Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4-5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57-65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Development of a Kribbella-specific isolation medium and description of Kribbella capetownensis sp. nov. and Kribbella speibonae sp. nov., isolated from soil.

Two actinobacterial strains were isolated from samples collected from the University of Cape Town, South Africa. A third actinobacterial strain was isolated from soil collected in the town of Stellenbosch, South Africa, using a newly-developed Kribbella-selective medium. Analysis of the 16S rRNA genes showed that the three strains belonged to the genus Kribbella. A multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strains YM55T and SK5 were most closely related to the type strains of Kribbella sindirgiensis and Kribbella soli, while strain YM53T was most closely related to the type strain of Kribbella pittospori. Digital DNA-DNA hybridisation and Average Nucleotide Identity (ANI) analyses showed that strains YM55T and SK5 belong to the same genomic species (OrthoANI value = 98.4%), but are distinct from the genomic species represented by the type strains of K. sindirgiensis (OrthoANI values < 95.6%) and K. soli (OrthoANI values < 91.4%). Strain YM53T is distinct from the genomic species represented by the type strain of K. pittospori (OrthoANI value = 94.0%). Phenotypic comparisons showed that strains YM55T and SK5 are distinct from the type strains of K. sindirgiensis and K. soli and that strain YM53T is distinct from the type strain of K. pittospori. Strains YM53T and YM55T are thus presented as the type strains of novel species, for which the names Kribbella capetownensis sp. nov. (= DSM 29426T = NRRL B-65062T) and Kribbella speibonae sp. nov. (= DSM 29425T = NRRL B-59161T), respectively, are proposed.

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners.

In this paper, we report on the chemistry of the rare South African Actinomycete Kribbella speibonae strain SK5, a prolific producer of hydroxamate siderophores and their congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine 1 and desoxy-desferrioxamine D1 2, have been isolated, together with four known hydroxamates, desferrioxamine D1 3, desferrioxamine B 4, desoxy-nocardamine 5 and nocardamine 6, and a diketopiperazine (DKP) 7. The structures of 1-7 were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives 8-10 were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella.

Streptomyces swartbergensis sp. nov., a novel tyrosinase and antibiotic producing actinobacterium.

As part of an antibiotic screening program, an actinobacterium, strain HMC13T, was isolated from soil collected from the banks of the Gamka River, Western Cape Province, South Africa. The isolate was found to produce branched mycelia that differentiated into spiral spore chains with spiny spores. 16S rRNA gene sequence analysis showed the strain to be closely related to Streptomyces caelestis NRRL 2418T (99.72%) and Streptomyces azureus NBRC 12744T (99.51%). Chemotaxonomic analyses confirmed the classification of the strain as a member of the genus Streptomyces: LL-DAP in the peptidoglycan, no diagnostic sugars in the whole cell sugar pattern, dominant menaquinones including MK9(H8), MK9(H6), and the polar lipids detected included phosphatidylethanolamine. The fatty acid profile revealed the presence of mostly branched, saturated fatty acids: iso-C15:0 (14.4%), anteiso-C15:0 (21.1%), iso-C16:0 (16.8%), C16:1ω7c/2-OH iso-C15:0 (5.8%), C16:0 (6.2%), iso-C17:1ω9c (5.8%), iso-C17:0 (5.9%), and anteiso-C17:0 (9.6%). Strain HMC13T is a tyrosinase producer and exhibits very strong antibiosis against Mycobacterium aurum A+ and Staphylococcus aureus subsp. aureus ATCC 33591 (methicillin resistant), while only weak activity was observed against Bacillus cereus ATCC 10876, Enterococcus faecium VanA (vancomycin resistant), Enterococcus faecalis ATCC 51299 (vancomycin resistant) and Candida tropicalis ATCC 750T. Strain HMC13T (= LMG 28849T = NRRL B-65294T) is proposed as the type strain of a new species, to be named Streptomyces swartbergensis sp. nov.

Kribbella podocarpi sp. nov., isolated from the leaves of a yellowwood tree (Podocarpus latifolius).

An endophytic actinobacterial strain was isolated from a yellowwood tree growing on the slope of Devil's Peak, Cape Town, South Africa. Analysis of the 16S rRNA gene showed that the strain belongs to the genus Kribbella. Phylogenetic analyses using the 16S rRNA gene and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strain YPL1T is closely related to the type strains of Kribbella karoonensis and Kribbella shirazensis. DDH experiments showed that strain YPL1T is a distinct genomic species from its close phylogenetic relative, K. karoonensis Q41T. Physiological comparisons further showed that strain YPL1T is phenotypically distinct from the type strains of Kribbella jejuensis, Kribbella aluminosa, K. karoonensis, K. shirazensis and Kribbella swartbergensis. Strain YPL1T is thus presented as the type strain of a novel species, for which the name Kribbella podocarpi sp. nov. (= DSM 29424T = NRRL B-65063T), is proposed.

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)).

Streptomyces fractus sp. nov., a novel streptomycete isolated from the gut of a South African termite.

