Identical 16S rDNA Sequences Research Articles

Paenibacillus isolates possess diverse dextran-degrading enzymes

To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced. Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70 degrees C were closely aligned to Paenibacillus spp. The three isolates purified at 60 degrees C had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50 degrees C produced dextranolytic activity in the spent media with maximal activity at 40 or 45 degrees C under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70 degrees C. The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity. Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.

The synthesis of the UV‐screening pigment, scytonemin, and photosynthetic performance in isolates from closely related natural populations of cyanobacteria (Calothrix sp.)

Two populations of the cyanobacterium Calothrix sp. found in Yellowstone thermal spring outflows differ greatly in their contents of scytonemin, a UV-screening pigment, and in their photosynthetic carbon assimilation rates. Clonal isolates from both populations were used to investigate these phenotypic differences. Identical partial 16S rDNA sequences ( approximately 900 bp) suggest a very close relationship between the two Calothrix populations and indicate that environmental differences may, in part, explain the field observations. The effects of native spring water on scytonemin synthesis and photosynthesis were tested during experiments using plated cells. Results show differences in the spring water environment were at least partly responsible for the differences in scytonemin content observed in the field. Furthermore, spring water effects on photosynthetic performance suggest adaptation in these strains to their spring of origin. Controlled experiments performed using cultures grown in artificial liquid medium showed no significant difference in photosynthetic carbon uptake between strains. However, significant differences were detected in their ability to synthesize scytonemin indicating genetic differences between populations. These findings suggest that both genetic and environmental differences are responsible for the naturally occurring variation in scytonemin content and photosynthetic ability in these two closely related populations.

Plesiocystis pacifica gen. nov., sp. nov., a marine myxobacterium that contains dihydrogenated menaquinone, isolated from the Pacific coasts of Japan.

Two strains of a novel myxobacterium (designated SIR-1T and SHI-1) were isolated from Japanese coasts located in the Pacific subtropical zone. Cells of both strains were Gram-negative, rod-shaped and motile by gliding. The strains were chemoheterotrophic and strictly aerobic. They had the common characteristics associated with myxobacteria, such as bacteriolytic action and fruiting-body formation. The characteristic feature of the strains was a NaCl growth requirement with an optimum concentration of 2.0-3.0 % (w/v), comparable to that of sea water. In addition, other cationic components of sea water, such as Mg2+, Ca2+ and K+, were needed for growth. The major respiratory quinone was MK-8(H2). The cellular fatty acid profile was characterized by the predominance of iso-C15:0. Characteristic fatty acids anteiso-C16:0 and anteiso-C17:0, and a long-chain polyunsaturated fatty acid (C20:4), were also detected. The G + C content of the genomic DNA of strains SIR-1T and SHI-1 was between 69.3 and 70.0 mol% (as determined by HPLC). Strains SIR-1T and SHI-1 shared almost identical 16S rDNA sequences, and clustered with the genus Nannocystis as their closest relative upon phylogenetic analysis. However, the phylogenetic distance between the novel strains and the genus Nannocystis was large enough to warrant their different generic allocation. This finding was supported by significant phenotypic differences between the novel strains and Nannocystis. Thus, strains SIR-1T and SHI-1 represent a novel genus and species, for which the names Plesiocystis and Plesiocystis pacifica, respectively, are proposed. The type strain of the species is SIR-1T (=JCM 11591T =DSM 14875T =AJ 13960T).

Enhygromyxa salina gen. nov., sp. nov., a Slightly Halophilic Myxobacterium Isolated from the Coastal Areas of Japan

Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan. All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically. They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v). In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth. The major respiratory quinone was MK-7. The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC). The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity). Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus. Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen. nov., sp. nov. The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).

Shewanella olleyana sp. nov., a marine species isolated from a temperate estuary which produces high levels of polyunsaturated fatty acids.

