- Volume 30, Issue 1, 1980

Hybrids between 14C-labeled ribosomal ribonucleic acid (rRNA) from either Gluconobacter oxydans subsp. oxydans NCIB 9013, Acetobacter aceti subsp. aceti NCIB 8621t1, or Zymomonas mobilis subsp. mobilis ATCC 29191 and deoxyribonucleic acid (DNA) from acetic acid bacteria and representative strains of possibly related and other gram-negative bacteria were prepared. Each hybrid was described by two parameters: Tm(e) , the temperature at which 50% of the hybrid was denatured, and the percentage of rRNA. binding, the amount of 14C-labeled rRNA (in micrograms) duplexed under stringent conditions per 100 µg of filter-fixed homologous or heterologous DNA. Each taxon occupied a definite area on the rRNA similarity maps. Parameters of hybrids formed with rRNA from G. oxydans subsp. oxydans NCIB 9013 showed that the acetic acid bacteria consist of two separate but closely related groups corresponding to the genera Acetobacter and Gluconobacter. When compared with rRNA from A. aceti subsp. aceti NCIB 8621t1, both genera were indistinguishable, showing that there were many strains of. Acetobacter whose rRNA cistrons are as different from the reference Acetobacter rRNA as from Gluconobacter rRNA. The rRNA cistrons of Acetobacter were more heterogeneous than these of Gluconobacter. The great similarities among the m(e) 's of the heterologous hybrids and among the numerous phenotypic features stress that both genera are more closely related to each other than to any other genus. The parameters of the DNA:rRNA hybrids located the acetic acid bacteria as a separate branch in an rRNA superfamily consisting of Rhodopseudomonas, Beijerinckia, Agrobacterium, Rhizobium, some Spirillum species, and Paracoccus denitrificans. Parameters of hybrids formed with rRNA from Z. mobilis subsp. mobilis ATCC 29191 showed that the genus Zymomonas forms a separate branch in the same rRNA superfamily. We detected a number of misnamed organisms. “Acetobacter” aceti subsp. xylinum NCIB 4112, “Acetobacter” aceti subsp. orleanensis NCIB 6426, and “Acetobacter” lermae NRRL B-1810 belong in the genus Gluconobacter. “Gluconobacter” industrius IFO 3261, “Gluconobacter” dioxyacetonicus IAM 1814, “Gluconobacter” sp. strains A4.1 and M28, “Pseudomonas” melophthora NCPPB 461 and 462, and “Pseudomonas” pomi NCPPB 463 are all regular members of Acetobacter. Our evidence is against the maintenance of “intermediate” strains of acetic acid bacteria. “Gluconobacter” liquefaciens NCIB 9505 and IAM 1834 and “G.” melanogenus IAM 1835 and IAM 1836 are genetically regular members of the genus Acetobacter. “Acetobacter” aurantius IFO 3246 is a Gluconobacter. “A.” aurantius IFO 3249, 3247, 13330, and 13333 are not acetic acid bacteria at all. We propose to unite Acetobacter and Gluconobacter in the family Acetobacteraceae. The ranges of the moles percent guanine plus cytosine of the DNAs have been determined for the different taxa in both genera.

Labeled [3H]deoxyribonucleic acid (DNA) from Lactobacillus jensenii ATCC 25258 yielded a substantial amount of heteroduplexes only with DNA molecules from other strains of L. jensenii. A low yield of heteroduplexing was obtained between DNAs of L. jensenii and L. acidophilus, although both species had the same guanine plus cytosine content (36 mol%). The striking phenotypic similarity between L. jensenii and L. leichmannii was not reflected in their DNA base composition (36 and 50 mol% guanine plus cytosine, respectively), and, as expected, they produced a very low yield of hybridized DNAs. Although the phylogenic relationship between these different species of Lactobacillus cannot be deduced from their DNA relatedness, it can nevertheless be established from the structural similarity of their D-lactic dehydrogenases.

Antigenic analysis of serovars belonging to saprophytic serogroup holland of Leptospira biflexa has revealed that both strain WazHolland and strain AM6 contain group-specific antigens and that either can be used as representative of serogroup holland in a preliminary classification of new saprophytic strains.

