- Volume 29, Issue 1, 1979

The abilities of various Enterobacteriaceae strains to grow on starch or pullulan and the occurrence of pullulanase in these strains were studied. Only the Klebsiella strains grew well on amylopectin and pullulan and produced pullulanase. None of the Enterobacter strains produced pullulanase. The pullulanase produced by various strains of Klebsiella was examined by the immunodiffusion method. Culture filtrates and cell extracts of all of the Klebsiella strains tested formed a precipitin line against antiserum prepared with crystalline pullulanase from K. pneumoniae IFO 3321. Except for K. pneumoniae IFO 3317 and ATCC 21073, these lines fused completely with the homologous line. No materials which crossreacted with the antiserum were produced by the other Enterobacteriaceae strains studied. The production of the enzyme is discussed as a character of potential use in the classification and identification of strains of Enterobacteriaceae.

The structural divergence of catalase derived from different mycobacteria has previously been studied in terms of immunological distances as determined by titration with antiserum against catalase extracted from Mycobacterium tuberculosis. This study has been extended by addition of a second reference antiserum: One raised against catalase from M. avium. The two systems show a high reciprocality of immunological distance against one another. The catalase from M. intracellulare is serologically very similar to that of M. avium, and the enzyme from M. bovis is very similar to that of M. tuberculosis. Catalase extracted from BCG, a pathovar of M. bovis, appears to exhibit structural modification which diverges from the parent M. bovis and converges on M. avium. Two classes of catalase have been separated from M. scrofulaceum: One is nonreactive with either reference antiserum, and the other exhibits immunological distances that suggest that M. avium is in a position between M. scrofulaceum and M. tuberculosis.

A study of the cellular and colonial morphologies, the deoxyribonucleic acid (DNA) base composition, the degree of lysis by lysozyme, and some fundamental biochemical reactions was carried out on 23 strains previously identified as members of Bacillus megaterium. Included among these was NCTC 10342, the neotype strain of B. megaterium. The results showed that, primarily on the basis of their biochemical reactions, two of the strains belong to B. cereus, two belong to B. subtilis, and 19 belong to B. megaterium. The first two strains mentioned above clearly differed from B. megaterium strains with respect to their DNA base composition (33.2 and 34.3 mol% guanine plus cytosine [G+C]), their lysis by lysozyme (5.6 and 6.6%), and the morphology of their macro-and microcolonies. These properties were not helpful in differentiating the other two strains from the B. megaterium strains, for the two strains showed high G+C values (45.9 and 47.4 mol%) and high values of lysis by lysozyme, (92.9 and 96.7%). The 19 remaining strains, confirmed as members of B. megaterium, had G+C contents which varied from 37.3 to 43.0 mol% and lysis percentages which varied from 67.0 to 94.9. For these strains, a significant (P < 0.05) correlation was found between the DNA base composition and lysis by lysozyme. Three biogroups were established on the basis of the biochemical reactions of the 19 strains. These biogroups are similar to the “aggregate” and the “intermediate” strains of B. megaterium established by Gordon et al.

An organism phenetically indistinguishable from Mycobacterium triviale was isolated from Mycobacterium gordonae (ATCC 14470) by successive ultraviolet irradiations after three selection steps. From the parent (a smooth-colony-forming, scotochromogenic strain) were isolated: (i) photochromogenic mutants; (ii) rough-colony-forming mutants; and (iii) non-photochromogenic (pigmentless) mutants. In the third step of mutation, some mutants showed simultaneous changes of other characters, e.g., loss of the ability to utilize several carbon sources. After ultraviolet irradiation, mutant colonies comprised 1 to 2 per 103 surviving colonies. It was impossible to isolate non-photochromogenic mutants directly from parent scotochromogenic bacteria. Mutants could be isolated only from photochromogenic mutants. This is in marked contrast to a previous work in which non-photochromogenic mutants were isolated directly from parent, scotochromogenic Mycobacterium scrofulaceum.

It is recommended by the ICSB Subcommittee on the Taxonomy of Enterobacteriaceae that the organism originally named Aerobacter liquefaciens by Grimes and Hennerty be regarded as a member of the genus Serratia rather than Enterobacter and that the organism originally named Pseudomonas proteamaculans by Paine and Stansfield be regarded as a member of the genus Serratia rather than Erwinia. In these positions, the correct names of these organisms are Serratia liquefaciens (Grimes and Hennerty) Bascomb et al. and Serratia proteamaculans (Paine and Stansfield) Grimont et al., respectively. The Subcommittee also proposes the following strains as neotypes for their respective species: Erwinia ananas NCPPB 1846; E. cancerogena NCPPB 2176; E. carotovora NCPPB 312; E. dissolvens NCPPB 1850; E. herbicola NCPPB 2971; E. milletiae NCPPB 2519; E. paradisiaca NCPPB 2511; E. stewartii NCPPB 2295; E. tracheiphila NCPPB 2452; Escherichia blattae CDC 9005-74; Klebsiella oxytoca ATCC 13182; K. ozaenae ATCC 11296; K. pneumoniae ATCC 13883; K. rhinoscleromatis ATCC 13884; Proteus mirabilis ATCC 29906; Providencia rettgeri ATCC 29944; P. stuartii ATCC 29914; Shigella flexneri ATCC 29903; and S. sonnei ATCC 29930.

