Antonie van Leeuwenhoek DOI 10.1007/s10482-015-0404-8
ORIGINAL PAPER
Streptomyces fractus sp. nov., a novel streptomycete isolated from the gut of a South African termite Jeffrey Rohland • Paul R. Meyers
Received: 24 November 2014 / Accepted: 3 February 2015 Ó Springer International Publishing Switzerland 2015
Abstract An actinobacterial strain, MV32T, 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 MV32T 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 MV32T within the genus Streptomyces, with 99.3 % sequence similarity to its closest relative, Streptomyces endophyticus YIM 65594T. Despite this high sequence similarity, DNA–DNA hybridisation analysis showed a DNA relatedness value of 62 ± 2 %, to S. endophyticus DSM 41984T (indicating that strain MV32T 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-16240T and Streptomyces cinnabarinus NRRL B-12382T. Based on these results and
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0404-8) contains supplementary material, which is available to authorized users. J. Rohland P. R. Meyers (&) Department of Molecular and Cell Biology, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa e-mail: [email protected]
supported by both chemotaxonomic data and a number of phenotypic differences, strain MV32T is proposed to represent a new species within the genus Streptomyces, with the name Streptomyces fractus (= DSM 42163T = NRRL B-59159T). Keywords Actinomycetes Streptomyces 16S rRNA gene Black-mound Termite Amitermes hastatus Fragmentation
Introduction The first report of an actinobacterium being isolated from a termite gut was in 1946, with the discovery of ‘‘Micromonospora propionici’’ by Hungate (1946). This name, however, has no standing in bacterial nomenclature. Since then there has been a lot more interest in the organisms that inhabit the gut systems of termites, mainly because of the potential biotechnological applications relating to cellulose digestion by termites and their gut commensals, as well as their oxidative enzymes (Varma et al. 1994; Le Roes-Hill et al. 2011). However, the majority of the work to date involves a broad determination of bacterial species richness, mainly from the higher order termite species from the family Termitidae (Bignell et al. 1991). Despite this, little attention has been paid to the actinobacterial population found in termite guts, and even less so for specific termite genera such as
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Amitermes. It is only recently that there has been renewed interest in the termite gut as a unique source of novel actinobacteria and their associated antibiotics (Carr et al. 2012; Matsui et al. 2012; Visser et al. 2012; Zhang et al. 2013). Actinomycetes are Gram positive, filamentous bacteria that belong to the class Actinobacteria, and are some of the most important producers of secondary metabolites, such as antibiotics. Only three other actinobacterial species isolated from termite guts have been previously described, namely Cellulosimicrobium variabile from Mastotermes darwinensis (Bakalidou et al. 2002), Saccharopolyspora pathumthaniensis from Speculitermes sp. (Sinma et al. 2011), and Gryllotalpicola reticulitermitis from Reticulitermes chinensis (Fang et al. 2014). The work presented here is the first description of a streptomycete isolated from the paunch region of the gut of a previously unexplored termite, Amitermes hastatus (family Termitidae), a termite species distributed through all the provinces of South Africa.
Materials and methods Isolation Twenty termites were collected from a mound in the Tygerberg Nature Reserve, Cape Town, South Africa. All twenty were euthanised by freezing before being surface sterilized in 70 % ethanol for 2 min before being washed twice in sterile distilled H2O. The paunch and colon regions were dissected from the termite abdomen and placed in separate tubes, each containing 2 ml phosphate buffer (10 mM Na2HPO42H2O, 1.8 mM KH2PO4; pH 7.0). The tubes were vigorously vortexed for 5 min, before a standard dilution series was set up using sterile phosphate buffer. Strain MV32T was isolated from the paunch region of an A. hastatus specimen on Medium II at pH 7 (Cazemier et al. 2003). The medium contained xylan from oat spelts as the carbon source, as well as 50 lg ml-1 cycloheximide and 10 lg ml-1 nalidixic acid. The plates were incubated under aerobic conditions at 30 °C for 4 weeks. Following isolation, strain MV32T was maintained on yeast-extract malt-extract agar (International Streptomyces Project medium 2; ISP#2, Shirling and Gottlieb 1966).
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Morphological and physiological characterisation Morphological characteristics were determined after growing strain MV32T on inorganic salts-starch agar (ISP#4) (Shirling and Gottlieb 1966) for 14 days at 30 °C. Spore chain morphology and spore surface ornamentation were determined using scanning electron microscopy. A determination of the aerial and substrate mycelium colours was performed on various media (see Supplementary Table S1 for details). Production of soluble pigment was determined on glycerol-asparagine agar (ISP#5), while melanin production was determined on peptone-yeast extract-iron agar (ISP#6) and tyrosine agar (ISP#7), as described by Shirling and Gottlieb (1966). Methods for growth determination at 30, 37, 45 °C and in the presence of NaCl (3, 5, 7 and 10 % w/v) were as described by Locci (1989). Testing for the reduction of nitrate and production of H2S, as well as the decomposition of allantoin, adenine, arbutin, casein, cellulose, aesculin, gelatin, hypoxanthine, starch, Ltyrosine, urea and xanthine, was performed as described previously (Locci 1989; Gordon et al. 1974). For cellulose degradation, carboxymethyl cellulose (CMC) was used; staining was performed using a 0.1 % Congo Red solution, followed by destaining with 1 M NaCl and then flooding the plates with 1 M HCl to visualise zones of degradation. Determination of sole carbon source utilisation and sole nitrogen source utilisation was performed as described by Shirling and Gottlieb (1966). All carbon sources and nitrogen sources were filter sterilised before being added to the sterile basal agar medium. All incubations were carried out at 30 °C, unless indicated otherwise. Chemotaxonomic analyses The isomer of diaminopimelic acid (DAP) and the whole cell sugar pattern were determined as described by Hasegawa et al. (1983), using freeze-dried cells grown in 100 ml ISP#2 broth for 4 days at 30 °C, with shaking. Phospholipid isolation was carried out as described by Minnikin et al. (1984) and analysed on silica gel 60 F254 plates (Merck), using two-dimensional thin-layer chromatography, according to Komagata and Suzuki (1987). Phospholipid detection was carried out using molybdenum blue, ninhydrin and a-naphthol reagents. Analysis of respiratory quinones was carried out by the Identification Service, Deutsche Sammlung
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von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. The whole cell fatty acid analyses were carried out by the BCCMLMG identification service, Gent, Belgium, using cells grown for 48 h at 30 °C on Tryptic Soy Agar (TSA) medium. Inoculation and harvesting of the cells, as well as the extraction and analysis of the fatty acids conformed to the recommendations of the commercial identification system MIDI (Microbial Identification System, Inc., Delaware U.S.A.; Sherlock version 3.10, database TSBA50 rev 5.0). Fatty acid methyl esters were separated by gas chromatography.
