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ORIGINAL PAPER
Streptomyces klenkii sp. nov., isolated from deep marine
sediment
Aysel Veyisoglu •Nevzat Sahin
Received: 23 September 2014 / Accepted: 30 October 2014
ÓSpringer International Publishing Switzerland 2014
Abstract A novel actinomycete strain, designated
S2704
T
, was isolated from a deep sediment sample,
collected from the southern Black Sea coast, Turkey
and characterized using a polyphasic approach. Com-
parison of 16S rRNA gene sequences showed that
strain S2704
T
is a member of the genus Streptomyces,
exhibiting highest similarity with Streptomyces spe-
cialis GW 41-1564
T
(97.1 %). Strain S2704
T
could be
distinguished from all other Streptomyces species with
validly published names by sequence similarity values
less than 97.0 %. The isolate was found to contain LL-
diaminopimelic acid as the diagnostic diamino acid.
Whole cell hydrolysates were found to contain
glucose, mannose and ribose. The polar lipids were
identified as diphosphatidylglycerol, phosphatidyleth-
anolamine, phosphatidylmethylethanolamine, phos-
phatidylinositol, glycophospholipid, a phospholipid
and two unknown glycolipids. The predominant
menaquinones were identified as MK-9(H
8
) and
MK-10(H
6
). The major fatty acids were found to be
anteiso-C
17:0
,iso-C
16:0
and anteiso-C
15:0
. The G?C
content of the DNA was determined to be 72.2 mol%.
On the basis of its phenotypic and molecular proper-
ties, strain S2704
T
is considered to be a novel species
of the genus Streptomyces, for which the name
Streptomyces klenkii sp. nov. is proposed. The type
strain is S2704
T
(=DSM 42104
T
=KCTC 29202
T
).
Keywords Streptomycetaceae Streptomyces
klenkii Polyphasic taxonomy Southern Black Sea
coast
Introduction
The Black Sea is a semi-enclosed basin with at least
three different microbial ecosystems. While the sur-
face layer of the Black Sea is rich in oxygen and driven
by oxygenic photosynthetic processes, the aphotic
deep layer is anoxic and sulfidic. There is a suboxic
zone (c. 50 m thick) at the boundary between the oxic
and anoxic layers (Murray et al. 1995). Many different
metabolisms of the same type exist in the suboxic
zone, which has \10 lM oxygen and undetectable
hydrogen sulfide (Kuypers et al. 2003; Manske et al.
2005; Lam et al. 2007; Oakley et al. 2007; Clement
et al. 2009). The Black Sea is not only an ideal place to
Electronic supplementary material The online version of
this article (doi:10.1007/s10482-014-0325-y) contains supple-
mentary material, which is available to authorized users.
A. Veyisoglu N. Sahin (&)
Department of Biology, Faculty of Art and Science,
Ondokuz Mayis University, Kurupelit,
55139 Samsun, Turkey
e-mail: nsahin@omu.edu.tr
Present Address:
A. Veyisoglu
Department of Molecular Biology and Genetics, Faculty
of Sciences, Canik Basari University,
55080 Samsun, Turkey
123
Antonie van Leeuwenhoek
DOI 10.1007/s10482-014-0325-y
study microbial community dynamics under oxic and
anoxic conditions but also provides rich sources of
both chemical and actinobacterial diversity (Xu et al.
2012; Veyisoglu et al. 2013; Veyisoglu and Sahin
2014). In this context, marine actinomycetes, partic-
ularly the members of the genus Streptomyces,
represent a major resource for biotechnological search
and discovery (Fiedler et al. 2005; Jensen et al. 2005).
In our continuing research on culturable actinobacte-
rial biodiversity of Black Sea deep sediment, a
putatively novel Streptomyces strain, S2704
T
, was
isolated from a sediment sample from Melet River
offshore, Turkey. On the basis of a polyphasic
taxonomic study, it is proposed here that isolate
S2704
T
be classified as the type strain of Streptomyces
klenkii sp. nov.
