Gareth J. Everest

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.

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.

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.

Nocardia rhamnosiphila sp. nov., isolated from soil.

In this study two actinomycete strains were isolated in Cape Town (South Africa), one from a compost heap (strain 202GMO(T)) and the other from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse (strain C2). Based on 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. Phylogenetic analysis showed that the strains clustered together and are most closely related to Nocardia flavorosea NRRL B-16176(T), Nocardia testacea JCM 12235(T), Nocardia sienata IFM 10088(T) and Nocardia carnea DSM 43397(T). This association was also supported by gyrB based phylogenetic analysis. The results of DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both strains 202GMO(T) and C2 from related species. However, their high DNA relatedness showed that they belong to the same species. Strain 202GMO(T) was selected as the type strain to represent this novel species, for which the name Nocardia rhamnosiphila is proposed (=DSM 45147(T)=NRRL B-24637(T)).

Description of Kribbella italica sp. nov., isolated from a Roman catacomb.

A novel actinobacterium, strain BC637(T), was isolated from a biodeteriogenic biofilm sample collected in 2009 in the Saint Callixstus Roman catacomb. The strain was found to belong to the genus Kribbella by analysis of the 16S rRNA gene. Phylogenetic analysis using the 16S rRNA gene and the gyrB, rpoB, relA, recA and atpD concatenated gene sequences showed that strain BC637(T) was most closely related to the type strains of Kribbella lupini and Kribbella endophytica. DNA-DNA hybridization experiments confirmed that strain BC637(T) is a genomic species that is distinct from its closest phylogenetic relatives, K. endophytica DSM 23718(T) (63 % DNA relatedness) and K. lupini LU14(T) (63 % DNA relatedness). Physiological comparisons showed that strain BC637(T) is phenotypically distinct from the type strains of K. endophytica and K. lupini. Thus, strain BC637(T) represents the type strain of a novel species, for which the name Kribella italica sp. nov. is proposed ( = DSM 28967(T) = NRRL B-59155(T)).

Amycolatopsis roodepoortensis sp. nov. and Amycolatopsis speibonae sp. nov.: antibiotic-producing actinobacteria isolated from South African soils

Two novel members of the genus Amycolatopsis were isolated from soil samples collected in South Africa. Strains JS72T and M29T clustered in the same clade in the 16S-rRNA, gyrB-16S-rRNA and gyrB-recN gene trees. Both strains showed anti-mycobacterial activity. The oxyB P450 monooxygenase B gene required for the production of glycopeptide antibiotics was detected in both strains, while strain JS72T was also shown to contain the 3-amino-5-hydroxy-benzoic acid synthase gene, which is required for the production of the ansamycin class of antibiotics. Genetic distance values (based on the gyrB and recN genes) were calculated between strains JS72T and M29T and their closest phylogenetic relatives. The values for strain JS72T were all above the threshold values of 0.02 and 0.04, respectively, that have been proposed to distinguish Amycolatopsis-type strains. The gyrB-based values for strain M29T were above the threshold for all but one strain; the recN-based values were all above the threshold. These data, along with DNA–DNA hybridization data, showed that strains JS72T and M29T belong to distinct genomic species. The physiological, phylogenetic and genetic distance data support the description of strains JS72T and M29T as the type strains of novel species, for which the names Amycolatopsis speibonae sp. nov. (=DSM 46660T=NRRL B-24958T) and Amycolatopsis roodepoortensis sp. nov. (=DSM 46661T=NRRL B-24959T) are proposed, respectively.