The growth characteristics, biochemical activities, and serological relationships of a group of mycoplasmas that were originally described as capable of splitting urea were reexamined. Most of the strains examined were isolated from the genital tract of mares and stallions or from the tissues of aborted equine fetuses, but one was obtained from the respiratory tract and another was obtained from the brain of a horse. The colonial morphology and growth characteristics of these isolates were those of the classical “large-colony” mycoplasmas rather than those of species of Ureaplasma. The mycoplasmas hydrolyzed arginine, were inhibited by sodium polyanethol sulfonate and digitonin, and failed to grow without cholesterol. Thus, they had the properties of a typical arginine-degrading species of Mycoplasma. The strains produced a slow but restricted increase in the pH of some types of urea-containing broth, but these pH changes were smaller and less rapid than those given by strains of Ureaplasma. Apart from these properties, the strains were unremarkable in their biochemical activities, although they could be distinguished from strains of M. equirhinis, an arginine-degrading species found in horses, by their failure to produce “film and spots” on serum-containing media. The serological results and electrophoresis of cell proteins suggest that the strains examined constitute a fairly homogeneous group. A representative strain, TB, was shown to be serologically distinct from strains of 48 species of Mycoplasma and of 3 species of Acholeplasma. These species included 8 found in horses and 24 which metabolized arginine or arginine and glucose. On the basis of these results, it is suggested that strain TB and related strains constitute a new species, for which the name Mycoplasma subdolum is proposed. Strain TB (= NCTC 10175 = ATCC 29870) is the type strain.

An extensive phenotypic characterization of an actinoplanete capable of producing the antimetabolite L-azetidine-2-carboxylic acid is presented. On the basis of the properties of this organism, it is concluded that it belongs to a new species, for which the name Actinoplanes ferrugineus is proposed. The type strain is X-14695 (= ATCC 29868).

Good separation of bacterial cell wall amino acids, including the isomers of diaminopimelic acid, was obtained by two-dimensional thin-layer chromatography, using commercial cellulose-coated aluminum sheets with isopropanol-acetic acid-water (75:10:15, vol/vol) and methanol-pyridine-10 N hydrochloric acid-water (64:8:2:14, vol/vol) as the first and second solvents, respectively.

The evolutionary affinities among several morphologically and physiologically similar species of the ascogenous yeast genus Hansenula were studied by techniques of genome comparison. Among the organisms studied, two clusters of related strains were identified by deoxyribonucleic acid base composition and sequence relatedness. These clusters showed little relatedness to each other or to the other strains studied. Several strains of H. wingei, as well as the type strain of H. canadensis, exhibited 78 to 98% base sequence complementarity with the type strain of H. wingei. These species are all associated with coniferous trees. An additional strain of H. wingei shared 62% of its nucleotide sequences with the type strain. Three other yeasts resembling H. wingei but isolated from exudates of alder trees showed >98% complementarity among themselves but <10% relatedness to the H. wingei type. These strains are considered to represent a new species, H. alni. The results suggest the inadequacy of current taxonomic criteria and the need for new approaches to yeast systematics.

A comparative study was made of the molecular weights of the exo-β-glucanases (EC 3.2.1.58) of species belonging to Kluyveromyces or to various other yeast genera. Their immunological distances were determined with the exo-β-glucanases from Kluyveromyces fragilis, K. phaseolosporus, K. aestuarii, and Saccharomyces cerevisiae as references. The molecular sizes of the enzymes were estimated by polyacrylamide gel exclusion chromatography, and the immunological comparison was done by the microcomplement fixation technique. The molecular weights of the enzymes from ascomycetous species ranged from 24 x 103 to 63 x 103, but the majority of the species, including most species of Kluyveromyces, contained exo-β-glucanases with molecular weights between 35 x 103 and 45 x 103. Immunological identity was observed among the exo-β-glucanases from the following species: K. fragilis (reference species), K. marxianus, K. bulgaricus, K. cicerisporus, and K. wikenii; closely related were K. phaseolosporus (reference species), K. delphensis, K. lactis, K. vanudenii, and K. drosophilarum; also closely related were Saccharomyces cerevisiae (reference species), S. italicus, S. uvarum, and S. chevalieri. The exo-β-glucanase from K. aestuarii showed low degrees of homology with those from all tested species of Kluyveromyces. It was concluded from these data that ascospore shape has little significance in the phylogeny of this genus. Other evolutionary implications are discussed.