- Volume 42, Issue 3, 1992
Volume 42, Issue 3, 1992
- Original Papers Relating To Systematic Bacteriology
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Phylogeny of Rapidly Growing Members of the Genus Mycobacterium
More LessThe 16S rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16S rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaurum and M. diernhoferi, (ii) M. gadium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. flavescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.
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Polyphasic Taxonomic Study of the Emended Genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an Aerotolerant Bacterium Isolated from Veterinary Specimens
The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and “Campylobacter butzleri” were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that “C. butzleri” should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.
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Mycoplasma penetrans sp. nov., from the Urogenital Tract of Patients with AIDS
An unusual mycoplasma, which was isolated from the urine of a human immunodeficiency virus-positive male homosexual patient, has an elongated flask shape and two unique sharply divided internal compartments. The tiplike compartment is densely packed with fine granules, and the body compartment is loosely filled with coarse granules consistent with ribosomal structures. The organism has properties of adherence, hemadsorption, and cytadsorption and invades many different types of mammalian cells. Adhesion and penetration apparently involve the terminally located tiplike structure. Cholesterol is required for growth, and the mycoplasma ferments glucose and hydrolyzes arginine, but does not hydrolyze urea. The results of DNA homology studies revealed that this organism is not genetically related to previously described mycoplasma species that have the same biochemical properties. The results of serologic studies demonstrated that this organism is antigenically distinct from all previously described mycoplasmas. We propose that this new mollicute species should be named Mycoplasma penetrans sp. nov. The type strain is strain GTU-54-6A1 (= ATCC 55252).
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Enterococcus flavescens sp. nov., a New Species of Enterococci of Clinical Origin
Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 439.
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Genomic Fingerprinting by Arbitrarily Primed Polymerase Chain Reaction Resolves Borrelia burgdorferi into Three Distinct Phyletic Groups
The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319,1982) and was isolated by Barbour et al. in 1983 (A. G. Burbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.
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Delineation of Borrelia burgdorferi Sensu Stricto, Borrelia garinii sp. nov., and Group VS461 Associated with Lyme Borreliosis
We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.
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rRNA Gene Restriction Patterns as Taxonomic Tools for the Genus Aeromonas
More LessIn the genus Aeromonas there are at least 13 DNA hybridization groups, which are difficult to differentiate biochemically. We investigated the usefulness of rRNA gene restriction patterns for characterization and identification of the various groups. Genomic DNA was digested with restriction endonuclease SmaI, transferred to a nylon membrane, and hybridized with biotinylated plasmid pKK3535 containing the rrnB operon of Escherichia coli. The SmaI bands at 0.8 to 4 kb but not those at positions corresponding to sizes larger than 4 kb showed a good correlation with hybridization groups, allowing identification of strains to the level of genetic species. We demonstrated that the 567-bp fragment localized between positions 80 and 647 of the 16S ribosomal gene of E. coli was essential for hybridization to the low-molecular-weight fragments, whereas the remainder of the operon did not hybridize to these fragments. On the basis of these results, we concluded that the Aeromonas chromosome contains multiple rRNA operons which may be used for species identification.
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Characterization of Anaerovibrio burkinabensis sp. nov., a Lactate Fermenting Bacterium Isolated from Rice Field Soils
More LessA strictly anaerobic, gram-negative bacterium was isolated from rice field soils by using lactate as a sole carbon and energy source. The cells were non-spore-forming, motile, curved rods. Optimal growth occurred at 35°C and pH 6.8. No NaCl requirement was observed. Vitamins were required for growth. Our isolate, strain B4B0 T (T = type strain), fermented pyruvate, fumarate, malate, citrate, dihydroxyacetone, fructose, 1,2-propanediol, glutamate, and aspartate to acetate, propionate, succinate, and traces of hydrogen. Strain B4B0 T did not use ribose or glycerol as an energy source, although glycerol degradation produced mainly 1,3-propanediol. Ferric iron was facultatively reduced. Nitrate and sulfate were not reduced. Cytochrome b was present. The guanine-plus-cytosine content of the DNA was 44.1 ± 0.1 mol%. We propose that strain B4B0 (= DSM 6283) should be the type strain of a new species in the genus Anaerovibrio, Anaerovibrio burkinabensis.
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Desulfovibrio longus sp. nov., a Sulfate-Reducing Bacterium Isolated from an Oil-Producing Well
More LessA novel type of sulfate-reducing bacteria with unusual morphology was isolated from an oil-producing well in the Paris Basin. The cells of this bacterium, strain SEBR 2582T (T = type strain), are long, thin, flexible rods, contain desulfoviridin, and are physiologically similar to members of the genus Desulfovibrio. On the basis of 16S rRNA sequence data, this strain should be included in the genus Desulfovibrio. However, strain SEBR 2582T differs from other members of this genus morphologically, physiologically, and phylogenetically. Thus, a new species, Desulfovibrio longus sp. nov., is proposed for this organism.