An actinobacterial strain, MV32(T), was isolated from the paunch region of the hindgut of a South African termite, Amitermes hastatus, as part of an investigation of the actinobacterial population residing within this higher order termite species. Strain MV32(T) was chosen for further study from amongst the many potentially novel actinomycete isolates because of its strong antibacterial activity against Mycobacterium aurum A+. 16S rRNA gene phylogenetic analyses clearly placed strain MV32(T) within the genus Streptomyces, with 99.3% sequence similarity to its closest relative, Streptomyces endophyticus YIM 65594(T). Despite this high sequence similarity, DNA-DNA hybridisation analysis showed a DNA relatedness value of 62 ± 2%, to S. endophyticus DSM 41984(T) (indicating that strain MV32(T) belongs to a different genomic species), as well as values of 14.4 ± 0.8 and 10.4 ± 2.9%, respectively, to its next closest relatives, Streptomyces kunmingensis NRRL B-16240(T) and Streptomyces cinnabarinus NRRL B-12382(T). Based on these results and supported by both chemotaxonomic data and a number of phenotypic differences, strain MV32(T) is proposed to represent a new species within the genus Streptomyces, with the name Streptomyces fractus (= DSM 42163(T) = NRRL B-59159(T)).

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.

Amycolatopsis umgeniensis sp. nov., isolated from soil from the banks of the Umgeni River in South Africa.

A novel member of the genus Amycolatopsis was isolated from soil collected from the banks of the Umgeni River, KwaZulu Natal province, South Africa. The strain, designated UM16(T), grouped with the type strains of Amycolatopsis alba, Amycolatopsis coloradensis and Amycolatopsis thailandensis by 16S rRNA gene based phylogeny. Genetic distance values, based on the gyrB and recN genes, between strain UM16(T) and its closest relatives were all above the threshold values of 0.02 and 0.04, respectively, that have been proposed to distinguish Amycolatopsis type strains. DNA-DNA hybridisation experiments confirmed that strain UM16(T) represents a unique genomic species, sharing 18.4 ± 5.1, 16.2 ± 1.8 and 45.8 ± 8.9 % DNA relatedness to the type strains of A. alba, A. coloradensis and A. thailandensis, respectively. The physiological, phylogenetic and DNA-relatedness data support the description of strain UM16(T) as the type strain of a novel species, for which the name Amycolatopsis umgeniensis sp. nov. is proposed (= DSM 45272(T) = NRRL B-24724(T)).

Oxalate utilisation is widespread in the actinobacterial genus Kribbella.

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (-OOC-COO-) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.

Evaluation of the use of recN sequence analysis in the phylogeny of the genus Amycolatopsis.

Partial recN gene sequences (>1 kb) were obtained from 35 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of recN sequence analysis as an alternative to DNA-DNA hybridization (DDH) for distinguishing closely related species was also assessed. The recN based phylogeny mostly confirmed the conventional 16S rRNA and gyrB gene-based phylogenies and thus provides further support for these phylogenetic groupings. As is the case for the gyrB gene, pairwise recN sequence similarities cannot be used to predict the DNA relatedness between type strains but the recN genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. A recN genetic distance of >0.04 between two Amycolatopsis strains is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).

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.

The use of gyrB sequence analysis in the phylogeny of the genus Amycolatopsis.

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA-DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).

Gordonia lacunae sp. nov., isolated from an estuary.

An actinomycete, strain BS2T, was isolated from a sand sample collected from an estuary in Plettenberg Bay, Western Cape Province, South Africa. Based on 16S rRNA gene sequence similarities and chemotaxonomy, strain BS2T was identified as a member of the genus Gordonia. It exhibited weak antibiosis against Mycobacterium aurum A+ and Mycobacterium smegmatis LR222. Phenotypic and phylogenetic results allowed for the differentiation of strain BS2T from other species within the genus Gordonia. DNA-DNA hybridization further differentiated strain BS2T from its nearest phylogenetic neighbour, Gordonia terrae NRRL B-16283T (57.5+/-4.4% DNA relatedness). Strain BS2T is therefore a novel species within the genus Gordonia, for which the name Gordonia lacunae is proposed, with the type strain being BS2T (=DSM 45085T=NRRL B-24551T).

Streptosporangium minutum sp. nov., isolated from garden soil exposed to microwave radiation.

