Streptomyces Leeuwenhoekii Sp. Nov., the Producer of Chaxalactins and Chaxamycins, Forms a Distinct Branch in Streptomyces Gene Trees
Total Page：16
File Type：pdf, Size：1020Kb
Antonie van Leeuwenhoek (2014) 105:849–861 DOI 10.1007/s10482-014-0139-y ORIGINAL PAPER Streptomyces leeuwenhoekii sp. nov., the producer of chaxalactins and chaxamycins, forms a distinct branch in Streptomyces gene trees Kanungnid Busarakam • Alan T. Bull • Genevie`ve Girard • David P. Labeda • Gilles P. van Wezel • Michael Goodfellow Received: 8 November 2013 / Accepted: 11 February 2014 / Published online: 7 March 2014 Ó Springer International Publishing Switzerland 2014 Abstract A polyphasic study was carried out to gene alleles underpinned the separation of strain C34T establish the taxonomic status of an Atacama Desert from all of its nearest phylogenetic neighbours, apart isolate, Streptomyces strain C34T, which synthesises from Streptomyces chiangmaiensis TA-1T and Strep- novel antibiotics, the chaxalactins and chaxamycins. tomyces hyderabadensis OU-40T which are not cur- The organism was shown to have chemotaxonomic, rently in the MLSA database. Strain C34T was cultural and morphological properties consistent with distinguished readily from the S. chiangmaiensis and its classification in the genus Streptomyces. Analysis S. hyderabadensis strains by using a combination of of 16S rRNA gene sequences showed that strain C34T cultural and phenotypic data. Consequently, strain formed a distinct phyletic line in the Streptomyces C34T is considered to represent a new species of the gene tree that was very loosely associated with the genus Streptomyces for which the name Streptomyces type strains of several Streptomyces species. Multilo- leeuwenhoekii sp. nov. is proposed. The type strain is cus sequence analysis based on five house-keeping C34T (= DSM 42122T = NRRL B-24963T). Analysis of the whole-genome sequence of S. leeuwenhoekii, with 6,780 predicted open reading frames and a total Electronic supplementary material The online version of genome size of around 7.86 Mb, revealed a high this article (doi:10.1007/s10482-014-0139-y) contains supple- potential for natural product biosynthesis. mentary material, which is available to authorized users. K. Busarakam Á M. Goodfellow (&) Keywords Streptomyces Á Polyphasic School of Biology, University of Newcastle, taxonomy Á Atacama Desert Á Natural products Á Newcastle upon Tyne NE1 7RU, UK Genome sequence e-mail: [email protected] A. T. Bull School of Biosciences, University of Kent, Introduction Canterbury CT2 7NJ, UK Novel actinomycetes, notably streptomycetes, from G. Girard Á G. P. van Wezel Molecular Biotechnology, Institute of Biology, Leiden extreme habitats are proving to be a potentially rich University, P.O. Box 9505, 2300RA Leiden, source of new bioactive natural products that can be The Netherlands developed as resources for healthcare. Streptomycetes account for about 40 % of all known natural products D. P. Labeda National Center for Agricultural Utilization Research, and their genomes typically contain over twenty USDA-ARS, Peoria, IL 61604, USA biosynthetic gene clusters that encode for known or 123 850 Antonie van Leeuwenhoek (2014) 105:849–861 predicted secondary metabolites (Goodfellow and from hyper-arid Atacama Desert soil, which produces Fiedler 2010;Be´rdy 2012). Although an underex- the chaxalactins and chaxamycins. A polyphasic plored resource, it is difficult to discover new chemical taxonomic study showed that this isolate belongs to entities from known streptomycetes as screening them a new species, for which we propose the name tends to lead to the costly rediscovery of known Streptomyces leeuwenhoekii sp. nov. compounds (Busti et al. 2006; Williams 2008). Consequently, innovative strategies are needed to selectively isolate, dereplicate and identify new Materials and methods Streptomyces species for pharmaceutical screening programmes, as exemplified by the taxonomic Selective isolation, maintenance and cultural approach to drug discovery recommended by Good- conditions fellow and Fiedler (2010). This strategy proved to be effective in the isolation of novel streptomycetes from Strain C34T was recovered from a hyper-arid soil deep-sea sediments, as illustrated by the discovery of sample collected from the Chaxa de Laguna, Salar de caboxamycin, a new benzoxazole antibiotic produced Atacama of the Atacama Desert (23°1700S, 68°1000W), by a Streptomyces strain that was isolated from near Tocanao (Okoro et al. 2009). The organism was sediment collected from the Canary Basin in the isolated on starch-casein agar supplemented with Atlantic Ocean (Hohmann et al. 2009). We have actidione and nystatin (each at 25 lgml-1)after recently applied a similar strategy to study actinomy- incubation at 28 °C for 21 days following inoculation cete communities in another neglected biome, the with a 10-1 soil suspension that had been heated at Atacama Desert in Northern Chile (Bull and Asenjo 55 °C for 6 min, as described by Okoro et al. (2009). 