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Legionella shakespearei sp. nov., Isolated From Cooling Tower Water
A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.
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DNA Relatedness among Some Thermophilic Members of the Genus Methanobacterium: Emendation of the Species Methanobacterium thermoautotrophicum and Rejection of Methanobacterium thermoformicicum as a Synonym of Methanobacterium thermoautotrophicum
DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain ▵HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum ▵HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.
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Phylogenetic Interrelationships of Members of the Genera Aeromonas and Plesiomonas as Determined by 16S Ribosomal DNA Sequencing: Lack of Congruence with Results of DNA-DNA Hybridizations
More LessThe phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.
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Low-Frequency Restriction Fragment Analysis of Frankia Strains (Actinomycetales
More LessLow-frequency restriction fragment analysis of more than 100 strains of the genus Frankia showed that restriction enzyme Dral (recognition site, TTT’AAA) gave rise to large DNA fragments (200 to 1,500 kb), which, when they were subjected to cluster analysis, reflected the host plants from which the strains were isolated. Our results support the conclusions of Lalonde and his colleagues (M. Lalonde, L. Simon, J. Bousquet, and A. Seguin, p. 671-680, in H. Bothe, F. J. de Bruijn, and W. E. Newton, ed., Nitrogen Fixation: Hundred Years After, 1988; P. Normand, P. Simonet, and R. Bardin, Mol. Gen. Genet. 213:238-246, 1988) and Fernandez et al. (M. P. Fernandez, H. Meugnier, P. A. D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989) that various biochemical and genomic analyses can give rise to groupings of Frankia strains that are consistent with the host plants from which the strains are isolated and that the resulting groups may form a basis for defining Frankia species.
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Description of Porphyromonas circumdentaria sp. nov. and Reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov.
More LessA new species, Porphyromonas circumdentaria, is proposed for pigmented, asaccharolytic strains that were isolated from the gingival margins or mouth-associated diseases of cats. This bacterium is an obligately anaerobic, gram-negative, brown- or black-pigmented, asaccharolytic, nonmotile, nonsporing, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 40 to 42 mol%. It produces major amounts of acetic, butyric, and isovaleric acids and minor amounts of propionic, isobutyric, and phenylacetic acids as end products of metabolism in cooked meat medium. Glutamate and malate dehydrogenases are present, while 6-phosphogluconate and glucose-6-phosphate dehydrogenases are absent. The major cellular fatty acid is 13-methyltetradecanoic acid (iso-C15:0 acid). P. circumdentaria strains are catalase positive and produce ammonia, and colonies fluoresce under short-wavelength UV light. These strains do not hemagglutinate erythrocytes, exhibit trypsinlike activity, or produce chymotrypsin or α-fucosidase. They are heavily piliated and produce a capsule. The type strain is strain VPB 3329 (= NCTC 12469). Bacteroides salivosus (D. N. Love, J. L. Johnson, R. F. Jones, and A. Calverley, Int. J. Syst. Bacteriol. 37:307-309, 1987) is an obligately anaerobic, gram-negative, pigmented, asaccharolytic, nonmotile, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 42 to 44 mol%. This organism produces major amounts of acetic, butyric, and phenylacetic acids and minor amounts of isobutyric and isovaleric acids as end products of metabolism in cooked meat medium. B. salivosus has been shown to contain 13-methyltetradecanoic acid (iso-C15:0 acid) as its major cellular fatty acid and to have glutamate and malate dehydrogenases, while 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase are absent. Nomenclature changes published in 1988 (H. N. Shah and M. D. Collins, Int. J. Syst. Bacteriol. 38:128-131, 1988) for the asaccharolytic, pigmented, anaerobic, rod-shaped bacteria require reassignment of this organism to the genus Porphyromonas as Porphyromonas salivosa comb. nov.
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Bacillus methanolicus sp. nov., a New Species of Thermotolerant, Methanol-Utilizing, Endospore-Forming Bacteria
The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60°C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5S rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16S and 5S rRNA sequences indicated that this new species is distinct from Bacillus brevis but closely related to B. firmus and B. azotoformans. The name proposed for this new species is B. methanolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.
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Differentiation of the Subspecies of Campylobacter fetus by Genomic Sizing
More LessCampylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SaiI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar “intermedius” and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.
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Flexibacter ovolyticus sp. nov., a Pathogen of Eggs and Larvae of Atlantic Halibut, Hippoglossus hippoglossus L.