Two polyunsaturated fatty acid (PUFA) producing strains (ACEM 6 and ACEM 9(T)) isolated from a temperate, humic-rich river estuary in Tasmania, Australia, were found to be members of the genus Shewanella. These strains were able to utilize humic compounds (tannic acid) and derivatives (2,6-anthraquinone disulfonate) as sole carbon sources and as electron acceptors for anaerobic respiration. The major fatty acids were typical of the genus Shewanella; however, PUFAs mostly made up of eicosapentaenoic acid were produced at high levels (10.2-23.6% of total fatty acids) and at relatively high incubation temperatures (10.2% at 24 degrees C). Sequence analysis indicated that ACEM 6 and ACEM 9(T) had identical 16S rDNA sequences and were most closely related to Shewanella japonica (sequence similarity 97.1%). DNA hybridization and phenotypic characteristics confirmed that the isolates constituted a novel species of the genus Shewanella, which is designated Shewanella olleyana sp. nov. (type strain ACEM 9(T) = ACAM 644(T) = LMG 21437(T)).

Diversity of grass-associated Microbacteriaceae isolated from the phyllosphere and litter layer after mulching the sward; polyphasic characterization of Subtercola pratensis sp. nov., Curtobacterium herbarum sp. nov. and Plantibacter flavus gen. nov., sp. nov.

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

Lactobacillus diolivorans sp. nov., a 1,2-propanediol-degrading bacterium isolated from aerobically stable maize silage.

Inoculation of maize silage with Lactobacillus buchneri (5 x 10(5) c.f.u. g(-1) of maize silage) prior to ensiling results in the formation of aerobically stable silage. After 9 months, lactic acid bacterium counts are approximately 10(10) c.f.u. g(-1) in these treated silages. An important subpopulation (5.9 x 10(7) c.f.u. g(-1)) is able to degrade 1,2-propanediol, a fermentation product of L. buchneri, under anoxic conditions to 1-propanol and propionic acid. From this group of 1,2-propanediol-fermenting, facultatively anaerobic, heterofermentative lactobacilli, two rod-shaped isolates were purified and characterized. Comparative 16S rDNA sequence analysis revealed that the newly isolated bacteria have identical 16S rDNA sequences and belong phylogenetically to the L. buchneri group. DNA-DNA hybridizations, whole-cell protein fingerprinting and examination of phenotypic properties indicated that these two isolates represent a novel species, for which the name Lactobacillus diolivorans sp. nov. is proposed. The type strain is LMG 19667T (= DSM 14421T).

Salana multivorans gen. nov., sp. nov., a novel actinobacterium isolated from an anaerobic bioreactor and capable of selenate reduction.

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).

Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid.

Four bacterial strains that oxidize L-sorbose to 2-keto-L-gulonic acid, a key intermediate in the synthesis of vitamin C, were isolated from soils of geographically distinct locations. All were Gram-negative, facultatively anaerobic, chemoheterotrophic rods. Comparative analysis revealed nearly identical 16S rDNA sequences amongst them (99.7-100% identical) and identified them as members of the alpha-subclass of the Proteobacteria. Phylogenetic analysis identified the closest taxonomically defined genus as Roseobacter (92.1-92.8% identical). On the basis of phylogenetic, phenotypic and genotypic analyses, a new genus is proposed, Ketogulonigenium gen. nov. Based upon these analyses, we also propose the reclassification of strain DSM 4025TP, originally identified as Gluconobacter oxydans, to the genus Ketogulonigenium. Two species are proposed: the type species Ketogulonigenium vulgare gen. nov., sp. nov., consisting of strains 62A-12APP, 266-13BPP and the type strain K. vulgare DSM 4025TP, and Ketogulonigenium robustum gen. nov., sp. nov., consisting of the type strain K. robustum X6LTP (= NRRL B-21627 = KCTC 0858BP). The species affiliation of the fifth strain (291-19PP) remains unresolved.

Diagnostics of neisseriaceae and moraxellaceae by ribosomal DNA sequencing: ribosomal differentiation of medical microorganisms.

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.

Differences between Subcultures of the Mesorhizobium loti Type Strain from Different Culture Collections

Because of differences in the reported 16S rRNA gene sequence of the Mesorhizobium loti type strain available from different culture collections, we collected different subcultures of this strain and compared them by 16S rDNA sequencing, SDS-PAGE of whole-cell protein extracts and RAPD-PCR. Our results indicate that the 16S rDNA sequence differences can be explained by the presence of two different organisms in one of the subcultures. In addition, even for subcultures of the type strain that had identical 16S rDNA sequences, small differences could be observed in the protein profiles and in the RAPD-PCR patterns. These latter observations indicate that maintenance procedures necessary for long-term preservation by freeze-drying can cause subcultures of the same original strain to undergo changes, effectively leading to different fingerprints even though 16S rDNA sequences remain identical.