cinnabarinus NRRL B-12382T and Streptomyces kunmingensis NRRL B-16240T by the DSMZ identification service. DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridisation was carried out as described by De Ley et al. (1970), incorporating the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VISspectrophotometer equipped with a Peltier-thermostatted 6 9 6 multicell changer and a temperature controller with an in situ temperature probe (Varian).
Genomic and phylogenetic analysis Results and discussion A rapid restriction enzyme digestion method described by Cook and Meyers (2003), was used in the identification of strain MV32T to the genus level. DNA extraction, PCR amplification and DNA sequencing were all carried out as described by Cook and Meyers (2003). After 16S rRNA gene amplification, the DNA was purified using a MSB Spin PCRapaceÒ PCR purification kit (STRATEC Molecular, Germany), before being submitted for sequencing. The resulting 1474-nt sequence was submitted to the GenBank database for comparison, using the BlastN search tool. The 16S rRNA gene sequences of the top hits resulting from this analysis, combined with the results obtained from submission to the EzTaxon database (Kim et al. 2012), were aligned with that of strain MV32T using the MEGA software program, version 6 (Tamura et al. 2013). Phylogenetic trees were constructed using the maximum likelihood (Felsenstein 1981), maximum parsimony (Takahashi and Nei 2000) and neighbourjoining (Saitou and Nei 1987) methods, based on a common 1420 bp region of the 16S rRNA gene. The robustness of the trees was assessed by bootstrapping based on 1,000 resampled data sets. The trees were rooted using the type strain of the type species of the genus Kitasatospora, Kitasatospora setae KM-6054T. DNA–DNA hybridization analysis was performed between strain MV32T and Streptomyces endophyticus DSM 41984T by the BCCM/LMG culture collection (Belgium). Genomic DNA was isolated using a modification of Gevers et al. (2001). Hybridisations were performed according to a modification (Goris et al. 1998; Cleenwerck et al. 2002) of the method described by Ezaki et al. (1989). DNA–DNA hybridisation was performed against Streptomyces
An actinobacterial strain, MV32T, was isolated from the paunch region of the hindgut of a South African termite, A. hastatus. The GenBank BLAST and EzTaxon analyses found strain MV32T to be most closely related to S. endophyticus DSM 41984T with 99.3 % 16S rRNA gene sequence similarity. Pairwise 16S rRNA gene sequence alignments between strain MV32T and its closest relatives (generated using the EzTaxon website) showed that the three most closely related species (in terms of sequence similarity) were S. endophyticus (99.3 %), S. kunmingensis (98.3 %) and S. cinnabarinus (98.2 %). Phylogenetic analyses showed that the type strains of S. endophyticus and S. kunmingensis are the closest phylogenetic neighbours of strain MV32T, forming a cluster supported by all three algorithms used as well as moderate bootstrap support (70 %; Fig. 1). There was very strong bootstrap support for the clustering of strain MV32T with S. endophyticus (96 %; Fig. 1). There was no association of strain MV32T with S. cinnabarinus, which clustered in a separate branch of the tree. The chemotaxonomy results support the placement of strain MV32T within the Streptomyces genus, as the DAP isomer was found to be LL-DAP, and the whole cell sugar pattern had no diagnostic sugars (cell-wall type I, whole cell sugar pattern C; Lechevalier and Lechevalier 1970). The diagnostic phospholipid was found to be phosphatidylethanolamine, indicating strain MV32T has phospholipid pattern PII (Locci 1989). The predominant menaquinones were identified as MK-9(H8) (46 %) and MK-9(H6) (39 %), with traces of MK-9(H4) (6 %), MK-10(H2) (4 %) and MK10(H0) (4 %) also detected. The fatty acid profile was
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Antonie van Leeuwenhoek Streptomyces griseoluteus NBRC 13375
* 99
T (AB184363)
Streptomyces seoulensis NBRC 16668 T (AB249970)
Streptomyces resistomycificus NBRC 12814 T (AB184166) Streptomyces chartreusis NBRC 12753 T (AB184839) Streptomyces aureocirculatus NBRC 13018 T (AB184260) 73
Streptomyces bobili JCM 4624 T (AB045876)
58 65
Streptomyces galilaeus JCM 4757 T (AB045878) Streptomyces phaeoluteigriseus NRRL ISP -5182 T (AJ391815) Streptomyces rhizophilus JR-41T (HQ267989) Streptomyces curacoi NRRL B -2901 T (EF626595) Streptomyces bungoensis NBRC 15711 T (AB184696)
* 58
Streptomyces fractus MV32 T (FJ857947)
*96 *70
Streptomyces endophyticus YIM 65594T (GU367154) Streptomyces kunmingensis NBRC 14463 T (AB184597) Streptomyces siamensis KC-038 T (AB773848) Streptomyces alboflavus NBRC 3438T (AB184775)