The actinobacterium, strain M26T, was isolated from garden soil that was pre-treated with microwave radiation. The soil sample was collected in Roodepoort, Gauteng Province, South Africa as part of an antibiotic-screening programme. The isolate produced branched vegetative mycelium with sporangiophores bearing small sporangia ranging from 3 to 6 µm in diameter. Rapid genus identification revealed that the isolate belongs to the genus Streptosporangium. To confirm this result, the strain was subjected to polyphasic taxonomic characterisation. Chemotaxonomic characteristics were as follows: meso-DAP in the peptidoglycan, the whole-cell hydrolysate yielded madurose, predominant menaquinones were MK9 (21%), MK9(H2) (40%), MK9(H4) (31%) and MK9(H6) (3%); the polar lipid profile included an aminolipid, phosphoglycolipids, phosphatidylethanolamine, and phosphatidylmonomethylethanolamine. In addition, the fatty acid profile showed the presence of C16:0 (12.8%), C17:1ω8c (14.2%), and 10-methyl-C17:0 (15.8%). Furthermore, 16S rRNA gene sequence phylogenetic analysis showed that the strain is closely related to members of the genus Streptosporangium, which supports its classification within the family Streptosporangiaceae. Strain M26T exhibited antibiosis against a range of pathogenic bacteria, including, but not limited to Acinetobacter baumannii ATCC 19606T, Enterobacter cloacae subsp. cloacae ATCC BAA-1143, Enterococcus faecalis ATCC 51299 (vancomycin resistant), Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19111, Mycobacterium tuberculosis H37RvT, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica subsp. arizonae ATCC 13314T, and the methicillin-resistant Staphylococcus aureus subsp. aureus ATCC 33591 (MRSA). The name Streptosporangium minutum is proposed with the type strain M26T (=LMG 28850T =NRRL B-65295T).

Molecular-signature analyses support the establishment of the actinobacterial genus Sphaerimonospora (Mingma et al. 2016).

The genera Microbispora and Sphaerimonospora were examined for GyrB and RecA amino-acid signatures to determine whether molecular-signature analyses support the recent establishment of the genus Sphaerimonospora. The creation of Sphaerimonospora was based mainly upon morphological differences between Microbispora and Sphaerimonospora and the clustering of the type strains of the two genera in phylogenetic trees based on a multilocus sequence analysis. The molecular-signature analyses showed that all members of Sphaerimonospora can be distinguished from all members of Microbispora at 14 amino acid positions in the GyrB protein and at four positions in the shorter RecA protein. These amino acid differences can be used as signatures to differentiate the members of these genera from each other and thus provide support for the establishment of the genus Sphaerimonospora. This is the first demonstration of the use of molecular signatures to support the establishment of a new genus in the family Streptosporangiaceae. Following the transfer of Microbispora mesophila and Microbispora thailandensis from Microbispora to Sphaerimonospora, all species in the genus Microbispora are characterised by the insertion of a small, hydrophobic amino acid after position 208 in the GyrB protein. This insertion is absent from the GyrB protein of members of the genus Sphaerimonospora.

Streptomyces pharetrae sp. nov., isolated from soil from the semi-arid Karoo region.

An actinomycete, strain CZA14T, was isolated from Worcester in the Western Cape province of South Africa. Based on rapid genus identification, 16S-rDNA sequence similarities and chemotaxonomy, strain CZA14T was identified as a member of the genus Streptomyces. It exhibited weak antibiosis against Bacillus coagulans ATCC 7050T, Mycobacterium aurum A+ and Acinetobacter calcoaceticus C91. The results of physiological tests and analysis of the 16S-rDNA sequence allowed for the differentiation of strain CZA14T from other species of the genus Streptomyces. Strain CZA14T therefore represents a new species for which the name Streptomyces pharetrae is proposed, with the type strain CZA14T (= DSM 41856T = NRRL 24333T).

Analysis of recombinase A (recA/RecA) in the actinobacterial family Streptosporangiaceae and identification of molecular signatures.

The family Streptosporangiaceae (suborder Streptosporangineae) comprises 13 genera and 100 species with validly published names. In a recent study, gyrB gene sequences were obtained for members of the family Streptosporangiaceae and the GyrB amino acid sequences were analysed for molecular signatures. In this study, recA gene sequences (895nt) were determined for the type strains of members of the family Streptosporangiaceae. The sequences used represent 81% of the full-length recA gene of Streptosporangium roseum DSM 43021(T). The recA gene sequences were used for phylogenetic analyses and the trees were compared to the corresponding 16S-rRNA and gyrB gene trees. RecA amino acid alignments (298 amino acids) were generated and inspected for unique amino acid signatures to distinguish the genera in the family from each other. As was observed for the gyrB gene trees, the recA gene trees generally supported the division of the members of the family Streptosporangiaceae into 13 genera. The genus Nonomuraea was not monophyletic in any of the recA gene trees, while the genera Planomonospora and Streptosporangium were not monophyletic in the maximum likelihood and maximum parsimony trees. The gyrB-recA concatenated-gene tree was more robust than the recA gene tree, with 63 nodes in the gyrB-recA tree having bootstrap values ≥95%. The only insertions in the recA gene sequences were inteins identified in the type strains of Acrocarpospora phusangensis, Acrocarpospora pleiomorpha and Microbispora mesophila. Examination of the RecA sequence alignments for genus-specific amino acid sequences showed that the genera Herbidospora, Planobispora, Planomonospora and Streptosporangium contain unique amino acid sequences that distinguish these genera from all other genera in the family Streptosporangiaceae. The results of this investigation extend the results of the GyrB study and will be useful in future taxonomic studies in the family Streptosporangiaceae by providing additional genus-specific molecular signatures.