More LessA psychrotrophic Flexibacter sp., Flexibacter ovolyticus sp. nov., was isolated from the adherent bacterial epiflora of Atlantic halibut (Hippoglossus hippoglossus L.) eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae. The strains which we isolated had the enzymatic capacity to dissolve both the chorion and the zona radiata of the egg shells. A total of 35 isolates were characterized by using morphological and biochemical tests. These strains were rod shaped, gram negative, Kovacs oxidase positive, and pale yellow and exhibited gliding motility. They did not produce acid from any of the wide range of carbohydrates tested. Our isolates had the ability to degrade gelatin, tyrosine, DNA, and Tween 80. Starch, cellulose, and chitin were not degraded. The strains were catalase and nitrate reductase positive, did not produce H2S, and did not grow under anaerobic conditions. F. ovolyticus resembles Flexibacter maritimus, but differs from the latter species in several biochemical and physiological characteristics. DNAs from F. ovolyticus strains had guanine-plus-cytosine contents which ranged from 30.3 to 32.0 mol% (strains EKC001, EKD002T [T = type strain], and VKB004), and DNA-DNA hybridization studies revealed levels of relatedness between F. ovolyticus EKD002T and F. maritimus NCMB 2154T and NCMB 2153 of 42.7 and 30.0%, respectively. Compared with previously described Cytophaga and Flexibacter spp. with low guanine-plus-cytosine contents, F. ovolyticus constitutes a new species. Strain EKD002 (= NCIMB 13127) is the type strain of the new species.
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Characterization of Three Thermophilic Strains of Methanothrix (“Methanosaeta”) thermophila sp. nov. and Rejection of Methanothrix (“Methanosaeta”) thermoacetophila
Three thermophilic Methanothrix (“Methanosaeta”) strains, strains PT T (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii (“Methanosaeta concilii”) GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila (“Methanosaeta thermoacetophila”), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.
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DNA Relatedness among Erysipelothrix rhusiopathiae Strains Representing All Twenty-Three Serovars and Erysipelothrix tonsillarum
The levels of relatedness among strains of Erysipelothrix rhusiopathiae (serovars 1 through 23 and type N) were estimated by performing DNA-DNA hybridization experiments with the type strains of E. rhusiopathiae and Erysipelothrix tonsillarum, which are the two Erysipelothrix species that have been described. Two distinct DNA relatedness groups were identified. The group 1 strains, representing serovars 1, 2, 4 through 6, 8, 9, 11, 12, 15 through 17, 19, and 21 and type N, exhibited more than 73% hybridization with the type strain of E. rhusiopathiae but less than 24% hybridization with the type strain of E. tonsillarum. Group 2 included serovar 3, 7, 10, 14, 20, 22, and 23 strains, and these strains exhibited more than 66% hybridization with the type strain of E. tonsillarum but less than 27% hybridization with the type strain of E. rhusiopathiae. Strains representing serovars 13 and 18 exhibited low levels of hybridization (16 to 47%) with both of the type strains, indicating that these serovars may be members of a new genomic species. The members of the E. rhusiopathiae and E. tonsillarum groups resembled each other in many phenotypic characteristics, but differed in their ability to produce acid from saccharose and in their pathogenicity for swine. Our results confirm that the genus Erysipelothrix contains two main genomic species, E. rhusiopathiae and E. tonsillarum, which can be differentiated into serovars.
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Chemotaxonomic Differentiation of Coryneform Bacteria Isolated from Biofilters
More LessCoryneform bacteria that were isolated from biofilters which are used for waste gas treatment of animal-rendering plant emissions were differentiated and partially identified by using chemotaxonomic methods. On the basis of the results of a numerical analysis of whole-cell fatty acid profiles, 79 isolates were divided into two major groups; the members of the first group contained saturated and monounsaturated fatty acids, whereas the members of the second group were characterized by iso- and anteiso-branched fatty acids. Division into subclusters was based mainly on quantitative differences in fatty acid composition and was confirmed by the results obtained for additional chemical markers (e.g., respiratory quinones, mycolic acids, polar lipids, cell wall amino acids, and whole-cell sugar patterns). By combining the results obtained for chemotaxonomic analyses that were performed for strains containing saturated and monounsaturated fatty acids, we were able to identify the genus Corynebacterium (two Corynebacterium species were differentiated on the basis of the occurrence of tuberculostearic acid), the genus Gordona, and the genus Mycobacterium. Among the strains that produced iso-anteiso fatty acid patterns, one subgroup was affiliated with the “nicotianae” group of the genus Arthrobacter; however, some strains contained a new combination of chemical markers. Peptidoglycan type A4α, L-Lys-Gly-L-Glu was combined with menaquinones MK-7 and MK-8, whereas peptidoglycan type A4α, L-Lys-L-Glu occurred together with MK-8 and MK-9. The second subgroup was characterized by a new type B peptidoglycan and MK-11, as well as small amounts of MK-12. Differentiation that was based first on chemotaxonomy and second on physiology gave reliable results. Thus, coryneform strains with new characteristics were isolated from biofilters.
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