Antonie van Leeuwenhoek

, Volume 101, Issue 1, pp 185–193

Verrucosispora maris sp. nov., a novel deep-sea actinomycete isolated from a marine sediment which produces abyssomicins

Authors

    • School of BiologyUniversity of Newcastle
  • James E. M. Stach
    • School of BiologyUniversity of Newcastle
  • Roselyn Brown
    • School of BiologyUniversity of Newcastle
  • Avinash Naga Venkata Bonda
    • School of BiologyUniversity of Newcastle
  • Amanda L. Jones
    • School of BiologyUniversity of Newcastle
    • School of Life Sciences, Ellison BuildingNorthumbria University
  • Joanne Mexson
    • School of BiosciencesUniversity of Kent
  • Hans-Peter Fiedler
    • Mikrobiologisches InstitutUniversität Tübingen
  • Tiago Domingues Zucchi
    • School of BiologyUniversity of Newcastle
    • Departamento de Entomologia e Acarologia, ESALQUniversidade de São Paulo
  • Alan T. Bull
    • School of BiologyUniversity of Newcastle
    • School of BiosciencesUniversity of Kent
Original Paper

DOI: 10.1007/s10482-011-9651-5

Cite this article as:
Goodfellow, M., Stach, J.E.M., Brown, R. et al. Antonie van Leeuwenhoek (2012) 101: 185. doi:10.1007/s10482-011-9651-5

Abstract

Verrucosispora isolate AB-18-032T, the abyssomicin- and proximicin-producing actinomycete, has chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora. The organism formed a distinct phyletic line in the Verrucosispora 16S rRNA gene tree sharing similarities of 99.7%, 98.7% and 98.9% with Verrucosispora gifhornensis DSM 44337T, Verrucosispora lutea YIM 013T and Verrucosispora sediminis MS 426T, respectively. It was readily distinguished from the two latter species using a range of phenotypic features and from V. gifhornensis DSM 44337T, its nearest phylogenetic neighbor, by a DNA G+C content of 65.5 mol% obtained by thermal denaturation and fluorometry and DNA:DNA relatedness values of 64.0% and 65.0% using renaturation and fluorometric methods, respectively. It is apparent from the combined genotypic and phenotypic data that strain AB-18-032T should be classified in the genus Verrucosispora as a new species. The name Verrucosispora maris sp. nov. is proposed for this taxon with isolate AB-18-032T (= DSM 45365T = NRRL B-24793T) as the type strain.

Keywords

Verrucosispora marisPolyphasic taxonomyMarine sedimentActinomycete

Introduction

The genus Verrucosispora was established by Rheims et al. (1998) as a member of the family Micromonosporaceae. It currently encompasses the species Verrucosispora gifhornensis (Rheims et al. 1998), Verrucosispora lutea (Liao et al. 2009) and Verrucosispora sediminis (Dai et al. 2010), the single members of which were isolated from a peat bog, marine sediment and a deep-sea sediment, respectively. The taxon forms a distinct branch in the 16S rRNA gene tree and is characterized by branching hyphae which form a well-developed substrate mycelium, absent or sparse aerial hyphae, non-motile, spores with smooth, warty or hairy surfaces which are borne singly, in pairs or clusters and by the presence of meso-diaminopimelic acid and glycine in the peptidoglycan (wall chemotype II sensu Lechevalier and Lechevalier 1970), a glycolyl type of muramic acid, tetrahydrogenated menaquinones with nine isoprene units (MK 9 [H]4) as the predominant isoprenologue, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol mannoside as major polar lipids (phospholipid type II after Lechevalier et al. 1977) and a DNA G+C base composition within the range 66.8–70.0%. This taxonomic profile serves to distinguish the genus Verrucosispora from the other genera classified in the family Micromonosporaceae (Xie et al. 2011).

