Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions,and description of Thauera phenylacetica sp. nov., Thauera aminoaromaticasp. nov., and Azoarcus buckelii sp. nov

Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.     

Ribosomal ribonucleic acid isolated from Salmonella typhimurium: absence of the intact 23S species.

Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA.     

Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.     

Genetic characterization of pathogenic Borrelia, group A14S, isolated in Ukraine

Borrelia Ir-5215, isolated from ticks Ixodes ricinus in Ukraine (the Crimean autonomous region), was identified by the method of the polymorphism of the fragment length of the restriction amplicon of rRNA spacer region 5S-23S. Its Msel-restriction profile was relatively similar to that of B. afzelii. The sequencing of spacer region rrf (5S)-rrl (23S) and 16S rRNA gene, as well as the analysis of the similarity of nucleotide sequences, obtained in the course of these study, revealed the differences between Borrelia sp, lr-5215 and six European species of Borrelia burgdorferi sensu lato and a high level of similarity (more than 95.1% for 5S-23S rRNA and 99.4% for 16S rRNA gene) to three known representatives of genome group A14S (Borrelia spp. A14S, I-77 and PC-Rq17). This suggests that isolated Borrelia lr-5215 is a new representative of pathogenic B. burgdorferi sensu lato genome group A14S, which is spread, together with Central Europe, also in southern Ukraine.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences analysis of the genus Myxococcus.

Phylogenetic relationships of the species belonging to the genus Myxococcus were elucidated based on the sequences of 16S rRNA genes and 16S-23S rRNA gene internal transcribed spacer (ITS) regions. The Myxococcus species were consequently classified into four distinct groups. The type strain of Myxococcus coralloides occupied an independent position (Group 1); it has been recently reclassified as Corallococcus coralloides. Group 2 comprised the type strains of both Myxococcus virescens and Myxococcus xanthus, and some strains assigned to Myxococcus flavescens. The type strain of M. flavescens was contained in Group 3 along with the strains of Myxococcus fulvus. Group 4 included the strains belonging to C. coralloides, M. fulvus, and M. stipitatus. The type strain of M. fulvus that was allocated outside Group 4 in the 16S rRNA gene tree belonged to Group 3 in the ITS tree. These results strongly suggest that the morphological characteristics of Myxococcus species are not consistent with the phylogenetic relationships. The Myxococcus species must therefore be redefined according to the phylogenetic relationships revealed in this study.     

Streptacidiphilus jiangxiensis sp. nov., a novel actinomycete isolated from acidic rhizosphere soil in China

The taxonomic position of three acidophilic actinomycetes isolated from acidic rhizosphere soil was established using a polyphasic approach. The morphological and chemical properties of the isolates were found to be consistent with their assignment to the genus Streptacidiphilus. Almost complete 16S rRNA gene sequences determined for the strains were aligned with corresponding sequences of representatives of the genera Kitasatospora, Streptacidiphilus and Streptomyces and phylogenetic trees inferred using three tree-making algorithms. The organisms formed a distinct subclade within the Streptacidiphilus 16S rRNA gene tree. They also shared nearly identical phenotypic profiles and rep-PCR fingerprint patterns that readily distinguished them from representatives of the established species of Streptacidiphilus. It is evident from the genotypic and phenotypic data that the three isolates form a new species in the genus Streptacidiphilus. The name proposed for this new species is Streptacidiphilus jiangxiensis, the type strain is isolate 33214T (= AS 4.1857T = JCM 12277T).     

A phylogenetic analysis of Bacillus thuringiensis serovars by RFLP-based ribotyping

AIMS: To determine the 23S and 5S rRNA gene fingerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. METHODS AND RESULTS: Eighty-six B. thuringiensis strains which include 80 serovar type strains, five intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95.5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene fingerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. CONCLUSION: This method permitted the classification and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes a further step to the definition of valid taxonomic sublevels for the B. thuringiensis species.     

Babesia leo n. sp. from lions in the Kruger National Park, South Africa, and its relation to other small piroplasms.

Babesia leo, a small piroplasm isolated from lions in South Africa is described as a distinct species based on a phylogenetic analysis of the 18S rRNA gene. Intraerythrocytic trophozoite and merozoite stages of B. leo are morphologically indistinguishable from other small piroplasms of felids. Previous studies showed that B. leo was biologically and antigenically distinct from B. felis, which is known to infect wild and domestic felids in South Africa. Molecular characterization showed strong support for the phylogenetic seperation of B. leo as a distinct species from B. felis and other felid piroplasms. Phylogenetic analysis also showed that Babesia microti and all of the felid piroplasms from Africa with known 18S rRNA gene sequences available, including B. leo, formed a single, separate clade, sister to the other babesial and theilerial piroplasm parasites.     

The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid.

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.     

Isolate B12, which harbours a virus-like element,represents a new species of the archaebacterial genus Sulfolobus,Sulfolobus shibatae,sp. nov.

The Sulfolobus isolate B12 and its endogenous virus-like element SSV1 have provided a fruitful system for detailed analysis of certain aspects of archaebacterial molecular biology, especially those concerning gene expression. In the course of clarifying this isolate's taxonomic position, we determined DNA base composition, ability to grow autotrophically, nucleotide sequence of 16S ribosomal RNA, and level of total genomic homology to other Sulfolobus strains. Although the results generally demonstrate a similarity to S. solfataricus, DNA-DNA hybridisation and 16S rRNA sequence data indicate that isolate B12 in fact represents a distinct species.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1 B, D-3300 Braunschweig, FRG - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Identical 5S rRNA nucleotide sequence of Vibrio cholerae strains representing temporal, geographical, and ecological diversity.