* 100 * 86 * 75
Streptomyces fulvissimus NBRC 3717 T (AB184787) Streptomyces antibioticus NBRC 12838 T (AB184184) Streptomyces flavofungini NBRC 13371 T (AB184359)
Streptomyces griseoruber NBRC 12873 T (AB184209) Streptomyces cinnabarinus NBRC 13028 T (AB184266)
84
* 60
Streptomyces atriruber NRRL B -24165 T (EU812169)
Streptomyces avermitilis MA-4680 T (BA000030) Streptomyces phaeofaciens NBRC 13372 T (AB184360)
*100
Streptomyces mirabilis NBRC 13450 T (AB184412) Streptomyces mauvecolor LMG 20100 T (AJ781358) Streptomyces hundungensis MBRL 251 T (JN560157)
*86 *97 *
Streptomyces xanthochromogenes NRRL B-5410 T (DQ442559)
Streptomyces psammoticus NBRC 13971 T (AB184554) Kitasatospora setae KM-6054 T (AB022868) 0.005
Fig. 1 Maximum likelihood phylogenetic tree based on 1420 bp of 16S rRNA gene sequence, showing the position of strain MV32T amongst its closest phylogenetic neighbours. GenBank sequence accession numbers are shown in parentheses. Bootstrap support for each node is indicated as a percentage,
calculated from 1,000 randomly re-sampled datasets (only values C50 % are shown). Nodes marked by asterisks were also obtained in the neighbour joining and maximum parsimony analyses. Bar 5 substitutions per 1,000 nucleotides
found to contain i-C14:0 (1.8 %), i-C15:0 (6.3 %), aiC15:0 (36.5 %), i-C16:1 H (2.78 %), i-C16:0 (15.63 %), i-C15:0 2-OH and/or C16:1x7c (0.94 %), C16:0 (2.96 %), i-C17:1x9c (2.0 %), ai-C17:1x9c (4.1 %), i-C17:0 (3.1 %), ai-C17:0 (20.3 %), cyclo-C17:0 (1.8 %) and C18:0 (1.8 %). All of these chemotaxonomic characteristics are consistent with the placement of strain MV32T within the genus Streptomyces. One distinctive difference exhibited by strain MV32T was the unusual characteristic of fragmentation in broth cultures of all media tested. No evidence of this was found when the strain was grown on agar media. A particular actinomycete-specific protein,
SsgA, has been shown to affect the level of mycelial fragmentation when strains are grown in submerged cultures. Van Wezel et al. (2006) showed that overexpression of this protein in a number of recombinant Streptomyces strains resulted in significant reduction in pellet formation. This observation may suggest that MV32T naturally produces high levels of SsgA and is worthy of further investigation. In standard agar overlay testing, strain MV32T shows antibacterial activity against Mycobacterium aurum A?, but not against Staphylococcus aureus ATCC 25923, Enterococcus faecium VanA and Escherichia coli ATCC 25922.
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Physiological differences were found between strain MV32T and S. endophyticus (Table 1). These include the inability of S. endophyticus to grow at 37 °C, as well as the ability of strain MV32T to degrade arbutin but not starch, while S. endophyticus is not able to degrade arbutin, but is able to degrade starch. They also differ in their ability to grow on meso-inositol as sole carbon source (as strain MV32T is unable to grow). S. cinnabarinus is able to grow on meso-inositol as a sole carbon source, and differs from strain MV32T in its production of melanin on ISP#6, as well as a lack of growth on 10 % (w/v) NaCl plates. S. cinnabarinus also exhibits a distinct red–orange substrate mycelium colour on two growth media (ISP#2 and ISP#5), as compared with the other strains tested (see Supplementary Table S1). Table 1 also shows a number of physiological differences between strain MV32T and S. kunmingensis, in particular the observation that S. kunmingensis produces sclerotia and has loose spirals for its spore chain morphology, whereas the spore chain morphology of strain MV32T is straight (Rectiflexibiles; Fig. 2). S. kunmingensis is
Fig. 2 Scanning electron micrograph of strain MV32T (grown on ISP#2 at 30 °C for 14 days) showing its smooth spore surface ornamentation and Rectiflexibiles spore chain morphology. The bar represents 1 lm
also unable to grow on adonitol as sole carbon source nor L-4-hydroxyproline as sole nitrogen source, whereas strain MV32T can grow on both. Traditionally, a threshold of 97 % 16S rRNA gene sequence similarity has been used to determine whether DNA–DNA hybridisation is necessary to determine if two strains belong to distinct genomic
Table 1 Phenotypic comparison between strain MV32T (1) and its closest phylogenetic relatives, S. endophyticus DSM 41984T (2), S. cinnabarinus NRRL B-12382T (3) and S. kunmingensis NRRL B-16240T (4) 1 Spore chain morphology
2
3 a
Flexuous
4 b,c
Sclerotia, loose spiralsd
Straight
Straight
-
-
?
-
?
-
?(w)
?(w)
?
?(w)
-
?(w)
Arbutin
?
-
?
?
Starch
-
?
-
-
Adonitol
?
?
?
-
meso-Inositol
-
?
?
?(w)
?
?