An additional Verrucosispora strain, isolate AB-18-032, was recovered from a deep-sea sediment and shown to synthesize novel secondary metabolites. This isolate, which has been provisionally labeled “Verrucosispora maris” (Goodfellow and Fiedler 2010), produces a family of novel polycyclic polyketides, the abyssomicins (Bister et al. 2004; Riedlinger et al. 2004; Keller et al. 2007a, b) and proximicin A, an aminofuran type antibiotic (Fiedler et al. 2008). In addition, it is known from the whole genome sequence of this organism that it contains around 23 biosynthetic gene clusters encoding for the production of known or predicted secondary metabolites (Roh et al. 2011). Partial characterization of the abyssomicin-producing strain showed that it had chemotaxonomic and morphological properties similar to that of the type strain of V. gifhornensis, but could be distinguished from the latter using a combination of genotypic and phenotypic properties (Riedlinger et al. 2004).

The aim of the present study was to establish the taxonomic status of isolate AB-18-032. To this end, it was compared with V. gifhornensis DSM 44337T, V. lutea YIM 013T and V. sediminis MS 426T in a polyphasic taxonomic analysis. The resultant data showed that the isolate merited recognition as a new species for which the name Verrucosispora maris sp. nov. is proposed.

Materials and methods

Strains and cultural conditions

Strain AB-18-032T was isolated on a colloidal chitin agar (Hsu and Lockwood 1975) plate which had been inoculated with a suspension of a sediment sample collected from the Sea of Japan and incubated at 30°C for 4 weeks (Mexson 2001). Both the isolate and the type strains of V. gifhornensis, V. lutea and V. sediminis were maintained on yeast extract-malt extract agar (ISP medium 2; Shirling and Gottlieb 1966) at 28°C and as suspensions of hyphal fragments in glycerol (20%, v/v) at −20 and −80°C. Biomass for the chemotaxonomic and molecular systematic studies was prepared by growing the isolate and V. gifhornensis DSM 44337T in shake flasks of yeast extract-malt extract broth for 15 days at 28°C prior to checking for purity and harvesting by centrifugation. Biomass for the chemical analyses was washed in distilled water and freeze-dried, and that for the molecular systematic studies in NaCl-EDTA buffer (0.1 M EDTA, pH 8.0, 0.1 M NaCl); in each case the resultant preparations were stored at −20°C until needed.

16S rRNA gene sequencing analyses

Isolation of chromosomal DNA, PCR amplification and direct sequencing of the PCR products of isolate AB-18-032T were carried out as described by Kim et al. (1999). The resultant almost complete 16S rRNA gene sequence (1440 nucleotides [nt]) was aligned manually with corresponding sequences of the type strains of the three Verrucosispora species and the type strains of the type species of genera classified in the family Micromonosporaceae, retrieved from the DDBJ/EMBL/GenBank databases, using the PHYDIT program (http://plaza.snu.ac.kr/~jchun/phydit/). Phylogenetic trees were inferred using the least-squares (Fitch and Margoliash 1967), maximum-likelihood (Felsenstein 1981), maximum-parsimony (Kluge and Farris 1969) and neighbor-joining (Saitou and Nei 1987) tree-making algorithms from the PHYLIP package (Felsenstein, 1993). Evolutionary distance matrices were generated for the least-squares and neighbor-joining methods, as described by Jukes and Cantor (1969). The resultant unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein 1985) based on 1000 resamplings of the neighbor-joining dataset, using the SEQBOOT and CONSENSE options from the PHYLIP package.

Chemotaxonomy

Standard chromatographic procedures were used to determine the menaquinone and polar lipid composition of isolate AB-18-032T (Minnikin et al. 1984; Collins 1994) and to establish if it contained mycolic acids (Minnikin et al. 1975). The G+C mol% content of isolate AB-18-032T and the type strain of V. gifhornensis were determined using the fluorometric method of Gonzalez and Saiz-Jimenez (2002). Briefly, genomic DNA was obtained for the strains using the Qiagen genomi-tip 20/G method, according to the manufacturer’s instructions (Qiagen, UK). DNA melting temperatures (Tm) were calculated using a DNA engine Opticon 2 (Biorad, UK) in the presence of 30% formamide. Tm was calculated from the minimum value of the slope tangent to the melting curve of fluorescence versus temperature (Fig. 3). The G+C mol% was calculated using the equation % GC = 1.99 Tm–71.08.