Materials and methods
Isolation and maintenance of the organism
Strain S2704
T
was isolated from a sediment sample
collected by a dredge at a depth of 45 m, along Melet
River offshore of the southern Black Sea coast, Turkey,
in August 2010 (GPS coordinates for the sampling site
are 41°00.3530N and 37°57.4890E). Sediment samples
were subsampled aseptically and stored at -20 °C
until use. Strain S2704
T
was isolated from the sediment
sample by using non-sporulating medium (20 g casa-
minoacids, 20 g soluble starch, 4 g yeast extract, 15 g
agar, pH 7.2–7.4 per litre distilled water; Sanglier et al.
1992), supplemented with filter sterilised rifampicin
(5 lgml
-1
) and nystatin (50 lgml
-1
) incubated at
28 °C for 30 days. The strain was isolated as a pure
culture and maintained on yeast malt extract agar
(International Streptomyces Project medium 2 [ISP 2];
Shirling and Gottlieb 1966) slopes at room temperature
and stored in glycerol suspensions (20 %, v/v) at
-20 °C. Streptomyces specialis GW 41-1564
T
was
obtained from DSMZ (German Collection of Micro-
organisms and Cell Cultures) and cultured under the
same conditions for comparative testing.
Morphological, cultural and physiological
characteristics
Cultural characteristics were investigated on ISP 2,
oatmeal agar(ISP 3), inorganic salts-starch agar (ISP 4),
glycerol-aspargine agar (ISP 5), peptone-yeast extract-
iron agar (ISP 6), tyrosine agar (ISP 7) (Shirling and
Gottlieb 1966), modified Bennett’s agar (MBA; Jones
1949), Czapek’s agar (Waksman 1967), nutrient agar
(Waksman 1967) and tryptic soyagar (TSA; Difco). The
degree of growth, aerial mycelium and pigmentation
were recorded after 14 days of incubation at 28 °C.
National Bureau of Standards (NBS) colour name charts
(Kelly 1964) were used for determining colour desig-
nation and names. Colony morphology and micromor-
phological properties of strain S2704
T
were determined
by examining gold coated dehydrated specimens of
14-day cultures from ISP 4 medium using a JEOL JSM
6060 instrument. Growth at different temperatures (4,
10, 20, 28, 37, 45 and 50 °C), pH 4.0–12.0 (at intervals
of 1.0 pH unit) and in the presence of NaCl (0–10 %;
w/v) was determined on yeast extract-malt extract (ISP
2). KH
2
PO
4
/HCl, KH
2
PO
4
/K
2
HPO
4
and K
2
HPO
4
/
NaOH buffer systems were used to maintain the pH
values of the media, while an NaCl buffer system was
used for NaCl tolerance. Anaerobic growth was tested
using Anaerocult A (Merck, Darmstad, Germany).
Established methods were used to determine whether
the strains degraded Tweens 40 and 80 (Nash and Krent
1991); the remaining degradation tests were examined
using methods described by Williams et al. (1983).
Carbon source utilization was tested using carbon
source utilization (ISP 9) medium (Shirling and Gottlieb
1966) supplemented with a final concentration of 1 %
(w/v) of the tested carbon sources. Nitrogen source
utilization was examined using the basal medium
recommended by Williams et al. (1983)supplemented
with a final concentration of 0.1 % (w/v) of the tested
nitrogen sources. Antimicrobial activity of strain
S2704
T
to inhibit the growth of nine microorganisms,
includingGram-positive and Gram-negative bacteria as
well as fungi, was observed using an overlay technique
described by Williams et al. (1983). Spot-inoculated
colonies on modified Bennett’s agar plates were
inverted over 2 ml chloroform for 40 min. Killed
colonies were overlaid with 5–7 ml sloppy modified
Bennett’s broth inoculated with the test organisms.
Zones of inhibition were scored as positive results after
24 h at 37 °C.
Chemotaxonomic characterization
Biomass for chemical studies was prepared by grow-
ing the strain in N-Z-Amine broth (DSMZ Medium
Antonie van Leeuwenhoek
123
554) in Erlenmayer flasks for 14 days at 28 °C. Cells
were harvested by centrifugation, washed twice in
distilled water and re-centrifuged and freeze-dried.
