- Volume 25, Issue 2, 1975

The deoxyribonucleic acid base composition of two strains of Thermoplasma acidophilum including the type strain 122-1B2 was determined by buoyant density and thermal denaturation temperature. The guanine plus cytosine content of the two strains examined was found by both methods to be about 46%. This result is strikingly at variance with the significantly lower values, about 25%, reported by the group of workers who first described this organism. The genome size, as determined by the renaturation method of Wetmur and Davidson, was found to be about 109 daltons. This is identical with the genome size of members of the family Acholeplasmataceae, order Mycoplasmatales, within which order T. acidophilum has been tentatively classified.

Nineteen strains of gram-negative organisms representing 10 Haemophilus species, Eikenella corrodens, and the so-called Haemophilus vaginalis were investigated for cytochromes and respiratory quinones. Phenotypical variation of cytochrome and quinone contents was checked using cells grown with oxygen and fumarate as alternative electron acceptors. The cytochromes were detected by difference spectra of briefly sonicated cells at room temperature, and the quinones were determined in lipid extracts by difference spectrophotometry and partly by thin-layer chromatography. In contrast to other conventional groups of chemo-organotrophic, gram-negative bacteria that have been found to be highly homogeneous with respect to the respiratory chain components, the organisms investigated here may be divided into three categories by their respiratory quinones, namely (i) bacteria containing desmethylmenaquinone as sole respiratory quinoneH. influenzae, H. aegyptius, H. paraphrophilus, H. parahaemolyticus, H. parainfluenzae (in part), (ii) bacteria containing both ubiquinone and desmethylmenaquinone-H. haemoglobinophilus, H. parasuis, H. paragallinarum, H. parainfluenzae (in part), and (iii) bacteria containing only ubiquinone-H. piscium, so-called H. vaginalis, and E. corrodens. The data presented may contribute to further characterization, independent of growth factor requirements, of the taxonomic entities investigated. In the absence of information on deoxyribonucleic acid relatedness between the organisms under study, a provisional reclassification of Haemophilus and related taxa on the basis of respiratory quinones is discussed.

Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (EC 1.6.99.1) was assessed in membrane and supernatant fractions obtained by osmotic lysis and differential centrifugation from Acholeplasma granularum, A. modicum, A. axanthum, and Mycoplasma strain 14. NADH:ferricyanide oxidoreductase activity (EC 1.6.99.1) was also assessed in fractions of A. laidlawii B and the mycoplasma. NADH oxidase and NADH:ferricyanide oxidoreductase activities were localized in the membrane fraction of the acholeplasmas and in the supernatant fraction of the mycoplasma. The localization of NADH oxidase in membrane fractions of cells of Acholeplasma and supernatant fractions of cells of Mycoplasma after osmotic lysis is useful in differentiating these genera.

Antiserum prepared against a Streptococcus faecalis fructose diphosphate aldolase (EC 4.1.2.13) was used to measure the extent of immunological homology between the reference enzyme and aldolases of various streptococci and gram-positive nonsporeforming anaerobic bacteria. The majority of streptococci surveyed were isolated from the oral cavity or blood samples. Strains within the species Streptococcus mutans Clarke and Streptococcus mitis (mitior) possessed aldolases that exhibited marked antigenic heterogeneity and thereby contrasted sharply with previously studied species of Streptococcus (London and Kline, 1973). Varying degrees of cross-reactivity were observed between the aldolases of certain species of Eubacterium, Butyribacterium, and Propionibacterium and the anti-streptococcal aldolase serum. Evidence is also presented confirming an earlier report by Neimark (1974) that aldolases of Acholeplasma species react with the anti-S. faecalis aldolase serum.

Thirty-four strains designated Nocardia farcinica, four strains of N. asteroides, and one of N. brasiliensis have been serologically analyzed by means of an immunodiffusion technique. These strains have also been studied concerning their ability to produce arylsulfatase, to grow in the presence of 7% NaCl, and to grow at 45 C. The N. farcinica strains were divided into two well-distinguished clusters. One of these clusters comprised 24 strains, which appear to belong to the genus Mycobacterium, whereas the other cluster comprised 10 strains, which seem to belong to the genus Nocardia.

The nomenspecies Pseudomonas avenae Manns 1909 and Pseudomonas alboprecipitans Rosen 1922, causal agents of leaf blight of oats and leaf blight of corn, have been compared with respect to their physiological and biological properties as described in the early literature. Except for a few minor physiological characters, most of the properties were identical to those determined herein with P. alboprecipitans isolates obtained in Georgia and Florida and with the "lecto" type strain (ATCC 19860). All isolates of P. alboprecipitans were closely related genetically as judged from deoxyribonucleic acid (DNA)-DNA reassociation experiments. On the other hand, DNA of P. alboprecipitans failed to reassociate with DNA of fluorescent Pseudomonas syringae 5D417 and Pseudomonas andropogonis PA146 isolated from sorghum or the nonfluorescent Pseudomonas atrofaciens PA13 isolated from wheat. P. alboprecipitans isolates also possessed DNA of relatively high guanine plus cytosine (G+C) content (72.2 mol%) as compared to the 57.8 mol% G+C of P. atrofaciens and P. andropogonis.