DNA:DNA relatedness studies

These studies were made between isolate AB-18-032T and V. gifhornensis DSM 44337T using two procedures. Initially, DNA:DNA hybridization between duplicated DNA preparations of the organisms was carried out by the identification service of the DSMZ (Braunschweig, Germany), as described by Kim et al. (1999). Next, DNA:DNA relatedness values (∆Tm) were determined using a fluorometric method (Gonzalez and Saiz-Jimenez 2005): the optimum temperature for renaturation (Tor) was calculated using Tor – 0.51 (%GC)+47. The melting temperature (Tm) at which 50% of the initial double stranded molecules denaturated into single-stranded DNA for isolate AB-18-032T gDNA and the isolate AB-18-032T/Verrucosispora gifhornensis hybrid DNA was compared and the difference (∆Tm) calculated.

Phenotypic tests

Isolate AB-18-032T and the type strains of the three Verrucosispora species (Table 2) were examined, in duplicate, for a range of phenotypic tests which were incubated at 28°C for 2–3 weeks, except for the temperature tests, following the addition of a standard inoculum equivalent to 2.5 on the McFarland scale. Allantoin hydrolysis was carried out after Gordon (1967), the degradation (%, w/v) of adenine (1.0), elastin (0.3), gelatin (0.4), guanine (0.5), hypoxanthine (0.4), starch (1.0), l-tyrosine (0.5), uric acid (0.5), xanthine (0.4) and xylan (0.4) using modified Bennett’s agar as the basal medium (Tan et al. 2006), the pH and tolerance tests on ISP medium 2 (Shirling and Gottlieb 1966), the antibiotic sensitivity studies using glucose-yeast extract agar as the basal medium (Gordon and Mihm 1962), and the use of sole carbon sources following the protocol established after Stevenson (1967). The remaining tests on the strains were performed using media and methods described by Williams et al. (1983). Isolate AB-18-032T was also examined to determine its ability to produce acid from a range of sugars using the procedure described by Gordon et al. (1974).

Results

16S rRNA sequencing and DNA:DNA relatedness studies

It can be seen from Fig. 1 that isolate AB-18-032T falls into the Verrucosispora 16S rRNA gene clade, an association which is supported by all of the tree-making algorithms and by a 99% bootstrap value. The isolate shared its highest 16S rRNA similarity with the type strain of V. gifhornensis, namely 99.6%, a value which corresponded to 5 nucleotide (nt) differences at 1432 locations. The corresponding 16S rRNA similarities with the type strains of V. lutea and V. sediminis were 98.7% and 98.9%, values equivalent to 19 and 15 nt differences, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs10482-011-9651-5/MediaObjects/10482_2011_9651_Fig1_HTML.gif
Fig. 1

Neighbor-joining tree (Saitou and Nei 1987) based on nearly complete 16S rRNA gene sequences showing relationships between strain AB-18-032T and representatives of the family Micromonosporaceae. Asterisks indicate branches of the tree that were also found using the least-squares (Fitch and Margoliash 1967), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Kluge and Farris 1969) tree-making algorithms. F, P and L indicate that the corresponding nodes were also recovered in trees generated with the least-squares, maximum-parsimony or maximum-likelihood algorithms, respectively. The numbers at the nodes indicate the levels of bootstrap support (%) based on a neighbor-joining analysis of 1000 re-sampled datasets; only values above 50% are shown. T = type strain

The DNA:DNA relatedness values between isolate AB-18-032T and V. gifhornensis DSM 44237T averaged out at 64.0% using the renaturation method conducted at the DSMZ, a value below the 70% cut-off point recommended for the delineation of bacterial species according to Wayne et al. (1987). The ∆Tm between isolate AB-18-032T gDNA and isolate AB-18-032TV. gifhornensis hybrid DNA was 5°C (Fig. 2), a value which represents a DNA:DNA relatedness value of 65% (Gonzalez and Saiz-Jimenez 2005).
https://static-content.springer.com/image/art%3A10.1007%2Fs10482-011-9651-5/MediaObjects/10482_2011_9651_Fig2_HTML.gif
Fig. 2