?(w)
-
Production of melanin on ISP#6 Growth at 37 °C Growth in presence of 10 % (w/v) NaCl Decomposition of
Growth on sole carbon source
Growth on sole nitrogen source L-4-hydroxyproline
Unless otherwise indicated, all results were obtained during this study ? positive, – negative; ?(w) weakly positive a
Li et al. (2013)
b
Kim and Goodfellow (2002)
c
Takahashi et al. (2002)
d
Ruan et al. (1985)
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species. However, there is a growing acceptance of a higher threshold (98.7–99 %) for species delineation (Stackebrandt and Ebers 2006; Meier-Kolthoff et al. 2013; Kim et al. 2014). Despite the high 16S rRNA gene sequence similarity to its closest phylogenetic relatives, DNA–DNA hybridisation analysis found strain MV32T to be a distinct genomic species. When compared to S. endophyticus DSM 41984T, the DNA reassociation value was found to be 62 ± 2 %, and when compared to S. cinnabarinus NRRL B-12382T and S. kunmingensis NRRL B-16240T, respectively, values of 10.4 ± 2.9 and 14.4 ± 0.8 % were determined. These values are well below the 70 % DNA relatedness cut-off point recommended by Wayne et al. (1987) to distinguish between genomic species of prokaryotes. Based on the phylogenetic and phenotypic data presented, strain MV32T is concluded to represent a new species within the genus Streptomyces, for which the name Streptomyces fractus is proposed, with the type strain MV32T (=DSM 42163T = NRRL B-59159T). Description of Streptomyces fractus sp. nov. Streptomyces fractus (frac’tus. L. masc. adj. fractus, meaning broken, shattered, having been broken, referring to the fragmentation of the hyphae in liquid media). Gram-positive actinobacterium that has Rectiflexibiles spore chains and a smooth spore surface ornamentation. Grows well on a number of different media (see Supplementary Table S1) and typically has beige/light brown substrate mycelium with wrinkled, crater-like colonies. Sporulation, when it occurs, appears as a white spore mass. Produces no diffusible pigments when grown on ISP#5 and no melanin when grown on ISP#6 and ISP#7 media. Grows well at both 30 and 37 °C but not at 45 °C and is able to grow in the presence of NaCl in the range from 3–10 % (w/v). Nitrate is reduced and hydrogen sulphide is produced very weakly. Catalase positive but oxidase negative. Degrades adenine, arbutin, casein, aesculin, gelatin, hypoxanthine, L-tyrosine and xanthine but not allantoin, carboxymethyl cellulose, guanine, starch, urea or xylan. Uses adonitol, L(?)-arabinose, D(?)-cellobiose, D(-)fructose, D(?)-galactose, D(?)-glucose, maltose, D(-)mannitol, D(?)-raffinose, L(?)-rhamnose, salicin, sucrose and D(?)-xylose as sole carbon sources but is unable to utilise meso-inositol. Grows on all sole
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nitrogen sources tested: L-asparagine, L-arginine (weakly), L-cysteine, L-histidine, L-4-hydroxyproline, L-methionine, L-phenylalanine (weakly), KNO3 (weakly), Lserine, L-threonine and L-valine. The cell wall contains LL-DAP and the whole cell hydrolysate contains no diagnostic sugars. The diagnostic phospholipid is phosphatidylethanolamine. Diphosphatidylglycerol and an unidentified aminolipid are also produced. The predominant menaquinones are MK-9(H8) and MK-9(H6), with traces of MK-9(H4), MK-10(H2) and MK-10(H0) also present. The major fatty acids present are ai-C15:0, i-C16:0 and ai-C17:0. The type strain, MV32T (=DSM 42163T = NRRL B-59159T), was isolated from the paunch region of the hindgut of the Black-mound Termite, Amitermes hastatus. The GenBank accession number for the 16SrRNA gene sequence of strain MV32T is FJ857947. Acknowledgments We thank Miranda Waldron for help with the scanning electron microscopy and Di James and Bruna Galva˜o for help with the sequencing. Thanks to Dr J. P. Euze´by for help with the Latin species name and to Dr D. Labeda for providing the type strains of S. cinnabarinus and S. kunmingensis. Jeffrey Rohland gratefully acknowledges funding provided by the South African National Research Foundation (NRF) in the form of a grant-holder linked bursary, as well as funding from the University Research Committee (URC), University of Cape Town. Paul Meyers was funded by the NRF (grant number: 85476), the Medical Research Council of South Africa and the URC. We would also like to thank Ms Hestelle Melville and Tygerberg Nature Reserve for providing permission and permits for collection of the termites.