Factor analysis of the saprophytic serogroups Semaranga and Andamana of Leptospira biflexa reveals their main and minor antigens, without deeply affecting their taxonomic status. The analysis shows that in the Semaranga group two subgroups can be recognized on the basis of two main antigens, Sem 2 and Sem 4. The new system for designation of the leptospiral agglutinogens, proposed by Kmety and Lataste-Dorolle, is adopted.

A method is described for the simulation of bacterial biochemical taxonomic data with randomly inserted variations from typical reactions. This method is applied to data from a group of Enterobacteriaceae. The simulated data thus generated show good agreement with expected results. The procedure will be useful in evaluating identification algorithms and in analysis of patterns of variation among bacteria.

Thermoactinomyces candidus, a new species isolated from home environments and other sources, is reported. T. candidus differs from T. vulgaris in that the former species hydrolyzes esculin, splits arbutin, and does not attack tyrosine, hypoxanthine, or starch, whereas the latter doeS. T. candidus differs from T. sacchari by producing fast-growing colonies, abundant aerial mycelia, and hemolysis in blood agar, by decomposing esculin and arbutin, and by failing to hydrolyze starch. The type strain of T. candidus is T-106 (= ATCC 27868).

A serological examination, by the indirect immunofluorescence test, was made of twenty-five bovine Ureaplasma (T-mycoplasma) strains. Two of the 25 strains were apparently identical and did not cross-react with any of the others. None of the other remaining 23 strains were identical but each cross-reacted with at least one other strain, sometimes by one-way reactions. The percentage of similarity of strains was calculated and a similarity matrix was constructed. By this method, the 25 strains were found to fall into three groups. One group contained 11 strains, the second contained 12 strains, whereas the third group contained the two strains that were apparently identical but unrelated to any of the other 23. Certain strains within the two larger groups had a low percentage of similarity to some strains in the other group. Based on these results, a serological method of typing bovine Ureaplasma strains is proposed, using antisera to eight strains with which all 25 strains react. None of these eight antisera reacted with any of the eight proposed serotypes of Ureaplasma urealyticum.

The deoxyribonucleic acid (DNA) relationship among Lactobacillus species of the subgenus Streptobacterium Orla-Jensen was assessed by means of DNA/DNA hybridization experiments. High genetic homologies were found among Lactobacillus casei subsp. casei, L. casei subsp. alactosus, L. casei subsp. pseudoplantarum, and L. casei subsp. tolerans; however, there was only a low level of homology between the DNA of L. casei subsp. rhamnosus and the DNA of other subspecies of L. casei. The DNAs of Lactobacterium zeae ATCC 15820 and L. casei subsp. casei ATCC 393 were nearly completely homologous. Although the latter strain has been proposed as the neotype for L. casei, it is not related to any of the other strains within the subspecies of L. casei. Lactobacillus plantarum and strains identified as belonging to L. plantarum subsp. rudensis and L. plantarum subsp. arabinosus formed a fairly homogeneous and easily recognized group. The DNA of Lactobacillus pentosus was unrelated to that of any known streptobacteria, and we therefore advocate the recognition of this organism as an independent species. There is a high degree of homology between Lactobacillus sake and Lactobacillus homohiochi; the name L. sake has priority, and L. homohiochi is very probably a synonym; for the same reason, Lactobacillus yamanashiensis has priority over its later subjective synonym, Lactobacillus mali. The general DNA/DNA homology relationships among Lactobacillus casei, L. plantarum, L. coryniformis, L. curvatus, L. xylosus, L. alimentarius, L. farciminis, and Sporolactobacillus inulinus justify their status as separate species.

Obligately anaerobic, filterable microorganisms have been isolated from the rumens of cattle and sheep. On the basis of typical colonial appearance, lack of cell wall, filterability through a 450-nm membrane filter, absence of reversion to a bacterial form, and inhibition of growth by homologous antiserum, these organisms fit the description for organisms in the order Mycoplasmatales. They exhibit unique cultural, biochemical, and serological properties, and we have thus placed them in a new genus, Anaeroplasma. A. abactoclasticum is the type species of the new genus. Strain 6-1, a sterol-requiring strain, has been designated as the type strain of A. abactoclasticum and has been deposited in the American Type Culture Collection as ATCC 27879; a non-sterol-requiring strain, number 161, has been deposited as ATCC 27880. The relationship of Anaeroplasma abactoclasticum to Acholesplasma bactoclasticum, an anaerobic mycoplasma possessing extracellular proteolytic and bactoclastic enzymes, is discussed.