Thermal denaturation of genomic DNA from Verrucosispora isolate AB-18-032T (solid black line) and the V. gifhornensis and Verrucosispora isolate AB-18-032T (solid black line) and the Verrucosispora isolate AB-18-032T/V. gifhornensis hybrid DNA mix (dotted line). The calculated ΔTm is 5°C

Chemotaxonomic, cultural, morphological and phenotypic characteristics

Isolate AB-18-032T contained menaquinones MK9(H4), MK-9 (H2) and MK-9 (H6) in a ratio of 60:1:6, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylserine as major polar lipids, but lacked mycolic acids. Using the fluorometric method, the G+C contents of the DNA of isolate AB-18-032T and V. gifhornensis DSM 44237T were recorded as 65.5% and 72%, respectively (Fig. 3). In addition, the isolate, like the three Verrucosispora type strains, produced a light to dark orange substrate mycelium on the ISP media (Table 1). All of the strains grew particularly well on tryptic-yeast extract and yeast extract-malt extract agars.
https://static-content.springer.com/image/art%3A10.1007%2Fs10482-011-9651-5/MediaObjects/10482_2011_9651_Fig3_HTML.gif
Fig. 3

Fluorimetric estimation of G+C mol% content in V. gifhornensis (dotted line) and Verrucosispora isolate AB-18-032T (solid line). The analysis was done according to Gonzalez & Saiz-Jimenez (2002). Tm was calculated from the minimum value of the slope tangent to the melting curve of fluorescence versus temperature and G+C mol% content using the formula %GC = 1.99 Tm−71.08

Table 1

Growth and cultural characteristics of the Verrucosispora strain

Medium

Strains

 

Isolate AB-18-032T

V. gifhornensis DSM 44337T

V. lutea YIM 013T

V. sediminis MS 426T

Glycerol-asparagine agar (ISP medium 5)

 Growth

++

++

+

++

 Colour of substrate mycelium

Light orange

Light orange

Light orange

Light orange

Inorganic salts-starch agar (ISP medium 4)

 Growth

+++

++

+++

+++

 Colour of substrate mycelium

Orange

Light orange

Light orange

Orange

Oatmeal agar (ISP medium 3)

 Growth

+++

++

+++

+++

 Colour of substrate mycelium

Orange

Orange

Orange

Orange

Peptone-yeast extract agar (ISP medium 6)

 Growth

+

+

+

+++

 Colour of substrate mycelium

Orange

Orange

Orange

Orange

Tryptic-yeast extract agar (ISP medium 1)

 Growth

+++

+++

+++

+++

 Colour of substrate mycelium

Dark brown orange

Orange

Light orange

Orange

Tyrosine agar (ISP medium 7)

 Growth

+

++

+++

++

 Colour of substrate mycelium

Orange

Orange

Light orange

Light orange

Yeast extract-malt extract agar (ISP medium 2)