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Le Roes-Hill M, Rohland J, Burton S (2011) Actinobacteria isolated from termite guts as a source of novel oxidative enzymes. Antonie Van Leeuwenhoek 100:589–605 Lechevalier MP, Lechevalier HA (1970) Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol 20:435–443 Li J, Zhao G-Z, Zhu W-Y, Huang H-Y, Xu L-H, Zhang S, Li W-J (2013) Streptomyces endophyticus sp. nov., an endophytic actinomycete isolated from Artemisia annua L. Int J Syst Evol Microbiol 63:224–229 Locci R (1989) Streptomyces and related genera. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 4. Williams and Wilkins, Baltimore, pp 2451–2508 Matsui T, Tanaka J, Namihira T, Shinzato N (2012) Antibiotics production by an actinomycete isolated from the termite gut. J Basic Microbiol 52:731–735 Meier-Kolthoff JP, Go¨ker M, Spro¨er C, Klenk H-P (2013) When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 195:413–418 ´ Donnell AG, Goodfellow M, Alderson G, Minnikin DE, O Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinines and polar lipids. J Microbiol Methods 2:233–241 Ruan J, Lechevalier MP, Jiang C, Lechevalier HA (1985) Chainia kunmingensis, a new actinomycete species found in soil. Int J Syst Bacteriol 35:164–168 Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 Sinma K, Ishida Y, Tamura T, Kitpreechavanich V, Tokuyama S (2011) Saccharopolyspora pathumthaniensis sp. nov., a novel actinomycetes isolated from termite guts (Speculitermes sp.). J Gen Appl Microbiol 57:93–100 Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33:152–155 Takahashi K, Nei M (2000) Efficiencies of fast algorithms of phylogenetic inference under the criteria of maximum parsimony, minimum evolution, and maximum likelihood when a large number of sequences are used. Mol Biol Evol 17:1251–1258 Takahashi Y, Matsumoto A, Seino A, Ueno J, Iwai Y, Komura S (2002) Streptomyces avermectinius sp. nov., an avermectinproducing strain. Int J Syst Evol Microbiol 52:2163–2168 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 Van Wezel GP, Krabben P, Traag BA, Keijser BJF, Kerste R, Vijgenboom E, Heijnen JJ, Kraal B (2006) Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering. Appl Environ Microbiol 72:5283–5288 Varma A, Kolli BK, Paul J, Saxena S, Ko¨nig H (1994) Lignocellulose degradation by microorganisms from termite hills and termite guts: a survey on the present state of art. FEMS Microbiol Rev 15:9–28 Visser AA, Nobre T, Currie CR, Aanen DK, Poulsen M (2012) Exploring the potential for actinobacteria as defensive
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ORIGINAL PAPER
Streptomyces fractus sp. nov., a novel streptomycete isolated from the gut of a South African termite Jeffrey Rohland • Paul R. Meyers
Received: 24 November 2014 / Accepted: 3 February 2015 Ó Springer International Publishing Switzerland 2015
Abstract An actinobacterial strain, MV32T, 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 MV32T 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 MV32T within the genus Streptomyces, with 99.3 % sequence similarity to its closest relative, Streptomyces endophyticus YIM 65594T. Despite this high sequence similarity, DNA–DNA hybridisation analysis showed a DNA relatedness value of 62 ± 2 %, to S. endophyticus DSM 41984T (indicating that strain MV32T 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-16240T and Streptomyces cinnabarinus NRRL B-12382T. Based on these results and
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0404-8) contains supplementary material, which is available to authorized users. J. Rohland P. R. Meyers (&) Department of Molecular and Cell Biology, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa e-mail: [email protected]
supported by both chemotaxonomic data and a number of phenotypic differences, strain MV32T is proposed to represent a new species within the genus Streptomyces, with the name Streptomyces fractus (= DSM 42163T = NRRL B-59159T). Keywords Actinomycetes Streptomyces 16S rRNA gene Black-mound Termite Amitermes hastatus Fragmentation
Introduction The first report of an actinobacterium being isolated from a termite gut was in 1946, with the discovery of ‘‘Micromonospora propionici’’ by Hungate (1946). This name, however, has no standing in bacterial nomenclature. Since then there has been a lot more interest in the organisms that inhabit the gut systems of termites, mainly because of the potential biotechnological applications relating to cellulose digestion by termites and their gut commensals, as well as their oxidative enzymes (Varma et al. 1994; Le Roes-Hill et al. 2011). However, the majority of the work to date involves a broad determination of bacterial species richness, mainly from the higher order termite species from the family Termitidae (Bignell et al. 1991). Despite this, little attention has been paid to the actinobacterial population found in termite guts, and even less so for specific termite genera such as
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Amitermes. It is only recently that there has been renewed interest in the termite gut as a unique source of novel actinobacteria and their associated antibiotics (Carr et al. 2012; Matsui et al. 2012; Visser et al. 2012; Zhang et al. 2013). Actinomycetes are Gram positive, filamentous bacteria that belong to the class Actinobacteria, and are some of the most important producers of secondary metabolites, such as antibiotics. Only three other actinobacterial species isolated from termite guts have been previously described, namely Cellulosimicrobium variabile from Mastotermes darwinensis (Bakalidou et al. 2002), Saccharopolyspora pathumthaniensis from Speculitermes sp. (Sinma et al. 2011), and Gryllotalpicola reticulitermitis from Reticulitermes chinensis (Fang et al. 2014). The work presented here is the first description of a streptomycete isolated from the paunch region of the gut of a previously unexplored termite, Amitermes hastatus (family Termitidae), a termite species distributed through all the provinces of South Africa.
Materials and methods Isolation Twenty termites were collected from a mound in the Tygerberg Nature Reserve, Cape Town, South Africa. All twenty were euthanised by freezing before being surface sterilized in 70 % ethanol for 2 min before being washed twice in sterile distilled H2O. The paunch and colon regions were dissected from the termite abdomen and placed in separate tubes, each containing 2 ml phosphate buffer (10 mM Na2HPO42H2O, 1.8 mM KH2PO4; pH 7.0). The tubes were vigorously vortexed for 5 min, before a standard dilution series was set up using sterile phosphate buffer. Strain MV32T was isolated from the paunch region of an A. hastatus specimen on Medium II at pH 7 (Cazemier et al. 2003). The medium contained xylan from oat spelts as the carbon source, as well as 50 lg ml-1 cycloheximide and 10 lg ml-1 nalidixic acid. The plates were incubated under aerobic conditions at 30 °C for 4 weeks. Following isolation, strain MV32T was maintained on yeast-extract malt-extract agar (International Streptomyces Project medium 2; ISP#2, Shirling and Gottlieb 1966).