Analysis of representative strains of the eight human serotypes of Ureaplasma urealyticum by polyacrylamide gel electrophoresis identified 36 to 40 polypeptides for each strain. At least 80% of the peptides were common among strains, but unique major peptides were identifiable in ureaplasmic types. Type 1 had a polypeptide of 85,000 daltons, type 3 had a polypeptide of 72,000 daltons, type 5 had a polypeptide of 64,000 daltons, and type 8 had a polypeptide of 95,000 daltons. The unique polypeptides in types 1 and 8 were identified as membrane components. Two common major components of 44,000 and 70,000 daltons were observed. Several components were common to some, but not all, serotypes. Patterns obtained from U. urealyticum strains were strikingly different from the patterns of Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, and Mycoplasma hominis. Isoelectric focusing demonstrated a unique membrane protein for type 1 at pK 6.4, whereas type 8 possessed an assembly of five unique proteins at pK 7.0. Ureaplasmata were strongly similar to each other by isoelectric focusing, but strikingly different from members of the other genera studied. Although a filtered, strongly buffered dialysate medium with 1% serum and 30 mM urea was used both to maximize yields and to minimize contamination, minor contaminants were detected, which comigrated with horse transferrin (pK 6.0) and cytochrome c (molecular weight, 14,000). The similarities of the polypeptide patterns of U. urealyticum strains affirm their close relationships to each other, in contrast to the diversity shown in the genus Mycoplasma, and our recognition of type-specific membrane peptides will enhance the identification of serotypes and the classification of strains.

Deoxyribonucleic acid homologies were determined among 27 strains of Rhizobium trifolii, 4 strains of Rhizobium leguminosarum, and 4 strains of Rhizobium phaseoli. Results from related strains indicated that deoxyribonucleic acid homologies correlate with serological relationships and that the ability to form nodules on legume roots can be lost without a detectable change in homology with an independent reference strain. All rhizobia which effectively nodulated Trifolium repens, Trifolium subterraneum, Trifolium ambiguum, and Vicia hirsuta formed one population with an average relatedness of 70% (range, 49 to 94%) and a ▵Tm(e) of 0.0 to 11.8°C with respect to reference strains capable of nodulating the first two clover species. Two strains from African Trifolium species and two strains from a northern Asiatic species were less closely related. The average relatedness of strains from Phaseolus vulgaris to clover rhizobia was 46% (range, 37 to 50%), and the Tm(e) was 6.5 to 11.8°C. Taxonomic revisions consistent with these observations are discussed. It is proposed that R. trifolii and R. leguminosarum should be combined under the name which has priority, Rhizobium leguminosarum Frank. Within this species various biovars should be designated according to plant specificity. R. phaseoli should be retained at present as a separate species and examined in more detail. The results are discussed in relation to proposed genetic bases for plant specificity.

A total of 89 strains designated Lactobacillus acidophilus were examined for physiological properties, type of lactic acid produced, cell wall sugar pattern, guanine plus cytosine content of deoxyribonucleic acid (DNA), and DNA homology values compared with selected reference strains. Immunological reactions among a group of the strains were determined by gel diffusion tests, using antiserum to purified lactic acid dehydrogenase (LDH) from a single strain (Sharpe strain A18). Antiserum to glyceraldehyde-3-phosphate dehydrogenase from strain ATCG 4356 was used in microcomplement fixation tests to determine relationships among some strains. DNA preparations from 78 of the 89 strains of L. acidophilus were distributed among six distinct homology groups, designated Al, A2, A3, A4, Bl, and B2. The A group strains had 20 to 30% intergroup homology but very low homology to groups Bl and B2. Likewise, the strains in the two B groups had 20 to 30% intergroup homology but very low homology to the A group strains. Nine strains did not fall into any of the six homology groups. The guanine plus cytosine contents of the DNAs in strains comprising the six homology groups varied from 32 to 38 mol%. In the nine strains not falling into any of the homology groups, the guanine plus cytosine contents were 39 to 47 mol%. Homology group Al, which includes the neotype strain of L. acidophilus (ATCC 4356), is very homogeneous, with most strains showing 95% or more homology to the reference strain. This group corresponds to LDH serogroup III. Strains in the other homology groups showed 60 to 90% homology to then-reference strains. Strains of LDH serogroup II were found in homology groups A2, A3, and A4, and those in LDH serogroup I were in homology groups Bl and B2. In general, the glyceraldehyde-3-phosphate dehydrogenase serology results correlated well with the LDH results. Other phenotypic test results were similar for all of the DNA homology groups. It is recommended that homology group Al be designated L. acidophilus and that strain ATCC 4356 remain the neotype strain.

