Paul R. Meyers

CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies.

ABSTRACT The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has Km and Vmax values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned and expressed. This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B. pumilus and is not a reflection of its origin at a mining waste site.

Anti-mycobacterial activity of five plant species used as traditional medicines in the Western Cape Province (South Africa)

ETHNOPHARMACOLOGICAL RELEVANCE Five plants used in traditional medicine in the Western Cape Province of South Africa, have been investigated for anti-mycobacterial activity: Olea capensis, Tulbaghia alliacea, Dittrichia graveolens, Leysera gnaphalodes and Buddleja saligna. AIM OF THE STUDY The aim was to assess antimycobacterial activity in plants used in treatment of symptoms of TB, and through activity-guided fractionation of extracts to isolate compounds or mixtures with potential as anti-TB drug leads. MATERIALS AND METHODS Extracts and derived fractions were assayed against strains of Escherichia coli, Staphylococcus aureus, and Mycobacterium aurum A+. Isolated pure compounds were further tested against Mycobacterium species M. avium ATCC 25291, M. scrofulaceum ATCC 19981, M. microti ATCC 19422 and Mtb H37Rv, and for cytotoxicity against Chinese hamster ovarian cells. RESULTS Extracts of B. saligna and L. gnaphaloides exhibited significant anti-mycobacterial activity, primarily associated with the presence of non-cytotoxic triterpenoids oleanolic acid in B. saligna and both oleanolic and ursolic acids in L. gnaphaloides. CONCLUSIONS Anti-mycobacterial activity of extracts of selected plants is consistent with their traditional use. The identification of oleanolic and ursolic acids in these plants, and verification of their activity, underlines the potential for exploring structure-activity relationships of derivatives of these ubiquitous triterpenoids.

Micromonospora tulbaghiae sp. nov., isolated from the leaves of wild garlic, Tulbaghia violacea.

A novel actinomycete, strain TVU1(T), was isolated from leaves of the indigenous South African plant Tulbaghia violacea. Applying a polyphasic approach, the isolate was identified as a member of the genus Micromonospora. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain TVU1(T) was most closely related to Micromonospora echinospora DSM 43816(T). However, phylogenetic analysis based on gyrB gene sequences showed that strain TVU1(T) was most closely related to the type strains of Micromonospora aurantiaca and Micromonospora chalcea. DNA-DNA relatedness values between strain TVU1(T) and the type strains of M. echinospora, M. aurantiaca and M. chalcea were 7.6+/-4.5, 45.9+/-2.0 and 60.9+/-4.5 %, respectively. Strain TVU1(T) could be distinguished from the type strains of all three of these species by several physiological characteristics, such as colony colour, NaCl tolerance, growth temperature range and sole carbon source utilization pattern. Strain TVU1(T) (=DSM 45142(T)=NRRL B-24576(T)) therefore represents a novel species for which the name Micromonospora tulbaghiae sp. nov. is proposed.

Phylogenetic analysis of the genus Kribbella based on the gyrB gene: proposal of a gyrB-sequence threshold for species delineation in the genus Kribbella

Given the advances in molecular biology, many microbial taxonomists feel that a sequencing based method should be developed that can replace DNA-DNA hybridisation for species delineation. The potential of the gyrB gene to be used for phylogenetic studies has been investigated within a number of actinobacterial genera, including Gordonia, Micromonospora and the whorl-forming Streptomyces species. This study aimed to determine whether the gyrB gene can discriminate between type strains of the genus Kribbella. Previous studies, in the genus Micromonospora, have found that a gyrB-based genetic distance of 0.014 correlates to a DNA relatedness of 70% and that those strains with a genetic distance of greater than 0.014 are likely to be distinct species. In this study, the gyrB-based genetic distances between Kribbella type strains were found to range from 0.0164 to 0.1495, supporting the use of the 0.014 genetic-distance value as the threshold for species delineation within this genus. Phylogenetic analysis based on the gyrB gene had improved resolution (longer branch lengths) compared to that based on the 16S rRNA gene sequence. Based on this study, the gyrB gene can be used to distinguish between Kribbella type strains. Furthermore, it is proposed that a 390-nucleotide sequence of the gyrB gene of a Kribbella isolate is sufficient to assess whether it is likely to represent a new species, before time and effort is invested in polyphasic taxonomic characterisation of the isolate.

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16

Aims:  To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species.