Bacterial Community Changes and Enrichment of Burkholderia-like Bacteria Induced by Chlorinated Benzoates in a Peat-Forest Soil-Microcosm

Bacterial community shifts in a peat-forest soil spiked with 3-chlorobenzoate (3CBA) or 2,5-dichlorobenzoate (2,5DCB) were monitored by PCR-amplification of the V6 to V8 regions of the 16S rRNA and rDNA, followed by separation of the amplicons by temperature gradient gel electrophoresis. 3CBA disappeared to non-detectable levels after 15 days by a biologically mediated process, while 2,5DCB remained at the initial concentration values. The experiments were conducted under microcosms systems. Addition of the chlorinated benzoates to the soil resulted in a rapid decrease of the microbial diversity, as judged by a time-dependent reduction in the number of amplicons detected by temperature gradient gel electrophoresis. Few amplicons specifically enriched in the spiked soils were cloned and characterised by sequence analysis. The identity of the cloned DNA and the corresponding soil amplicons was confirmed by hybridisation with a radioactively labelled V6-probe. Analysis of the 16S rDNA sequences indicated that Burkholderia-related bacteria dominated the enriched soil populations under 3CBA stress. In addition, enrichment cultures growing on 3CBA as sole C-source were obtained from the respective spiked soil, which were found to contain bacteria with identical 16S rDNA sequences as those induced by 3CBA stress in soil.

Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly β-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.

Gordonia desulfuricans sp. nov., a benzothiophene-desulphurizing actinomycete.

The taxonomic position of two actinomycetes isolated from soil was established using a polyphasic approach. The organisms, designated 213ET and 213F, were found to have chemical and morphological properties consistent with their assignment to the genus Gordonia. Nearly complete sequences of the 16S rDNA genes of the two strains were determined following the isolation and direct sequencing of the amplified genes. The tested strains were found to have identical 16S rDNA sequences and formed a phylogenetic line within the evolutionary radiation occupied by the genus Gordonia that was most closely related to Gordonia rubropertincta DSM 43197T. However, DNA-DNA relatedness data showed that strain 213ET and Gordonia rubropertincta DSM 43197T belonged to distinct genomic species. Strains 213ET and 213F also shared an identical phenotypic profile which distinguished them from representatives of validly described Gordonia species. The combined genotypic and phenotypic data show that strains 213ET and 213F merit recognition as a new species of Gordonia. The name proposed for the new species is Gordonia desulfuricans, for which the type strain is 213ET (= NCIMB 40816T).

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.

Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies.

Tsetse flies (Diptera: Glossinidae) harbour three different symbiotic organisms in addition to the pathogenic African trypanosomes they transmit. The two gut-associated symbionts (primary, P; secondary, S) are enteric and are nutritionally required, whereas the third microorganism Wolbachia (family Rickettsiaceae) affects the reproductive biology of the insects it infects. The bacteriome-associated P-symbiont (Wigglesworthia glossinidia) displays a concordant phylogeny with its host tsetse species, whereas midgut S-symbionts characterized from distant tsetse have identical 16S rDNA sequences and therefore may either represent recent independent acquisitions or horizontal transfer between species. The S-symbionts have been cultured in vitro and a genetic transformation system has been developed. Here we report on their density and tissue tropism in different species (G. m. morsitans, G. p. palpalis, G. austeni and G. brevipalpis) and on their maternal route of transmission to tsetse progeny. Using a bacterium-specific PCR-assay, the S-symbionts were found primarily in the midgut, haemolymph, milk gland and in G. palpalis also in salivary glands of teneral flies. In older flies these infections were found to spread to other tissues including muscle, testes and fat body. The S-symbionts were transformed to express the marker gene product, Green Fluorescent Protein (GFP) in vitro. When the recombinant symbionts were introduced into the haemoceal of fertile female flies via intrathoracic microinjection, they were detected in the intrauterine progeny, indicating that haemolymph may provide a possible route for their transmission. The implications of these results for symbiont-host interactions and for transgenic strategies in tsetse are discussed.