The protein patterns of sodium dodecyl sulfate-solubilized whole cells of porcine strains of Haemophilus were studied by using polyacrylamide gel electrophoresis. The pattern of Haemophilus pleuropneumoniae was very homogenous and was independent of the serological type. At least two different patterns could be distinguished in Haemophilus parasuis, suggesting some heterogeneity in this species. The results were highly reproducible and were not affected by growth conditions. Comparable patterns were obtained after solubilization with sodium taurocholate and sodium carbonate and after ultrasonic treatment, but not after phenol-acetic acid extraction. In addition, we compared porcine strains with human strains (Haemophilus influenzae, Haemophilus parahaemolyticus) and also with some bacteria of the genera Pasteurella, Actinobacillus, Brucella, Moraxella, and Bordetella. The species-specific picture is given mostly by the pattern of a group of proteins with molecular weights just above 68,000 (Haemophilus) and in the region of molecular weights between 23,000 and 40,000 and between 15,000 and 17,000 (Haemophilus, Pasteurella, Actinobacillus). These observations suggest the possible use of this method as an aid in studying the taxonomy of these bacteria.

From the sphagnum vegetation of moor biotopes in northwestern Germany and Scandinavia, 183 strains of a new type of rapidly growing, scotochromogenic Mycobacterium have been isolated. Of these, 50 were randomly selected and subjected to a taxonomic analysis. The tested strains split urea and pyrazinamide (37 strains), hydrolyzed Tween 80, had phosphatase activity, and possessed putrescine oxidase and nitrate reductase. They produced acid from glucose, fructose, inositol, mannitol, and mannose and usually from sorbitol. Their internal similarity is 98.23 ± 2.29%. A comparison of their properties with those of strains of 22 taxa (clusters) of rapidly growing mycobacteria was made. The mycolic acid production and the micromorphology of these strains confirmed that the strains belong to the genus Mycobacterium. They have unique lipid and immunodiffusion patterns and form special sensitins. Hence, they are considered as belonging to a new species of nonpathogenic, rapidly growing mycobacteria for which the name Mycobacterium sphagni is proposed. Sph 38 is the type strain, a culture of which has been deposited in the American Type Culture Collection under the number 33027.

The name Bacteroides melaninogenicus subsp. macacae is proposed for a subspecies to accommodate strains of gram-negative, anaerobic, catalase-producing rods which produce black colonies on blood agar plates. The organisms weakly ferment galactose, glucose, lactose, and mannose and exhibit no fermentation of 24 other carbohydrates. The metabolic acid end products formed in peptone-yeast extract-glucose broth cultures include acetic, propionic, isobutyric, butyric, isovaleric, and succinic acids. The organisms utilize pyruvate, digest gelatin, and produce indole and hydrogen sulfide. B. melaninogenicus subsp. macacae appears to be serologically distinct from B. asaccharolyticus, B. melaninogenicus subsp. levii, B. melaninogenicus subsp. intermedius, and B. melaninogenicus subsp. melaninogenicus. The type strain of this new subspecies is ATCC 33141.

A total of 190 strains of Yersinia enterocolitica from environmental sources, humans, and other animals were scored on biochemical, nutritional, and resistance tests. These were run in duplicate at 30 and 35°C, and the results were analyzed by numerical taxonomy. The computer-generated phenograms derived from simple matching coefficients and unweighted average linkage analysis delineated seven phenons from the 30°C data and five phenons from the 35°C data at a similarity value of 85%. A centrostrain was derived from each cluster in both matrixes, and these centrostrains were used as reference strains for subsequent deoxyribonucleic acid hybridizations. The relative binding ratios of the centrostrains to one another, as well as to other organisms of their own phenons, were determined. The genetic relationships as denoted by relative binding ratios were compared with the phenetic relationships developed by numerical taxonomy. Our results demonstrate that taxonomic relationships generated by numerical taxonomy do not necessarily correlate highly with those derived by deoxyribonucleic acid hybridizations in either a linear or a numerical sense (correlation coefficients, 0.48 and 0.40; mean correlation ratio, 0.60). In addition, it was found that numerical taxonomies may show considerable fluctuations in phenomic composition, which are dependent on the temperature of incubation. Deoxyribonucleic acid hybridizations among the various strains of this study divided the species Y. enterocolitica into at least four deoxyribonucleic acid relatedness groups. The level of intergroup homology was high enough (relative binding ratio, >20%) to justify the inclusion of all groups as species in the genus Yersinia.

