Reinhard Hensel

Chemotaxonomic and molecular-genetic studies of the genus Thermus: evidence for a phylogenetic relationship of Thermus aquaticus and Thermus ruber to the genus Deinococcus

For a detailed pheno- and genotypic characterization of the genus Thermus, the following seven representative strains were analyzed: Three extremely thermophilic strains (optimal temperature for growth, 65 to 75°C), Thermus aquaticus DSM 625, “Thermus flavus” DSM 674, and “Thermus thermophilus” DSM 579; and four moderately thermophilic strains (optimal temperature for growth 55 to 60°C), Thermus ruber DSM 1279 and three new isolates (strains H1, H2, and H3) from sewage. All of these strains exhibited isobranched pentadecanoic and heptadecanoic acids as principal components of their fatty acids, possessed unsaturated menaquinones with eight isoprene units, and had deoxyribonucleic acid guanine-plus-cytosine contents of 59 to 65.5 mol%, and their cell walls contained a murein structure of the A3β variation (interpeptide bridge, Gly2). The clustering of the organisms into extremely and moderately thermophilic strains correlated well with molecular properties, such as the absorption spectra of their pigments and the ratio of pentadecanoic acid to heptadecanoic acid. This grouping was confirmed by the results of 16S ribosomal ribonucleic acid cataloging and deoxyribonucleic acid-deoxyribonucleic acid hybridization. Our data confirm current proposals for classification of the genus Thermus into the following two distinct species: T. aquaticus for the extremely thermophilic strains and T. ruber for the moderately thermophilic strains. Phylogenetically, representatives of the genus Thermus show a remote but significant relationship to Deinococcus species (similarity coefficients, 0.22 to 0.29); members of these two genera share a common peptidoglycan type.

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn2+ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn2+ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.

Bacillus pallidus sp. nov., a new thermophilic species from sewage

Summary Nine closely related thermophilic spore-forming strains were isolated from thermophilically treated waste water. On the basis of morphological and biochemical features, the organisms belong to the genus Bacillus but differ from the previously described thermophilic Bacillus species in phenotypical and genotypical properties. They were considered as representing a new species, Bacillus pallidus sp. nov.

Bacillus thermocloaceae sp. nov., a New Thermophilic Species from Sewage Sludge

Summary Three closely related strains belonging to the dominant flora in aerobe-thermophilically treated sludge were isolated and characterized. Although the isolates show the classical features of the genus Bacillus, their phenotypic and genotypic properties do not allow an assignment to any known species within this genus. They were described as representatives of a new species, Bacillus thermocloacae sp. nov. The type strain is the strain S 6025 (= DSM 5250).

Sphaerobacter thermophilus gen. nov., sp. nov. A Deeply Rooting Member of the Actinomycetes Subdivision Isolated from Thermophilically Treated Sewage Sludge

Summary The phenotypic and genotypic properties of a Gram-positive non-spore-forming strain belonging to the dominant flora grown on aerobe-thermophilically treated sewage sludge were characterized. The isolate tends to irregular shape, exhibits a new peptidoglycan type of the murein variation A3β, with β-alanine crosslinking the adjacent peptide subunits, a principal menaquinone of type MK-8/0 and a DNA G + C content of 66 mol%. Its taxonomic position within the actinomycetes subdivision of Gram-positive eubacteria was established by 16S rRNA sequence studies, which characterize the isolate as the most ancient member of that subdivision. The isolate is described as Sphaerobacter thermophilus sp. nov. gen. nov.

The Microflora Involved in Aerobic-Thermophilic Sludge Stabilization

Summary The microflora adapted to the habitat of aerobic-thermophilically treated sludge was investigated in a laboratory-scale fermenter as well as in plants working in practice. As demonstrated by the immunofluorescence technique, the dominant microflora of the laboratory model plant working under steady-state conditions largely corresponds to that of several thermophilically operated municipial sludge stabilization plants, indicating that the microflora of the model plant can be regarded as representative of the habitat of aerobic-thermophilic sludge stabilization in general. As dominant members of this microbial community, strains of Bacillus thermocloacae were identified; strains of Sphaerobacter thermophilus, Bacillus sphaericus, Bacillus pallidus and Bacillus stearothermophilus are less abundant. The influence of the operational parameters of the thermophilic process (e. g. quality of the substrate, discontinuous or continuous operation mode, hydraulic retention time) on the microflora is discussed.