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Morphological and physiological characterisation Morphological characteristics were determined after growing strain MV32T on inorganic salts-starch agar (ISP#4) (Shirling and Gottlieb 1966) for 14 days at 30 °C. Spore chain morphology and spore surface ornamentation were determined using scanning electron microscopy. A determination of the aerial and substrate mycelium colours was performed on various media (see Supplementary Table S1 for details). Production of soluble pigment was determined on glycerol-asparagine agar (ISP#5), while melanin production was determined on peptone-yeast extract-iron agar (ISP#6) and tyrosine agar (ISP#7), as described by Shirling and Gottlieb (1966). Methods for growth determination at 30, 37, 45 °C and in the presence of NaCl (3, 5, 7 and 10 % w/v) were as described by Locci (1989). Testing for the reduction of nitrate and production of H2S, as well as the decomposition of allantoin, adenine, arbutin, casein, cellulose, aesculin, gelatin, hypoxanthine, starch, Ltyrosine, urea and xanthine, was performed as described previously (Locci 1989; Gordon et al. 1974). For cellulose degradation, carboxymethyl cellulose (CMC) was used; staining was performed using a 0.1 % Congo Red solution, followed by destaining with 1 M NaCl and then flooding the plates with 1 M HCl to visualise zones of degradation. Determination of sole carbon source utilisation and sole nitrogen source utilisation was performed as described by Shirling and Gottlieb (1966). All carbon sources and nitrogen sources were filter sterilised before being added to the sterile basal agar medium. All incubations were carried out at 30 °C, unless indicated otherwise. Chemotaxonomic analyses The isomer of diaminopimelic acid (DAP) and the whole cell sugar pattern were determined as described by Hasegawa et al. (1983), using freeze-dried cells grown in 100 ml ISP#2 broth for 4 days at 30 °C, with shaking. Phospholipid isolation was carried out as described by Minnikin et al. (1984) and analysed on silica gel 60 F254 plates (Merck), using two-dimensional thin-layer chromatography, according to Komagata and Suzuki (1987). Phospholipid detection was carried out using molybdenum blue, ninhydrin and a-naphthol reagents. Analysis of respiratory quinones was carried out by the Identification Service, Deutsche Sammlung
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von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. The whole cell fatty acid analyses were carried out by the BCCMLMG identification service, Gent, Belgium, using cells grown for 48 h at 30 °C on Tryptic Soy Agar (TSA) medium. Inoculation and harvesting of the cells, as well as the extraction and analysis of the fatty acids conformed to the recommendations of the commercial identification system MIDI (Microbial Identification System, Inc., Delaware U.S.A.; Sherlock version 3.10, database TSBA50 rev 5.0). Fatty acid methyl esters were separated by gas chromatography.
cinnabarinus NRRL B-12382T and Streptomyces kunmingensis NRRL B-16240T by the DSMZ identification service. DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridisation was carried out as described by De Ley et al. (1970), incorporating the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VISspectrophotometer equipped with a Peltier-thermostatted 6 9 6 multicell changer and a temperature controller with an in situ temperature probe (Varian).
Genomic and phylogenetic analysis Results and discussion A rapid restriction enzyme digestion method described by Cook and Meyers (2003), was used in the identification of strain MV32T to the genus level. DNA extraction, PCR amplification and DNA sequencing were all carried out as described by Cook and Meyers (2003). After 16S rRNA gene amplification, the DNA was purified using a MSB Spin PCRapaceÒ PCR purification kit (STRATEC Molecular, Germany), before being submitted for sequencing. The resulting 1474-nt sequence was submitted to the GenBank database for comparison, using the BlastN search tool. The 16S rRNA gene sequences of the top hits resulting from this analysis, combined with the results obtained from submission to the EzTaxon database (Kim et al. 2012), were aligned with that of strain MV32T using the MEGA software program, version 6 (Tamura et al. 2013). Phylogenetic trees were constructed using the maximum likelihood (Felsenstein 1981), maximum parsimony (Takahashi and Nei 2000) and neighbourjoining (Saitou and Nei 1987) methods, based on a common 1420 bp region of the 16S rRNA gene. The robustness of the trees was assessed by bootstrapping based on 1,000 resampled data sets. The trees were rooted using the type strain of the type species of the genus Kitasatospora, Kitasatospora setae KM-6054T. DNA–DNA hybridization analysis was performed between strain MV32T and Streptomyces endophyticus DSM 41984T by the BCCM/LMG culture collection (Belgium). Genomic DNA was isolated using a modification of Gevers et al. (2001). Hybridisations were performed according to a modification (Goris et al. 1998; Cleenwerck et al. 2002) of the method described by Ezaki et al. (1989). DNA–DNA hybridisation was performed against Streptomyces
An actinobacterial strain, MV32T, was isolated from the paunch region of the hindgut of a South African termite, A. hastatus. The GenBank BLAST and EzTaxon analyses found strain MV32T to be most closely related to S. endophyticus DSM 41984T with 99.3 % 16S rRNA gene sequence similarity. Pairwise 16S rRNA gene sequence alignments between strain MV32T and its closest relatives (generated using the EzTaxon website) showed that the three most closely related species (in terms of sequence similarity) were S. endophyticus (99.3 %), S. kunmingensis (98.3 %) and S. cinnabarinus (98.2 %). Phylogenetic analyses showed that the type strains of S. endophyticus and S. kunmingensis are the closest phylogenetic neighbours of strain MV32T, forming a cluster supported by all three algorithms used as well as moderate bootstrap support (70 %; Fig. 1). There was very strong bootstrap support for the clustering of strain MV32T with S. endophyticus (96 %; Fig. 1). There was no association of strain MV32T with S. cinnabarinus, which clustered in a separate branch of the tree. The chemotaxonomy results support the placement of strain MV32T within the Streptomyces genus, as the DAP isomer was found to be LL-DAP, and the whole cell sugar pattern had no diagnostic sugars (cell-wall type I, whole cell sugar pattern C; Lechevalier and Lechevalier 1970). The diagnostic phospholipid was found to be phosphatidylethanolamine, indicating strain MV32T has phospholipid pattern PII (Locci 1989). The predominant menaquinones were identified as MK-9(H8) (46 %) and MK-9(H6) (39 %), with traces of MK-9(H4) (6 %), MK-10(H2) (4 %) and MK10(H0) (4 %) also detected. The fatty acid profile was