Standard procedures were used to determine the
isomers of diaminopimelic acid and diagnostic
whole-cell sugars (Hasegawa et al. 1983). Cellular
fatty acids were extracted, methylated and separated
by gas chromatography using an Agilent Technologies
6,890 N instrument, fitted with an autosampler and a
6,783 injector, according to the standard protocol of
the Sherlock Microbial identification (MIDI) system
(Sasser 1990;Ka
¨mpfer and Kroppenstedt 1996); the
fatty acid methyl ester peaks were quantified using
TSBA 5.0 database. Polar lipid and respiratory
quinone analyses were carried out by the Identification
Service of the DSMZ, Braunschweig, Germany.
Respiratory quinones were extracted from 100 mg of
freeze dried cells based on the two stage method
described by Tindall (1990a,b). Respiratory quinones
were separated into their different classes (menaqui-
nones and ubiquinones) by thin layer chromatography
on silica gel (Macherey–Nagel Art. NO. 805 023),
using hexane: tert-butylmethylether (9:1 v/v) as sol-
vent. UV absorbing bands corresponding to menaqui-
nones or ubiquinones were removed from the plate and
further analysed by HPLC. This step was carried out
on a LDC Analytical (Thermo Separation Products)
HPLC fitted with a reverse phase column (Macherey–
Nagel, 2 9125 mm, 3 lm, RP18) using methanol as
the eluant. Respiratory lipoquinones were detected at
269 nm. The DNA G?C content of the isolate was
determined following the procedure of Gonzalez and
Saiz-Jimenez (2005).
DNA preparation, amplification and determination
of 16S rRNA gene sequence
Genomic DNA extraction, PCR-mediated amplifica-
tion and sequencing of the 16S rRNA gene were
performed as described by Chun and Goodfellow
(1995), using an ABI PRISM 3730 XL automatic
sequencer. The resultant 16S rRNA gene sequence
was aligned with corresponding sequences of repre-
sentative type strains of the genus Streptomyces
(retrieved from the EzTaxon-e server; Kim et al.
2012) by using CLUSTAL W in MEGA5 (Tamura
et al. 2011). Phylogenetic analysis was carried out by
using three tree-making algorithms: neighbour-joining
(Saitou and Nei 1987), maximum-likelihood (Felsenstein
1981) and maximum parsimony (Fitch 1971) with
MEGA version 5.0 (Tamura et al. 2011). Evolutionary
distances were calculated using model of Jukes and
Cantor (1969). Topologies of the resultant trees were
evaluated by bootstrap analyses (Felsenstein 1985)
based on 1,000 resamplings.
Results and discussion
Morphological observation of a 14 days old culture of
strain S2704
T
grown on ISP 4 (inorganic salt starch)
agar revealed that strain S2704
T
has typical charac-
teristics of members of the genus Streptomyces. Aerial
and substrate mycelia were observed to be well-
developed without fragmentation. The aerial mycelia
produced long spore chains which were spiral or
retinaculum-apertum (looped at the top) in nature
(Fig. 1). The spore surface was observed to be smooth.
Strain S2704
T
was found to grow well on ISP 2, 3 and
4 but no growth was observed on ISP 5, 6, 7, modified
Bennett’s, Czapek’s, nutrient and tryptic soy agar. No
diffusible pigment was detected on any of the tested
media. Melanoid pigments were not produced on ISP 6
or ISP 7 medium. White aerial mycelium was
observed to be produced on ISP 3, 4 and 7 but no
aerial mycelium was formed on any of the other tested
media. The morphological, physiological and bio-
chemical properties of strain S2704
T
are given in
Table 1, Supplementary Table S1 and the species
description.