Crystallization and preliminary crystallographic analysis at low resolution of the allosteric l-lactate dehydrogenase from Lactobacillus casei☆

Abstract The allosteric l -lactate dehydrogenase from Lactobacillus casei has been crystallized in its complex with the activators fructose-1,6-diphosphate and Co 2+ . The enzyme crystallizes in space group C 2 with six tetramers in the unit cell. At very low resolution, 00 l reflexions are absent for l ≠ 3 n . The orientation of the molecular axes has been determined using the rotation function. All tetramers in the unit cell exhibit excellent 222 symmetry, and the overall arrangement resembles the packing that would be expected in the higher symmetry space group P 3 1 21. Comparison with the apo-enzyme structure of M4-lactate dehydrogenase from dogfish indicates high structural similarity between these enzymes and allowed us to identify the molecular axes of L. casei l -lactate dehydrogenase in terms of the “standard” molecular co-ordinate system P , Q , R . The similarity of both enzymes is good enough to allow the structure determination of L. casei l -lactate dehydrogenase by molecular replacement using the dogfish enzyme as a model. Sequencing results show that L. casei l -lactate dehydrogenase is lacking the N-terminal arm of vertebrate lactate dehydrogenases and electron density maps at 5 A resolution indicate that ligands might possibly bind in the region of the missing arm. The active site loop is involved in intermolecular contacts and its structure might be different from both, apo- and ternary dogfish l -lactate dehydrogenase.

Subunit composition and substrate binding region of potato l-lactate dehydrogenase

Abstract Four of the six electrophoretically distinguishable isoenzymes of the l -lactate dehydrogenase (EC 1.1.1.27) from potato tubers were purified from crude extracts. The isoenzymes are tetrameric and exhibit MWs around 145000. They are composed of mixtures of different subunits. Two of the isoenzymes together contain at least three, the other two together contain six different subunits indicating that the actual number of isoenzymes may be even greater than the number of electrophoretically detectable isoenzymes. Since the isoenzymes agree largely with respect to their enzymatic properties and to their primary structure as suggested from fingerprinting and amino acid analysis, it is suggested that the variation of the subunits is caused by proteolytic processing in vivo rather than by different genetic coding. The amino acid sequence of the substrate-binding region (Arg 6 peptide) shows a high homology to that of the l -lactate dehydrogenases of animals and bacteria indicating a common origin of plant, animal and bacterial enzymes.

Three New Murein Types in Coryneform Bacteria Isolated from Activated Sludge

Summary The cell walls of 4 strains of coryneform bacteria (designated S1, S2, S4, S27) isolatedfrom activated sludge contain three new murein types of the cross-linking group B. In all cases the interpeptide bridge contains a D-diamino acid. In the murein of strains S1 and S2, D-2,4-diaminobutyric acid residues function as interpeptide bridges between the α-carboxyl group of D-glutamic acid and the carboxyl group of the terminal D-alanine residue of an adjacent peptide subunit. The interpeptide bridge in the murein of strain S4 consists, however, of the dipeptide N 5 -(Gly)-D-Orn, and that in strain S27 of the dipeptide N 2 (L-Thr)-D-Dab. Position 3 of the peptide subunit is taken by homoserine residues in strains S1 and S2, by L-ornithine residues in strain S4 and by L-alanine in strain S27. According to the classification of Schleifer and Kandler (1972) the murein types L-HsrD-Glu-D-Dab (S1, S2) and L-Orn D-Glu-Gly-D-Orn (S4) belong to the known murein variations B2s and B2α respectively. For the classification of the new murein type of strain S27 (L-Ala D-Glu-D-Dab-Thr), the new murein variation B2δ is suggested.