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Antonie van Leeuwenhoek Streptomyces griseoluteus NBRC 13375
* 99
T (AB184363)
Streptomyces seoulensis NBRC 16668 T (AB249970)
Streptomyces resistomycificus NBRC 12814 T (AB184166) Streptomyces chartreusis NBRC 12753 T (AB184839) Streptomyces aureocirculatus NBRC 13018 T (AB184260) 73
Streptomyces bobili JCM 4624 T (AB045876)
58 65
Streptomyces galilaeus JCM 4757 T (AB045878) Streptomyces phaeoluteigriseus NRRL ISP -5182 T (AJ391815) Streptomyces rhizophilus JR-41T (HQ267989) Streptomyces curacoi NRRL B -2901 T (EF626595) Streptomyces bungoensis NBRC 15711 T (AB184696)
* 58
Streptomyces fractus MV32 T (FJ857947)
*96 *70
Streptomyces endophyticus YIM 65594T (GU367154) Streptomyces kunmingensis NBRC 14463 T (AB184597) Streptomyces siamensis KC-038 T (AB773848) Streptomyces alboflavus NBRC 3438T (AB184775)
* 100 * 86 * 75
Streptomyces fulvissimus NBRC 3717 T (AB184787) Streptomyces antibioticus NBRC 12838 T (AB184184) Streptomyces flavofungini NBRC 13371 T (AB184359)
Streptomyces griseoruber NBRC 12873 T (AB184209) Streptomyces cinnabarinus NBRC 13028 T (AB184266)
84
* 60
Streptomyces atriruber NRRL B -24165 T (EU812169)
Streptomyces avermitilis MA-4680 T (BA000030) Streptomyces phaeofaciens NBRC 13372 T (AB184360)
*100
Streptomyces mirabilis NBRC 13450 T (AB184412) Streptomyces mauvecolor LMG 20100 T (AJ781358) Streptomyces hundungensis MBRL 251 T (JN560157)
*86 *97 *
Streptomyces xanthochromogenes NRRL B-5410 T (DQ442559)
Streptomyces psammoticus NBRC 13971 T (AB184554) Kitasatospora setae KM-6054 T (AB022868) 0.005
Fig. 1 Maximum likelihood phylogenetic tree based on 1420 bp of 16S rRNA gene sequence, showing the position of strain MV32T amongst its closest phylogenetic neighbours. GenBank sequence accession numbers are shown in parentheses. Bootstrap support for each node is indicated as a percentage,
calculated from 1,000 randomly re-sampled datasets (only values C50 % are shown). Nodes marked by asterisks were also obtained in the neighbour joining and maximum parsimony analyses. Bar 5 substitutions per 1,000 nucleotides
found to contain i-C14:0 (1.8 %), i-C15:0 (6.3 %), aiC15:0 (36.5 %), i-C16:1 H (2.78 %), i-C16:0 (15.63 %), i-C15:0 2-OH and/or C16:1x7c (0.94 %), C16:0 (2.96 %), i-C17:1x9c (2.0 %), ai-C17:1x9c (4.1 %), i-C17:0 (3.1 %), ai-C17:0 (20.3 %), cyclo-C17:0 (1.8 %) and C18:0 (1.8 %). All of these chemotaxonomic characteristics are consistent with the placement of strain MV32T within the genus Streptomyces. One distinctive difference exhibited by strain MV32T was the unusual characteristic of fragmentation in broth cultures of all media tested. No evidence of this was found when the strain was grown on agar media. A particular actinomycete-specific protein,
SsgA, has been shown to affect the level of mycelial fragmentation when strains are grown in submerged cultures. Van Wezel et al. (2006) showed that overexpression of this protein in a number of recombinant Streptomyces strains resulted in significant reduction in pellet formation. This observation may suggest that MV32T naturally produces high levels of SsgA and is worthy of further investigation. In standard agar overlay testing, strain MV32T shows antibacterial activity against Mycobacterium aurum A?, but not against Staphylococcus aureus ATCC 25923, Enterococcus faecium VanA and Escherichia coli ATCC 25922.
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Physiological differences were found between strain MV32T and S. endophyticus (Table 1). These include the inability of S. endophyticus to grow at 37 °C, as well as the ability of strain MV32T to degrade arbutin but not starch, while S. endophyticus is not able to degrade arbutin, but is able to degrade starch. They also differ in their ability to grow on meso-inositol as sole carbon source (as strain MV32T is unable to grow). S. cinnabarinus is able to grow on meso-inositol as a sole carbon source, and differs from strain MV32T in its production of melanin on ISP#6, as well as a lack of growth on 10 % (w/v) NaCl plates. S. cinnabarinus also exhibits a distinct red–orange substrate mycelium colour on two growth media (ISP#2 and ISP#5), as compared with the other strains tested (see Supplementary Table S1). Table 1 also shows a number of physiological differences between strain MV32T and S. kunmingensis, in particular the observation that S. kunmingensis produces sclerotia and has loose spirals for its spore chain morphology, whereas the spore chain morphology of strain MV32T is straight (Rectiflexibiles; Fig. 2). S. kunmingensis is
Fig. 2 Scanning electron micrograph of strain MV32T (grown on ISP#2 at 30 °C for 14 days) showing its smooth spore surface ornamentation and Rectiflexibiles spore chain morphology. The bar represents 1 lm
also unable to grow on adonitol as sole carbon source nor L-4-hydroxyproline as sole nitrogen source, whereas strain MV32T can grow on both. Traditionally, a threshold of 97 % 16S rRNA gene sequence similarity has been used to determine whether DNA–DNA hybridisation is necessary to determine if two strains belong to distinct genomic
Table 1 Phenotypic comparison between strain MV32T (1) and its closest phylogenetic relatives, S. endophyticus DSM 41984T (2), S. cinnabarinus NRRL B-12382T (3) and S. kunmingensis NRRL B-16240T (4) 1 Spore chain morphology
2
3 a
Flexuous
4 b,c
Sclerotia, loose spiralsd
Straight
Straight
-
-
?