Enzymatic determination of the nucleotide sequence of 5S rRNA prepared from a Vibrio cholerae non-O1 serovar isolated from a canal in Louisiana in 1978 revealed it to be identical to that of V. cholerae O1 isolated in 1905 from a pilgrim at the E1 Tor quarantine station. The two strains are associated with sharply contrasting habitats. Ranges of variation of 5 to 15% in procaryotic 5S rRNA sequences between species are typical; variation within species may be 2% or greater. Because the 5S rRNA sequences were identical, it is concluded that they have a common phylogenic origin.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.     

Lactobacillus perolens sp. nov., a soft drink spoilage bacterium.

Lactic acid bacteria that are able to spoil soft drinks with low pH comprise a limited number of acidotolerant or acidophilic species of the genera Lactobacillus, Leuconostoc and Weissella. Various Gram-positive rods causing turbidity and off-flavour were isolated from orange lemonades. Physiological and biochemical studies including SDS-PAGE whole-cell protein analysis showed a homogeneous group of organisms. The 16S rRNA gene sequence analysis of two representatives revealed that they formed a phylogenetically distinct line within the genus Lactobacillus. All strains were facultatively heterofermentative, producing L-lactic acid. Based on the data presented a new species L. perolens is proposed. The name refers to the off-flavour caused by high amounts of diacetyl. The type strain of L. perolens is DSM 12744 (LMG 18936). A rRNA targeted oligonucleotide probe was designed that allows a fast and reliable identification of L. perolens.     

Gordonia namibiensis sp. nov., a novel nitrile metabolising actinomycete recovered from an African sand

A polyphasic approach was used to establish the taxonomic position of two actinomycetes isolated from a Namibian soil and shown to utilise nitrile compounds as growth substrates. The organisms, strains NAM-BN063AT and NAM-BN063B, had chemical and morphological properties consistent with their assignment to the genus Gordonia. Direct 165 rRNA sequencing studies confirmed the taxonomic position of the strains following the generation of phylogenetic trees using four different algorithms. The strains consistently formed a distinct phylogenetic line within the evolutionary radiation occupied by gordoniae and were most closely related to Gordonia rubropertincta DSM 43197T. DNA:DNA relatedness studies indicated that the two organisms belonged to a genomic species that was readily distinguished from G. rubropertincta. The unique phenotypic profile of the strains sharply separated them from representatives of all of the validly described species of Gordonia. The combination of genotypic and phenotypic data indicates that the two strains should be classified in the genus Gordonia as a new species. The name proposed for this taxon is Gordonia namibiensis, the type strain is NAM-BN063AT (= DSM 44568T = NCIMB 13780T).     

Criblamydia sequanensis, a new intracellular Chlamydiales isolated from Seine river water using amoebal co-culture

Accumulating evidence supports a role for Chlamydia-related organisms as emerging pathogens for human and animals. Assessment of their pathogenicity requires strain availability, at least for animal models and serological studies. As these obligate intracellular species are able to grow inside amoebae, we used co-culture with Acanthamoeba castellanii in an attempt to recover new Chlamydia-related species from river water. We isolated two strains from eight water samples. The first strain is a new Parachlamydia acanthamoebae strain that differs from previously described isolates by only two bases in the complete 16S rRNA gene sequence. The second isolate is the first representative of a new Chlamydiales family, as demonstrated by genetic and phylogenetic analyses of the 16S rRNA, 23S rRNA, ADP/ATP translocase and RnpB encoding genes. Using fluorescent in situ hybridization and electron microscopy, we demonstrated that it grows in high numbers in amoebae, where it exhibits a Chlamydia-like developmental cycle with reticulate bodies and star-like elementary bodies. Based on these results, we propose to name this new species 'Criblamydia sequanensis'. This work confirmed that amoebal co-culture is a relevant method to isolate new chlamydiae, and that it can be successfully applied to ecosystems colonized with a complex microbial community.     

Description of a new species, Bifidobacterium crudilactis sp. nov., isolated from raw milk and raw milk cheeses

A new Bifidobacterium species is described based on the study of ten Gram-positive strains with fructose-6-phosphate phosphoketolase activity. They are part of a phenotypic group comprising 141 strains isolated from raw milk and raw milk cheeses in French raw milk cheese factories. This group was separated by a numerical analysis based on API 50CH, API 32A tests and growth at 46 degrees C. A strong similarity of 16S rRNA sequences (99.8%) was shown between strain FR62/b/3(T) and Bifidobacterium psychraerophilum LMG 21775(T). However, low DNA-DNA relatedness was observed between their DNAs (31%). The new isolates are able to grow at low temperatures (all ten strains up to 5 degrees C) and strain FR62/b/3(T) grows under aerobic conditions, as does B. psychraerophilum. However, contrary to B. psychraerophilum, they do not ferment L-arabinose, D-xylose, arbutin or melezitose, but they do acidify lactose. The DNA G+C content of FR62/b/3(T) is 56.4mol%. Therefore, the name Bifidobacterium crudilactis sp. nov. is proposed, with its type strain being FR62/b/3(T) (=LMG 23609(T)=CNCM I-3342(T)).