. In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved. The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species. Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes. No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning. These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor. Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms.     

Kribbella yunnanensis sp. nov., Kribbella alba sp. nov., two novel species of genus Kribbella isolated from soils in Yunnan, China

Two actinomycete strains, designated YIM 30006(T) and YIM 31075(T), were isolated from soil samples in Yunnan, China and subjected to a polyphasic taxonomic study. Morphological and chemotaxonomic analysis revealed that the two isolates should be consistent with the nocardioform actinomycetes. Comparative 16S rDNA sequences confirmed that the two unknown isolates to be members of the genus Kribbella. Based on the results of phenotypic characteristics, phylogenetic studies and DNA-DNA hybridization results, strains YIM 30006(T) and YIM 31075(T) should be classified as two novel species of the genus Kribbella, for which the names Kribbella yunnanensis sp. nov. and Kribbella alba sp. nov. are proposed. The type strains for them are YIM 30006(T) (=CCTCC AA001019(T)=DSM 15499(T)) and YIM 31075(T) (=CCTCC AA 001020(T)=DSM 15500(T)), respectively. The 16S rDNA sequences of strains YIM 30006(T), YIM 31075(T) have been deposited in GenBank under the accession numbers AY 082061 and AY 082062, respectively.     

Identification of Acetobacter strains isolated from Indonesian sources,and proposals of Acetobacter syzygii sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis sp. nov

Forty-six strains of acetic acid bacteria newly isolated from flowers, fruits, and fermented foods collected in Indonesia were taxonomically studied. They were Gram-negative rods, produced acetic acid from ethanol, oxidized acetate and lactate to CO(2) and H(2)O, and had Q-9 as the major ubiquinone system. On the basis of DNA-DNA similarity, all strains studied, including type strains and reference strains of the genus Acetobacter, were separated into eleven groups (Groups I to XI). Of the 46 isolates, two isolates were included in Group II and identified as Acetobacter pasteurianus, five in Group IV as A. orleanensis, 16 in Group V as A. lovaniensis, five in Group VII as A. indonesiensis, and three in Group VIII as A. tropicalis. The remaining 15 isolates constituted three new groups based on DNA-DNA similarity; four isolates were included in Group IX, two in Group X, and nine in Group XI. No isolates were identified as A. aceti (Group I), A. peroxydans (Group III), and A. estunensis (Group VI). Phylogenetic analysis based on 16S rDNA sequences of representative strains of the Groups indicated belonging to the strains of the genus Acetobacter. On the basis of DNA base composition, DNA-DNA similarity, and 16S rDNA sequences, three new species of the genus Acetobacter are proposed: Acetobacter syzygii sp. nov. for Group IX, Acetobacter cibinongensis sp. nov. for Group X, and Acetobacter orientalis sp. nov. for Group XI. The distribution of Acetobacter strains in Indonesia is discussed in light of isolation sources.     

Sporobolomyces diospyroris sp. nov., Sporobolomyces lophatheri sp. nov. and Sporobolomyces pyrrosiae sp. nov., three new species of ballistoconidium-forming yeasts in the Agaricostilbum lineage isolated from plants in Taiwan

Three strains of xylose-lacking and ubiquinone-10-having ballistoconidium-forming yeasts isolated from plant leaves collected in Taiwan were found to represent respective new species. In phylogenetic trees constructed based on the nucleotide sequences of 18S rDNA and D1/D2 domain of 26S rDNA, they were located in the Agaricostilbum lineage (Agaricostilbum/Bensingtonia cluster). Since the taxonomic properties of these species coincide with those of the genus Sporobolomyces, they are described as Sporobolomyces diospyroris sp. nov., Sporobolomyces lophatheri sp. nov. and Sporobolomyces pyrrosiae sp. nov., respectively.     

Characterization of some strains from human clinical sources which resemble "Leptotrichia sanguinegens": description of Sneathia sanguinegens sp. nov., gen. nov

Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens". Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T.     

Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khoito-Gol hydrogen-sulfide springs, Buryatia

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8-9.5, with an optimum at pH 9.3 and in a temperature range of 5-39 degrees C, with an optimum at 28-32 degrees C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3-64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21-29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).     

Leifsonia gen. nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli subsp. cynodontis Davis et al. 1984

"Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980. This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11. A close relationship of "C. aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences. An analysis of DAB isomers of the peptidoglycan distinguished "C. aquaticum" clearly from these genera by possessing both L-DAB and D-DAB. We also found that the type strain of Clavibacter xyli subsp. cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C. aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae. Considering these results, we propose a new genus Leifsonia to accommodate the four strains. The four species, Leifsonia aquatica sp. nov., nom. rev., comb. nov. (type species, type strain=JCM 1368), Leifsonia shinshuensis sp. nov. (type strain=DB102=JCM 10591), Leifsonia naganoensis sp. nov. (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb. nov. (type strain=JCM 9733=ICMP 8790), were proposed here for the strains.     

Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms.

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 microns. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4',6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.