 Growth

+++

+++

+++

+++

 Colour of substrate mycelium

Orange

Orange

Dark brown

Dark orange

None of the strains formed aerial hyphae or produced diffusible pigments

Key: +++, abundant growth; ++, moderate growth; +, poor growth

Identical results were obtained with the duplicated phenotypic tests. It can be seen from Table 2 that isolate AB-18-032T can be readily distinguished from all of the Verrucosispora type strains using a broad range of phenotypic properties. All of the organisms hydrolyzed allantoin, degraded l-tyrosine and xanthine, grew on D-fucose, mannose, dextran, α-methyl-D-glucoside, sorbose, trehalose, xylitol and xylose as sole carbon sources; on gelatin, l-leucine, Dl-methionine, monoethanolamine, l-proline, l-threonine and l-tyrosine as sole carbon and nitrogen sources; at 30 and 37°C, at pH 7–9 and in the presence of novobiocin (8 μg ml). In contrast, none of them hydrolyzed urea; degraded adenine, casein, cellulose, chitin, gelatin, hypoxanthine or tributyrin; used meso-inositol as a sole carbon source; l-asparagine as a sole nitrogen source; l-glutamic acid, l-norvaline or uric acid as sole carbon and nitrogen sources; grew at 4, 10 or 45°C, at pH 5 or 6 or in the presence of bacitracin (8 μg ml), impenem (8), streptomycin (4), tetracycline (8), vancomycin (2) or NaCl (7.5%, w/v).
Table 2

Phenotypic properties which distinguish between strain AB-18-022T and the type strains of Verrucosispora species

Characteristic

Strains

Isolate AB-18-032T

V. gifhornensis DSM 44337T

V. lutea YIM 013T

V. sediminis NS 426T

Spores

 Arrangement

Single, clusters

Single, pairs clusters*

Single, pairs, clusters*

Single, clusters*

 Ornamentation

Warty

Smooth, warty, hairy*

Smooth*

Warty*

Biochemical tests

 Aesculin hydrolysis

+

 Arbutin hydrolysis

+

+

 

 H2S production

+

+

 Nitrate reduction

+

+

Degradation tests

 Elastin

+

 Guanine

+

+

+

 Starch

+

+

+

 Uric acid

+

+

 Xylan

+

Growth on sole carbon sources at 1%, w/v

 Adonitol, d-arabitol, l-fucose, glycerol, lactose, mannitol, melibiose

+

+

+

 l-Arabinose

+

+

 Amygdalin, arbutin, erythritol, ethanol, maltitriose

+

+

 Cellobiose, galactose, rhamnose, ribose

+

+

 Dulcitol

+

+

 Raffinose

+

+

+

 Sorbitol

+

+

 Turanose

+

+

Growth on sole carbon and nitrogen sources

 l-Asparagine, l-ornithine, d-phenylalanine

+

+

+

 l-Glycine

+

+

+

 l-Histidine

+

+

+

Growth on sole nitrogen sources

 l-Alanine, l-phenylalanine

+

+

 l-Arginine

+

+

+

 l-Glutamic acid

+

+

 l-Histidine

+

+

+

 l-Methionine

+

+

 l-Serine

+

 l-Valine

+

+

+

Sensitivity to antibiotics (μg ml)

 Ampicillin (8), cyprofloxacin (4)

+

+

+

 Cephaloridine (4)

+

 Chloramphenicol (8)

+

+

 Clindamycin (8)

+

+

 Gentamicin (8)

+

 Lincomycin (8)

+

+

+

 Oxytetracycline (16)

+

 Rifamicin (16)

+

+

+

Tolerance tests

 Growth in presence of 5% w/v, NaCl

+

+

 pH range for growth

7–10

7–9

7–10

7–10

 DNA G+C content (mol %)

69.5a/70.9b

72.0a/70.0c

69.3d

66.8e

Key: +, positive; −, negative

* These data were taken from Rheims et al. (1998), Liao et al. (2009) and Dai et al. (2010), respectively

aDetermined in this study by thermal denaturation and fluorometry (Gonzalez and Saiz-Jimenez 2005)

bDetermined by whole-genome sequencing (Roh et al. 2011)

cDetermined by HPLC (Rheims et al. 1998)

dDetermined by HPLC (Liao et al. 2009)

eDetermined by termal denaturation and UV/Vis spectrometry (Dai et al. 2010)

Discussion

The present results confirm and extend those of the earlier study of Riedlinger et al. (2004) in showing that strain AB-18-032T has chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora (Rheims et al. 1998). It is also evident from the totality of the genotypic and phenotypic data that Verrucosispora strain AB-18-032T can be readily distinguished from the type and only representative strains of recognized Verrucosispora species. It is, therefore, proposed that strain AB-18-032T be recognized as a new Verrucosispora species, Verrucosispora maris sp. nov.

Description of Verrucosispora maris sp. nov.