-
?
-
?(w)
?(w)
?
?(w)
-
?(w)
Arbutin
?
-
?
?
Starch
-
?
-
-
Adonitol
?
?
?
-
meso-Inositol
-
?
?
?(w)
?
?
?(w)
-
Production of melanin on ISP#6 Growth at 37 °C Growth in presence of 10 % (w/v) NaCl Decomposition of
Growth on sole carbon source
Growth on sole nitrogen source L-4-hydroxyproline
Unless otherwise indicated, all results were obtained during this study ? positive, – negative; ?(w) weakly positive a
Li et al. (2013)
b
Kim and Goodfellow (2002)
c
Takahashi et al. (2002)
d
Ruan et al. (1985)
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species. However, there is a growing acceptance of a higher threshold (98.7–99 %) for species delineation (Stackebrandt and Ebers 2006; Meier-Kolthoff et al. 2013; Kim et al. 2014). Despite the high 16S rRNA gene sequence similarity to its closest phylogenetic relatives, DNA–DNA hybridisation analysis found strain MV32T to be a distinct genomic species. When compared to S. endophyticus DSM 41984T, the DNA reassociation value was found to be 62 ± 2 %, and when compared to S. cinnabarinus NRRL B-12382T and S. kunmingensis NRRL B-16240T, respectively, values of 10.4 ± 2.9 and 14.4 ± 0.8 % were determined. These values are well below the 70 % DNA relatedness cut-off point recommended by Wayne et al. (1987) to distinguish between genomic species of prokaryotes. Based on the phylogenetic and phenotypic data presented, strain MV32T is concluded to represent a new species within the genus Streptomyces, for which the name Streptomyces fractus is proposed, with the type strain MV32T (=DSM 42163T = NRRL B-59159T). Description of Streptomyces fractus sp. nov. Streptomyces fractus (frac’tus. L. masc. adj. fractus, meaning broken, shattered, having been broken, referring to the fragmentation of the hyphae in liquid media). Gram-positive actinobacterium that has Rectiflexibiles spore chains and a smooth spore surface ornamentation. Grows well on a number of different media (see Supplementary Table S1) and typically has beige/light brown substrate mycelium with wrinkled, crater-like colonies. Sporulation, when it occurs, appears as a white spore mass. Produces no diffusible pigments when grown on ISP#5 and no melanin when grown on ISP#6 and ISP#7 media. Grows well at both 30 and 37 °C but not at 45 °C and is able to grow in the presence of NaCl in the range from 3–10 % (w/v). Nitrate is reduced and hydrogen sulphide is produced very weakly. Catalase positive but oxidase negative. Degrades adenine, arbutin, casein, aesculin, gelatin, hypoxanthine, L-tyrosine and xanthine but not allantoin, carboxymethyl cellulose, guanine, starch, urea or xylan. Uses adonitol, L(?)-arabinose, D(?)-cellobiose, D(-)fructose, D(?)-galactose, D(?)-glucose, maltose, D(-)mannitol, D(?)-raffinose, L(?)-rhamnose, salicin, sucrose and D(?)-xylose as sole carbon sources but is unable to utilise meso-inositol. Grows on all sole
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nitrogen sources tested: L-asparagine, L-arginine (weakly), L-cysteine, L-histidine, L-4-hydroxyproline, L-methionine, L-phenylalanine (weakly), KNO3 (weakly), Lserine, L-threonine and L-valine. The cell wall contains LL-DAP and the whole cell hydrolysate contains no diagnostic sugars. The diagnostic phospholipid is phosphatidylethanolamine. Diphosphatidylglycerol and an unidentified aminolipid are also produced. The predominant menaquinones are MK-9(H8) and MK-9(H6), with traces of MK-9(H4), MK-10(H2) and MK-10(H0) also present. The major fatty acids present are ai-C15:0, i-C16:0 and ai-C17:0. The type strain, MV32T (=DSM 42163T = NRRL B-59159T), was isolated from the paunch region of the hindgut of the Black-mound Termite, Amitermes hastatus. The GenBank accession number for the 16SrRNA gene sequence of strain MV32T is FJ857947. Acknowledgments We thank Miranda Waldron for help with the scanning electron microscopy and Di James and Bruna Galva˜o for help with the sequencing. Thanks to Dr J. P. Euze´by for help with the Latin species name and to Dr D. Labeda for providing the type strains of S. cinnabarinus and S. kunmingensis. Jeffrey Rohland gratefully acknowledges funding provided by the South African National Research Foundation (NRF) in the form of a grant-holder linked bursary, as well as funding from the University Research Committee (URC), University of Cape Town. Paul Meyers was funded by the NRF (grant number: 85476), the Medical Research Council of South Africa and the URC. We would also like to thank Ms Hestelle Melville and Tygerberg Nature Reserve for providing permission and permits for collection of the termites.
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