The thymidine kinase activities present in nine strains (including four type strains and one neotype strain) of nine species of the genus Mycoplasma and two strains (including one type strain) of two species of the genus Acholeplasma were characterized with respect to migration in polyacrylamide gels and isoelectric point as determined by electrofocusing. There was a marked heterogeneity in the enzymes, not only between the two genera but also within each genus.

14C-labeled ribosomal ribonucleic acid (rRNA) was prepared from Azotobacter chroococcum NCIB 8002, Azotobacter paspali 8A, Azomonas agilis NCIB 8636, Azomonas insignis WR 30, Beijerinckia indica NCIB 8712, and Azospirillum brasilense ATCC 29145. These rRNA’s were hybridized under stringent conditions with filter-fixed deoxyribonucleic acid from a great variety of gram-negative bacteria. Each hybrid was described by: (i) the temperature at which 50% of the hybrid was denatured, and (ii) the percent rRNA binding (amount in micrograms of rRNA duplexed to 100 µg of deoxyribonucleic acid). These data were used to construct rRNA similarity maps. The following conclusions could be drawn concerning rRNA cistron similarities. (i) Bacterial genera with free-living, aerobic, nitrogen-fixing members are very diverse and belong to different rRNA superfamilies. The present family Azotobacteriaceae is not a biological unit, and its status as a family is highly questionable. (ii) Azotobacter chroococcum, Azotobacter vinelandii, Azotobacter beijerinckii, Azotobacter paspali, Azotobacter miscellum, Azotobacter armeniae, and Azotobacter nigricans belong in the genus Azotobacter. Any synonymy of these names remains to be determined. Azomonas agilis, Azomonas insignis, and Azomonas macrocytogenes constitute independent branches, which are about equidistant from Azotobacter and section I of Pseudomonas as presented in Bergey's Manual of Determinative Bacteriology, 8th ed. Xanthomonas, Alteromonas vaga, and Alteromonas communis are located in the same rRNA superfamily. (iii) The genus Beijerinckia appears to be rather heterogeneous. Its closest relatives appear to be Xanthobacter autotrophics, “Mycobacterium” flavum, “Pseudomonas” azotocolligans, “Pseudomonas” diminuta, the authentic rhodopseudomonads, and some other organisms. These organisms belong in the same rRNA superfamily as Azospirillum, Agrobacterium, Rhizobium, Acetobacter, Gluconobacter, and Zymomonas. (iv) Derxia belongs in still another rRNA superfamily, together with Chromobacterium, Janthinobacterium, the Pseudomonas acidovorans and Pseudomonas solanacearum groups, Alcaligenes, and a few other taxa. (v) The following organisms were generically misnamed: “Azomonas insignis” ATCC 12523, “Mycobacterium” flavum 301, “Pseudomonas” azotocolligans ATCC 12417, “Pseudomonas” diminuta CCEB 513, and “Rhodopseudomonas” gelatinosa (all strains examined).

Optical determination of deoxyribonucleic acid (DNA)-DNA reassociation kinetics was applied to the classification of 32 selected strains of hydrogen-oxidizing bacteria belonging to the genera Pseudomonas, Alcaligenes, and Paracoccus. The renaturation studies revealed a high intraspecies DNA homology for some strains of the species Pseudomonas palleronii, Pseudomonas pseudoflava, and Alcaligenes paradoxus, supporting former taxonomic concepts of different authors. The 12 denitrifying strains belonging to the genus Paracoccus were shown to be interrelated at various levels of percent degree of binding, and at least two clusters of very high genome-DNA relatedness have been found. DNA-DNA reassociation kinetics were also used for the calculation of the average molecular weight of genome DNA. The genome molecular weight of the hydrogen-oxidizing bacteria investigated in this study ranged from 3 X 109 to 5 X 109.

The phytopathogenic bacterium which causes soft rot of the pseudostem of Musa paradisiaca Linnaeus has been designated previously as Erwinia carotovora subsp. paradisiaca, E. paradisiaca, E. carotovora subsp. chrysanthemi, and E. chrysanthemi. Thirty strains were examined and compared with strains of closely related Erwinia species and their subspecies. The strains from M. paradisiaca were found to be phenotypically similar to E. chrysanthemi strains on the basis of the following characteristics: pectate degradation; production of phosphatase, indole, acetoin, and acid from ethanol; growth at 36 to 37°C; susceptibility to erythromycin; gas from glucose; utilization of malonate; no growth in 5% NaCl; and no production of acid from α-methyl-d-glucoside, trehalose, maltose, lactose, or palatinose. They could be distinguished from 322 strains of E. chrysanthemi isolated from 22 other plant hosts by the following phenotypic properties: production of acid from d-arabinose and raffinose; utilization of sodium tartrate; no production of lecithinase or of acid from inulin, mannitol, or sorbitol; and inability to liquefy gelatin.