Verrucosispora maris (mar’is. L. gen. n. maris of the sea, denoting the source of the isolate).

The description is based on data taken from this and previous studies (Riedlinger et al. 2004; Goodfellow and Fiedler 2010; Roh et al. 2011). Aerobic, Gram-positive, non-acid-fast actinomycete which forms an extensively branched, light to dark orange pigmented substrate mycelium on ISP media. Neither aerial hyphae nor spore vesicles are formed. Single or clusters of non-motile, black sessile spores with warty surfaces are produced on oatmeal agar. Grows at 20 and 37°C, at pH 5–10 and in the presence of 2.5%, w/v NaCl. Acid is produced from cellobiose, glucose, maltose, sucrose and xylose, but not from l-arabinose, arbutin, fructose, galactose, lactose, mannitol, raffinose, rhamnose, salicin or sorbitol. Glucose, starch, butan-1-ol, butan-1, 3 diol, butan-1, 4-diol, propan-2-ol and 1,2-propanediol are used as sole carbon sources (at 1%, w/v or 1%, v/v), as are citric acid, d-gluconic acid, p-hydroxybutyric acid, propionic acid, pyruvic acid, sodium-n- butyrate, suberic and l-tartaric acids (at 0.1%, w/v). Does not use adipic acid, glutaric acid, l-lactic acid, m-hydroxybenzoic acid, malonic acid, d-mandelic acid, oxalic acid, sebacic acid, sodium acetate, sodium benzoate or valeric acid as sole sources of carbon (at 0.1%, w/v). l-tryptophane is used as a sole nitrogen source. Additional phenotypic properties are cited either in the text or in Tables 1 and 2.

The peptidoglycan is rich in meso-diaminopimelic acid and contains N-glycolated muramic acid. Mannose and xylose are the major sugars in whole-organism hydrolysates and tetrahydrogenated menaquinones with nine isoprene units the predominant isoprenologue. The phospholipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylserine and unknown glyco-and phospholipids. The genome comprises a single circular chromosome (6.7 Mb) and a circular plasmid (58 kb). The G+C content of the chromosome is 70.9% and that of the plasmid 70.3%. Produces abyssomicins B, C, atrop-C, D, G and H and proximicin A.

The type and only strain, AB-18-032T (=DSM 45365T = NRRL B-24793T) was isolated from a sediment sample collected from the Sea of Japan at a depth of 289 m in August 1991.

Additional Verrucosispora strains need to be isolated, characterized, dereplicated and screened to determine the full potential of these organisms to synthesize novel secondary metabolites given the discoveries of atrop-abyssomicin from the type strain of V. maris (Keller et al. 2007a, b) and proximicins A, B and C from a Verrucosispora strain isolated from a sediment sample taken from the Raune Fjord, Norway (Schneider et al. 2008). It is clear from a culture-independent study that the genus Verrucosispora is underspeciated (Xie et al. 2011). These authors also found that verrucosisporae were widely distributed in the marine ecosystem, a result which tallies with the isolation of such organisms from fjord sediments (Bredholdt et al. 2008; Fiedler et al. 2008), mangrove soils (Hong et al. 2009; Liao et al. 2009), marine sediments (Riedlinger et al. 2004; Dai et al. 2010) and a marine sponge (Jiang et al. 2007). An effective taxon-specific procedure needs to be developed to isolate verrucosisporae from such habitats.

Acknowledgments

ATB and MG were supported by the UK Natural Environmental Research Council (grants NER/T/S/2000/00614 and NER/T/S/2000/00616), JEMS by the Biological and Biotechnological Research Council (BBSRC grant BB/EO17053/1) and TDZ by a Conselho Nacional de DesenvolvImento Cientifico e Tecnológico Fellowship to study in the UK (grant 201066/2009-2). ATB thanks The Leverhulme Trust for an Emeritus Fellowship. The authors are indebted to Professor Koki Horikoshi (JAMSTEC) for the sediment sample.

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© Springer Science+Business Media B.V. 2011