2013). The strain was maintained on modified Bennett’s agar The Atacama Desert is the oldest and driest desert (Jones 1949) slopes and as suspensions of hyphal on Earth having evolved over several million years of fragments and spores in 20 % (v/v) glycerol at -80 °C. aridity and hyper-aridity (Go´mez-Silva et al. 2008). Biomass for the molecular systematic and most of the Environmental conditions in the desert have been chemotaxonomic studies was scraped from 14 day-old considered too extreme to support any form of life modified Bennett’s agar plates incubated at 28 °Cand given the dearth of liquid water, paucity of organic washed twice in distilled water; biomass for most of the matter, presence of inorganic oxidants and high levels chemotaxonomic analyses was freeze-dried and that for of UV radiation. Despite these harsh conditions, the molecular systematic work stored at -20 °C. Cells phylogenetically novel actinomycetes, especially for the fatty acid analysis were harvested from yeast streptomycetes, have been isolated from soils col- extract-malt extract broth (International Streptomyces lected from hyper- and extreme hyper-arid regions of Project [ISP] medium 2; Shirling and Gottlieb 1966) the desert (Okoro et al. 2009; Bull and Asenjo 2013). after 3 days at 25 °C. The names of three of the putatively novel strepto- mycetes have been validly published as Streptomyces Chemotaxonomy and morphology atacamensis, Streptomyces bullii and Streptomyces deserti (Santhanam et al. 2012a, b, 2013) and two Isolate C34T was examined for chemotaxonomic and additional Streptomyces strains have been shown to morphological properties considered to be typical of synthesise new antibiotics and anti-cancer com- the genus Streptomyces (Ka¨mpfer 2012). The arrange- pounds, the atacamycins, chaxalactins and chaxam- ment of aerial hyphae and spore chains were observed ycins (Nachtigall et al. 2011; Rateb et al. 2011a, b; on oatmeal agar (ISP medium 3; Shirling and Gottlieb Bull and Asenjo 2013). Another putatively novel 1966) after 14 days at 28 °C, using the coverslip Streptomyces strain from high altitude Atacama Des- technique described by Kawato and Shinobu (1959). ert soil produces novel aminobenzoquinones, the Spore chain morphology and spore surface ornamen- abenquines, which show inhibitory activity against tation were detected by examining gold-coated, bacteria and dermatophilic fungi (Schulze et al. 2011). dehydrated specimens taken from the oatmeal agar The present study was designed to establish the plate, by using an electron microscope (Cambridge taxonomic status of Streptomyces strain C34T isolated Stereoscan 240 instrument) and the procedure 123 Antonie van Leeuwenhoek (2014) 105:849–861 851 described by O’Donnell et al. (1993). Cultural char- streptomycetes (Ka¨mpfer 2012). The enzyme profile of acteristics of the isolate were determined using ISP the strain was determined using API ZYM strips media (Shirling and Gottlieb 1966) following incuba- (BioMerieux) and its ability to use a broad range of tion at 28 °C for 14 days. Standard protocols were carbon sources determined using Biolog GEN III Micro used to detect the isomers of diaminopimelic acid Plates, in each case following the manufacturer’s (Hasegawa et al. 1983), menaquinones (Collins et al. instructions; a standard inoculum equivalent to 5.0 on 1985) and whole organism sugars (Hasegawa et al. the McFarland scale (Murray et al. 1999) was used to 1983). Cellular fatty acids were extracted, methylated inoculate both the microplates and the API ZYM strips. and analysed by gas chromatography (Hewlett Pack- All of the tests were carried out in duplicate. ard, model 6890) following the recommended proce- dure of the Sherlock Microbial Identification System Generation of whole-genome sequence of isolate (MIDI, Sasser 1990). The resultant fatty acid methyl C34T and genome analysis esters were identified and quantified using the MIDI ACTINO 1 database (version 6.10). The isolate was grown on tryptone soya broth supplemented with 10 % sucrose-yeast extract-malt Phylogenetic analyses extract medium (1:1, v/v) with 5 mM MgCl2 and 0.5 % glycine at 30 °C for 48 h. Cells were resus- Genomic DNA was extracted from isolate C34T pended in 10 mM NaCl, 20 mM Tris–HCl (pH 8.0), biomass and PCR-mediated amplification of a 16S 1 mM EDTA and incubated with lysozyme at 37 °C rRNA purified gene product was achieved, as described for 1 to 30 min until they were lysed. SDS (0.5 % final by Kim and Goodfellow (2002). The resultant almost concentration) and proteinase K (40 lg) were added complete 16S rRNA gene sequence (1,416 nucleotides and the cell extract incubated at 50 °C for 6 h when a [nt]) was submitted to the EzTaxon-e server (http:// standard phenol/chloroform extraction was performed eztaxon-e.ezbiocloud.net/; Kim et al. 2012) and aligned on the lysate. The extract was adjusted to 0.3 M with corresponding 16S rRNA gene sequences of the sodium acetate (pH 5.5) and DNA was spooled with a type strains of the most closely related Streptomyces glass rod upon addition of 2 volumes of 96 % ethanol. species using CLUSTAL W version 1.8 software After washing and drying, the DNA was dissolved in (Thompson et al. 1994). Phylogenetic trees were gen- TE buffer. DNA quality was verified by Sall digestion erated from the aligned sequences using the maximum- and agarose gel electrophoresis.