Strain CMU-1, identified as a member of Angiococcus disciformis (Thaxter) Jahn 1924, was isolated from aquatic habitats in the fossa region of Davis Lake (Vestaburg Bog), Michigan. The myxobacter produced orange fruiting bodies consisting of numerous disk-shaped to oblate spheroid-shaped sporangia (average, 40 by 27 µm; 15 µm thick). The sporangia comprising the fruiting body occurred singly or were clumped in irregular masses upon a solid substrate. Mature myxospores ranged in shape from irregular spheres (1 µm in diameter) to ovals (1 by 1.5 µm). Vegetative rods were motile by gliding and measured 0.5 to 0.75 by 3 to 14 µm. The guanine plus cytosine content of the deoxyribonucleic acid extracted from A. disciformis CMU-1 was 68.4 mol%. Strain CMU-1 (ATCC 33172) has the properties recorded in the original description of Myxococcus disciformis by Thaxter, and it is herein proposed as the neotype strain of this organism. Because the myxospores were contained within sporangia, the organism cannot belong to the genus Myxococcus, and we propose that it properly belongs in the genus Angiococcus Jahn 1924.

The taxonomy of the typhus biogroup (Rickettsia prowazekii and R. mooseri [R. typhi]) of the genus Rickettsia and Rochalimaea quintana has been studied on the basis of the guanine plus cytosine content of the respective deoxyribonucleic acids (DNAs), the genome size, and the degree of DNA-DNA hybridization. The previously published guanine plus cytosine values for certain strains are confirmed, and the guanine plus cytosine values are described for some additional strains in each species. The genome sizes of four strains of R. prowazekii, three strains of R. mooseri, and two strains of R. quintana were very similar and averaged approximately 109 daltons. The various strains within the species boundaries hybridized at nearly the 100% level. The DNAs of various strains of R. prowazekii hybridized with the DNAs of various strains of R. mooseri rather consistently at the level of 70 to 77%. The DNAs of two strains of Rochalimaea quintana hybridized with those of R. prowazekii Breinl and R. mooseri Wilmington in the range of 25 to 33%.

The morphology, growth characteristics, biochemical activities, and serological relationships of a group of eight slow glucose-metabolizing mycoplasmas isolated from nasopharyngeal swabs from horses are described. Representatives of the group possessed a triple-layered limiting membrane and had the essential cultural and biological properties of mycoplasmas. They were highly filamentous, but unusual morphological features were the twisting of the filaments and the regular diagonal striations revealed by electron microscopy. Also notable were the somewhat fastidious nutritional requirements of the slow glucose-metabolizing mycoplasmas and their susceptibility to erythromycin (0.1 μg/ml). Serologically, a representative strain, 4822, was distinct from strains of 58 species of Mycoplasma and of Spiroplasma citri when tested by the disk growth inhibition test or the indirect fluorescent-antibody test. A cross-reaction with certain strains of M. pulmonis was detected in some growth inhibition tests on a medium (LH agar) subsequently shown to be suboptimal for growth. Comparison of 4822 with several strains of M. pulmonis in growth inhibition, metabolism inhibition, and indirect fluorescent-antibody tests with HuS medium failed to show any cross-reactions. Thus, serological methods based on inhibition of growth or metabolism must be used with caution when identifying nutritionally fastidious mycoplasmas that are likely to be inhibited by nonspecific factors. The evidence presented here suggests that the slow glucose-metabolizing strains constitute a new species, for which the name Mycoplasma fastidiosum is proposed. Strain 4822 (= NCTC 10180) is the type strain.

Three new species of Eubacterium, E. timidum, E. brachy, and E. nodatum, which were isolated principally from subgingival areas associated with periodontitis, are described. All are anaerobic, gram-positive, non-saccharolytic rods. They are differentiated from each other and from other non-saccharolytic species of eubacteria by acid and gas products in broth cultures and by morphological and growth characteristics. The type strains for the species are as follows: E. timidum ATCC 33093; E. brachy, ATCC 33089; and E. nodatum, ATCC 33099. These species were detected in 35 to 42% of the subgingival samples examined and, where present, constituted 3 to 57% of the cultivable flora. Strains of these species were isolated only occasionally from the adjacent supragingival areas sampled.