Marian Mordarski

Chemotaxonomic characters and classification of some nocardioform bacteria.

SUMMARY: Simple chemical analyses were carried out on 198 nocardioform bacteria by means of paper- and thin-layer chromatography. The strains considered to belong to the genus Nocardia contained the lipid LCN-A, arabinose and meso-diaminopimelic acid. All the representative strains from the ‘Mycobacterium’ rhodochrous complex possessed this lipid though in certain cases the characteristic spot had a slightly lower R F value than that of the reference lipid LCN-A from the standard strain of Nocardia asteroides. The genera Actinomadura, Mycobacterium, Oerskovia and Streptomyces did not contain lipid LCN-A and the distribution of the other two chemical characters varied. The method used to detect lipid LCN-A is simple and reliable and permits the separation of nocardias and ‘M.’ rhodochrous strains from allied taxa. These results correlate well with other trends in the taxonomy of nocardioform bacteria and confirm the value of chemotaxonomic characters, especially lipids, in the classification and identification of these organisms.

Ribosomal ribonucleic acid similarities in the classification of Rhodococcus and related taxa.

Duplexes were prepared between 14C-labelled rRNA from both Rhodococcus equi C7 and Rhodococcus rhodochrous N54 and DNA from 16 actinomycetes representing the genera Rhodococcus, Mycobacterium, Nocardia, Saccharopolyspora and Streptomyces. The relationships between the organisms were determined by plotting the temperature at which 50% of the duplex was denatured (Tm(e)) against the percentage of rRNA binding (microgram 14C-labelled rRNA duplexed per 100 micrograms filter-bound DNA). All of the strains formed stable duplexes but each organism occupied a definite area on the rRNA similarity map. All of the organisms share a close phylogenetic relationship but representatives of the genera Rhodococcus, Mycobacterium, Nocardia and Streptomyces fell into four recognizable clusters on the similarity map. These data support and extend current trends in the classification of Rhodococcus and allied taxa. The guanine plus cytosine content of the DNA from the test strains was within the range 69.3 to 76.9 mol %.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topoisomerase II inhibitors.

A series of new 5H-indolo[2,3-b]quinoline derivatives bearing methoxy and methyl groups at C-2 and C-9 was synthesized (according to the modified Graebe-Ullmann reaction). These compounds were evaluated for their antimicrobial and cytotoxic activity and tested as inhibitors of DNA topoisomerase II. Lipophilic and calf thymus DNA binding properties of these compounds were also established. In the SAR studies we used quantum-mechanical methodology to analyze the molecular properties of the drugs. All of the 5H-indolo[2,3-b]quinolines tested were found to inhibit the growth of gram-positive bacteria and pathogenic fungi at MIC ranging between 2.0 and 6.0 microM. They showed also cytotoxic activity in vitro against several human cancer cell lines of different origin (ID50 varied from 0.6 to 1.4 microM), and stimulated the formation of topoisomerase-II-mediated pSP65 DNA cleavage at concentration between 0.2 and 0.5 microM. The most active indolo[2,3-b]quinolines which had the greatest contribution to the increase in the Tm of DNA displayed also the highest DNA binding constants and the highest cytotoxic activity. The differences in DNA binding properties and cytotoxic activity seem to be more related to steric than electrostatic effects.

Deoxyribonucleic acid reassociation in the classification of the 'rhodochrous' complex and allied taxa.

The degree of binding was determined between DNA preparations from gordonae and rhodochrous strains and uracil-labelled DNA from five reference strains, Nocardia asteroides NK20, N. pellegrino PII, Gordona bronchialis TI, G. terrae T5 and rhodochrous strain R90. Most of the rhodochrous and pellegrino strains fell into one of two genetically homogeneous taxa. The nucleotide sequence homology data also suggested that G. bronchialis, G. rubra, and more equivocally G. terrae, formed distinct species. However, the values for DNA relatedness between these species, and between then and the rhodochrous homology groups, were comparatively low. Only a small degree of nucleotide sequence homology was found between the N. asteroides reference system and the rest of the taxa studied. The nucleotide composition of the DNA preparations from 29 of the 30 test strains fell between 63 and 69 mol% guanine plus cytosine.

DNA base composition and homology values in the classification of some Rhodococcus species

Similar DNA homology values were recorded when a modified S1 nuclease technique and a standard nitrocellulose membrane filter method were applied to representative strains of Rhodococcus. The DNA homology data showed that R. globerulus, R. luteus and R. sputi form distinct genomic species. The congruence between the DNA homology and earlier numerical phenetic data was good, but there was evidence that some strains had been misclassified in the previous studies. In particular, the type strains of R. obuensis and R. sputi belong to a single genomic species. The former name is thus a later, subjective synonym of the latter. The guanine plus cytosine content of the DNA of the rhodococci fell within the range 61 to 72 mol%.

Staphylococcus pulvereri sp. nov., isolated from human and animal specimens

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus pulvereri, was isolated from human and animal specimens. The complete 16S rRNA sequence of the type strain of S. pulvereri, NT215, was determined and compared with the sequences of 16S rRNAs from the other staphylococci. Strains of S. pulvereri were differentiated from other novobiocin-resistant Staphylococcus species by their biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is NT215 (= PCM 2443T).

Classification of the "Rhodochrous" Complex and Allied Taxa Based upon Deoxyribonucleic Acid Reassociation

The degree of binding was determined between deoxyribonucleic acid (DNA) preparations from nocardiae and “rhodochrous” strains and uracil-labeled DNA from three reference strains, Nocardia asteroides N668 and rhodochrous strains N11 and N54. In all cases, good congruence was found between the DNA reassociation data and that from numerical phenetic studies. Only a small degree of nucleotide sequence homology was found between the N. asteroides reference system and the other taxa studied, and there was evidence that N. asteroides is genetically heterogeneous. The moles percent guanine plus cytosine for the rhodochrous strains was within the range 58 to 67; the corresponding range for nocardiae was 64 to 68 mol%.

Deoxyribonucleic Acid Reassociation in the Classification of the Genus Rhodococcus Zopf 1891 (Approved Lists, 1980)

We determined the degree of binding between deoxyribonucleic acid (DNA) preparations from representative rhodococci and thymine-labeled DNAs (derived from labeled uracil) from the following six reference strains: Rhodococcus coprophilus (Approved Lists, 1980) strain N744, R. corallinys (Bergey et al. 1923) Goodfellow and Alderson 1979 (Approved Lists, 1980) strain N657, R. equi (Approved Lists, 1980) strain C7, R. rhodochrous (Approved Lists, 1980) strain N54, R. ruber (Approved Lists, 1980) strain N361, and R. rubropertinctus (Hefferan 1904) Goodfellow and Alderson 1979 (Approved Lists, 1980) strain N4. Although the congruence between the DNA reassociation and earlier numerical phenetic data was good, there was evidence that some strains had been misclassified in the numerical phenetic study. In particular, strains labeled R. corallinus (including the type strain of R. corallinus) and R. rubropertinctus belonged to a single DNA homology group. The former name is thus a later, subjective synonym of the latter. The guanine plus cytosine contents of the DNAs of the rhodococci fell within the range of 65 to 73 mo1%.

A carboline derivative as a novel mammalian DNA topoisomerase II targeting agent

The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5H-indol[2,3-b]quinoline, is able to stabilize in vitro the topoisomerase II-DNA cleavable complex and to induce DNA breaks in BPV I episome in rat fibroblasts. Cytotoxicity studies with DC3F cells resistant to ellipticine strongly suggest that topoisomerase II is a cellular target involved in the mechanism of cytotoxic action of this carboline derivative.