An anaerobic bacteriolytic mycoplasma was isolated by Robinson and Hungate and designated Acholeplasma bactoclasticum sp.n. We found that this strain and four similar isolates from the ovine rumen have a requirement for cholesterol and thus do not belong in the genus Acholeplasma. The morphological, cultural, and biochemical properties of these organisms are similar to those of a group of non-bacteriolytic anaerobic mycoplasmas that do not fit well in genera previously established for mycoplasmas and thus have been placed in a newly described genus, Anaeroplasma. We propose that the organisms isolated by Robinson and Hungate be transferred to the new genus as Anaeroplasma bactoclasticum (Robinson and Hungate) comb. nov., of which ATCC 27112 is the type strain.

It has been reported that the names Pseudomonas multivorans, P. kingae, and P. cepacia are synonymous and that P. cepacia has priority. We have confirmed this synonymy by subjecting a larger number of strains (15 each) of P. multivorans and P. kingae to a wider variety of morphological, nutritional, physiological, and biochemical tests than employed in other studies. A total of 127 tests performed on each strain showed that the two groups of strains were 94% similar, differing only in the following nine characters: growth at 41 C; pigmentation; growth on cetrimide agar; nitrate reduction; acid from sucrose and maltose on O-F medium; utilization of nicotinate and n-dodecane as sole carbon sources; and susceptibility to nalidixic acid. Salient properties for both groups of strains are nutritional versatility and accumulation of poly-β-hydroxybutyrate as a cellular reserve material.

A species of strictly anaerobic, carbohydrate-fermenting, formic-and butyric acid-producing, gram-negative to gram-variable bacteria is described on the basis of 35 isolates from human feces and one isolate from chicken cecal contents. Members of this species produce morphological forms with the appearance of buds. This species cannot be assigned to any known genus, and therefore a new genus, Gemmiger (L. n. gemma a bud; L. v. gero to bear; M. L. masc. n. gemmiger bud bearer), is proposed with G. formicilis n.sp. (M. L. adj. formicilis pertaining to formic acid) as the type species. The type strain of this species is Virginia Polytechnic Institute strain X2-56 (= ATCC 27749).

A comparison of the phenotypic and genotypic characters of the type strain of Photobacterium leiognathi Boisvert et al. 1967 with those of the type strain of Photobacterium mandapamensis Hendrie et al. 1970 indicates that these organisms belong to one and the same species. The correct name of this species, on the basis of priority, is P. leiognathi. American Type Culture Collection strain 25521 is designated as the type strain of P. leiognathi.

Additional bacteriophages were obtained from pooled sewage samples for the genus Levinea. On the basis of plaque morphology and host specificity, three different phages were isolated and purified by serial single-plaque isolations. Cross-neutralization tests were performed on each of the new phage isolates as well as on three previously reported Levinea phages using homologous and heterologous antisera. The data indicated no serological relatedness among the six phages and, therefore, three additional serological groups were established. The phages were designated DM-41, DM-51, and DM-61. Electron micrographs revealed that DM-41 and DM-51 possess a contractile tail whereas DM-61 has a short, noncontractile tail. The phages contained double-stranded deoxyribonucleic acid as determined by the acridine orange-fluorescent staining technique. The value of the phages for use in defining Levinea species and determining relatedness between levineae and the genus Citrobacter is discussed.

Phage-host specificity tests were accomplished utilizing six serologically unrelated phages with 177 isolates of the genus Levinea and 44 Citrobacter freundii isolates. A total of 84% of the levineae were infected by at least one of the phages, and 21 phage-host patterns were obtained. Only 9% of the C. freundii isolates were infected. One phage, DM-21, only infected L. malonatica whereas the other phages showed no species specificity. Most of the infectivity patterns were specific for either L. amalonatica or L. malonatica, but some patterns showed no host specificity. A correlation was found between dulcitol fermentation and infectivity with DM-21. There was no evidence of a relationship between infectivity and source of isolation. The recommendation that the proposed genus Levinea be accepted rather than the inclusion of these organisms within the genus Citrobacter is discussed.

Hoshina, in 1962, described a new species, Paracolobacterum anguillimortiferum. A comparison of the characteristics given in the original description by Hoshina of the new bactejium with those of Edwardsiella tarda Ewing and McWhorter 1965 indicates that these organisms are identical. Although the original strain of P. anguillimortiferum was lost, the name was validly published, and therefore the specific epithet "anguillimortiferum" has priority over "tarda". The new combination Edwardsiella anguillimortifera is proposed for this organism. The type strain of Edwardsiella tarda ATCC 15947 is designated as the neotype strain of Edwardsiella anguillimortifera.