Intraspecific variation of unusual phospholipids from Corynebacterium spp. containing a novel fatty acid.

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132(T) and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132(T) and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.

Two new Mycobacterium strains and their role in toluene degradation in a contaminated stream.

Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus Mycobacterium. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to Mycobacterium aurum and Mycobacterium komossense, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.

Discrimination of Streptomyces albidoflavus strains based on the size and number of 16S-23S ribosomal DNA intergenic spacers.

In an attempt to develop a rapid and accurate method for discrimination of streptomycetes at the strain level, 21 strains identified by fatty acid analysis as Streptomyces albidoflavus and the type strains of nine subjective synonyms of S. albidoflavus were selected for a full or partial 16S ribosomal DNA (rDNA) sequence analysis and an investigation of the 16S-23S rDNA intergenic spacer region. 16S rDNA sequence analysis showed that 27 of the strains exhibited 100% sequence similarity; these strains included the type strain of S. albidoflavus and the type strains of the subjective synonyms Streptomyces canescens, Streptomyces coelicolor, Streptomyces felleus, Streptomyces limosus, Streptomyces odorifer, and Streptomyces sampsonii. The type strains of the other subjective synonyms of S. albidoflavus (i.e., Streptomyces gougerotii, Streptomyces intermedius, and Streptomyces rutgersensis) were found to have levels of 16S rDNA sequence difference of 1.0 to 1.1% when they were compared to the type strain of S. albidoflavus. In order to discriminate between the strains which had identical 16S rDNA sequences, the 16S-23S rDNA intergenic spacer regions were amplified and cloned, and the sequences of the spacer regions were determined for four S. albidoflavus strains, including the type strain. The 16S-23S rDNA intergenic spacer region was found to vary in length and sequence composition among the strains and within each strain. The sizes and numbers of 16S-23S rDNA intergenic spacer regions for the 27 strains with identical 16S rDNA sequences were determined by high-resolution electrophoresis of FAM-labeled PCR products and a subsequent size analysis with GeneScan 672 software. This was shown to be a useful method for discrimination of S. albidoflavus strains. Strains with the same 16S-23S rDNA intergenic spacer band pattern, as determined by high-resolution electrophoresis of FAM-labeled PCR products, could be further discriminated on the basis of the sequence composition of the spacer region.

Restriction Fragment Length Polymorphism of Lactobacillus reuteri and Lactobacillus fermentum, Originating from Intestinal Mucosa, Based on 16S rRNA Genes

Restriction Fragment Length Polymorphisms (RFLPs) of the 16S rRNA gene were determined for fourteen field strains originally labelled Lactobacillus reuteri , four Lactobacillus fermentum strains, and eight type and reference strains of Lactobacillus . The strains were mainly originating from intestinal mucosa. The probe was a 420 bp internal fragment of a Lactobacillus reuteri 16S rRNA gene, amplified by Polymerase Chain Reaction (PCR). The patterns were obtained by cleavage of the chromosomal DNAs by the restriction endonucleases E co RI and Hind III separately. Unweighted Pair Group Method, using arithmetic Averages (UPGMA) based on Jaccard coefficients (S J ) of the ribopatterns resolved five clusters at the 30% similarity level. The L. reuteri field strains formed two clusters according to their source of isolation; strains originating from rat formed one cluster while those from pig and human strains formed the other cluster. These strains could not be affiliated to the type strain of L. reuteri . However, two field strains, classified by phenotypically means as L. fermentum , clustered together with the type strain of this species. The numbers of 16S rRNA genes of the L. reuteri strains were, depending on the strain, determined to be at least five through seven. The ribopatterns were compared to the results of DNA:DNA hybridization and partial sequencing of the 16S rDNA genes. All but one of the L. reuteri field strains had more than 70% DNA similarity and identical 16S rDNA sequence to the type strain L. reuteri DSM 20016 T . Two of the L. fermentum field strains had identical 16S rDNA sequence and more than 70% DNA similarity to L. fermentum ATCC 14931 T . Ribotyping appears not to be a means to identify L. reuteri at the species level but eventually this is possible with L. fermentum .