Multilocus sequence analysis of the actinobacterial genus Kribbella

Multilocus sequence analysis (MLSA) was used to refine the phylogenetic analysis of the genus Kribbella, which currently contains 17 species with validly-published names. Sequences were obtained for the 16S rRNA, gyrB, rpoB, recA, relA and atpD genes for 16 of the 17 type strains of the genus plus seven non-type strains. A five-gene concatenated sequence of 4099 nt was used to examine the phylogenetic relationships between the species of the genus Kribbella. Using the concatenated sequence of the gyrB-rpoB-recA-relA and atpD genes, most Kribbella type strains can be distinguished by a genetic distance of >0.04. Each single-gene tree had an overall topology similar to that of the concatenated sequence tree. The single-gene relA tree, used here for the first time in MLSA of actinobacteria, had good bootstrap support, comparable to the rpoB and atpD gene trees, which had topologies closest to that of the concatenated sequence tree. This illustrates that relA is a useful addition in MLSA studies of the genus Kribbella. We propose that concatenated gyrB-rpoB-recA-relA-atpD gene sequences be used for examining the phylogenetic relationships within the genus Kribbella and for determining the closest phylogenetic relatives to be used for taxonomic comparisons.

Kribbella hippodromi sp. nov., isolated from soil from a racecourse in South Africa.

A novel actinomycete, designated strain S1.4(T), was isolated from a soil sample collected from Kenilworth Racecourse in the Western Cape, South Africa. The strain was able to grow in the presence of 5 % NaCl. It contained ll-diaminopimelic acid and glycine in the cell-wall peptidoglycan with glucose present in the whole-cell sugar profile. Strain S1.4(T) was shown to be a member of either the genus Kribbella or the genus Nocardioides based on a rapid molecular identification method by using single-enzyme restriction endonuclease digestion of the PCR-amplified 16S rRNA gene with MboI, VspI, SphI, SnaBI, SalI and AgeI. Analysis of the 16S rRNA gene sequence indicated that strain S1.4(T) belonged to the genus Kribbella. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain S1.4(T) was related most closely to Kribbella solani DSA1(T). Strain S1.4(T) was phenotypically distinct from K. solani DSA1(T) and was shown to be a separate genomic species based on DNA-DNA hybridization experiments (40.4+/-3.8 % DNA-DNA relatedness between the two). Strain S1.4(T) (=DSM 19227(T) =NRRL B-24553(T)) is thus presented as the type strain of a novel species, for which the name Kribbella hippodromi sp. nov. is proposed.

Evaluation of the antibiotic biosynthetic potential of the genus Amycolatopsis and description of Amycolatopsis circi sp. nov., Amycolatopsis equina sp. nov. and Amycolatopsis hippodromi sp. nov

Aims:  To describe three new Amycolatopsis strains and assess the antibiotic biosynthetic potential of the genus.

Micromonospora equina sp. nov., isolated from soil from a racecourse.

Two actinomycete strains were isolated from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse in Cape Town, South Africa. Rapid molecular identification indicated that the isolates belonged to the family Micromonosporaceae. Based on 16S rRNA gene sequence blast analysis, the isolates were identified as members of the genus Micromonospora. Phylogenetic analysis showed that the isolates clustered with each other and were most closely related to Micromonospora viridifaciens DSM 43909(T). Further 16S rRNA gene sequence analysis using EzTaxon revealed that the isolates are closely related to Micromonospora auratinigra TT1-11(T), Micromonospora chaiyaphumensis MC5-1(T), Micromonospora eburnea LK2-10(T), Micromonospora nigra DSM 43818(T) and Micromonospora olivasterospora DSM 43868(T). DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both isolates from related species; however, their high DNA-DNA relatedness showed that they belong to the same genomic species. Strain Y22(T) ( = DSM 45644(T) = NRRL B-24859(T)) was selected as the type strain to represent this novel species, for which the name Micromonospora equina sp. nov. is proposed.

Kribbella albertanoniae sp. nov., isolated from a Roman catacomb, and emended description of the genus Kribbella.

A novel actinobacterium, strain BC640(T), was isolated from a biofilm sample collected in 2009 in the Saint Callistus Roman catacombs. Analysis of the 16S rRNA gene sequence showed that the strain belonged to the genus Kribbella. Phylogenetic analysis using the 16S rRNA gene and concatenated gyrB, rpoB, relA, recA and atpD gene sequences showed that strain BC640(T) was most closely related to the type strains of Kribbella yunnanensis and Kribbella sandramycini. Based on gyrB genetic distance analysis, strain BC640(T) was shown to be distinct from all Kribbella type strains. DNA-DNA hybridization experiments confirmed that strain BC640(T) represents a genomic species distinct from its closest phylogenetic relatives, K. yunnanensis DSM 15499(T) (53.5±7.8 % DNA relatedness) and K. sandramycini DSM 15626(T) (33.5±5.0 %). Physiological comparisons further showed that strain BC640(T) is phenotypically distinct from the type strains of K. yunnanensis and K. sandramycini. Strain BC640(T) ( = DSM 26744(T) = NRRL B-24917(T)) is thus presented as the type strain of a novel species, for which the name Kribbella albertanoniae sp. nov. is proposed.