Fig. 1 Scanning electron micrograph of strain S2704
T
grow on
Inorganic salts-starch medium (ISP medium no. 4) at 28 °C for
14 days
Antonie van Leeuwenhoek
123
Cells of strain S2704
T
were observed to contain LL-
diaminopimelic acid as diamino acid. Whole-cell
hydrolysates were found to contain glucose, mannose
and ribose. Strain S2704
T
was found to exhibit a
quinone system with the predominant compounds
MK-9(H
8
) (65.0 %) and MK-10(H
6
) (20.0 %). Minor
amounts of MK-9(H
6
) (3.0 %), MK-11 (1.0 %) and an
unidentified component were also detected. The polar
lipid profile was found to consist of diphosphatidyl-
glycerol, phosphatidylethanolamine, phosphatidyl-
methylethanolamine, phosphatidylinositol, glyco-
phospholipid, a phospholipid and two unknown gly-
colipids (i.e. phospholipid pattern type 2 sensu
Lechevalier et al. 1977; Supplementary Fig. S1). The
major fatty acids found were identified as anteiso-
C
17:0
(43.3 %), iso-C
16:0
(26.9 %) and anteiso-C
15:0
(13.5 %) (Supplementary Table S2). The G?C con-
tent of the DNA was determined to be 72.2 mol%.
The almost complete 16S rRNA gene sequence of
strain S2704
T
(1,478 nt) was compared with those of
members of the genus Streptomyces and related taxa.
Strain S2704
T
was found to share highest 16S rRNA
gene sequence similarity with Streptomyces specialis
GW 41-1564
T
(97.1 %), Streptomyces hainanensis
YIM 47672
T
(96.5 %), Streptomyces avicenniae
MCCC 1A01535
T
(96.4 %) and \96 % to that of
other Streptomyces species. The phylogenetic tree
based on the 16S rRNA gene sequences of strain
S2704
T
and the most closely related type strains of
species of the genus Streptomyces is shown in Fig. 2.
Topologies of phylogenetic trees built using the
maximum-likelihood and maximum-parsimony algo-
rithms were similar to the tree constructed by neigh-
bor-joining analysis (Supplementary Fig. S2 and S3).
Strain S2704
T
was found to show antimicrobial
activity against Staphylococcus aureus ATCC
33862, Enterobacter aerogenes NRRL B-3567 and
Aspergillus parasiticus NRRL-465
T
, but not against
Aspergillus flavus NRRL-1957
T
,Candida albicans
ATCC 10231
T
,Escherichia coli ATCC 25922,
Pseudomonas aeruginosa NRRL B-2679, Bacillus
subtilis NRRL B-209 and Bacillus licheniformis
NRRL B-1001.
It is evident from the combination of genotypic and
phenotypic data that strain S2704
T
can be distin-
guished from all species of Streptomyces with validly
published names. It shares less than 97.1 % 16S rRNA
gene sequence similarity with other Streptomyces
species, and it could be clearly distinguished from S.
specialis GW 41-1564
T
based on physiological and
biochemical characteristics as summarized in Table 1.
Furthermore, the predominant menaquinones of strain
S2704
T
are MK-9(H
8
) and MK-10(H
6
), which are
significantly different from those of S. specialis GW
Table 1 Phenotypic properties of strain S2704
T
and S. spe-
cialis, the most closely related type strain of the genus
Streptomyces
Characteristics 1 2
Biochemical tests
Arbutin hydrolysis -?
pH tolerence
6-?
11 ?-
Temperature
45 °C?-
NaCl (%, w/v)
2.0 -?
3.0 -?
Carbon source utilization (1.0 %, w/v)
Myo-inositol -?
D-arabinose -?
D-sorbitol -?
D-ribose -?
Dextrin -?
Lactose -?
Xylitol -?
Nitrogen source utilization (0.1 %, w/v)
L-tyrosine -?
Strains were positive for degradation starch (1 %), tween 40
(1.0 %) and tween 80 (1.0 %), ability of growth at adonitol, D-
mannitol and sucrose as sole carbon sources (1.0 %), ability of
growth at L-arginine as sole nitrogen sources (0.1 %), and
growth at pH: 7, pH: 8, 9 and 10, temperature 28 °C and 37 °C,
0 % and 1 % NaCl concentrations. But negative for
degradation adenin (0.5 %), casein (1 %), elastin (0.3 %),
guanine (0.5 %), hypoxanthine (0.4 %), xanthine (0.4 %) and
xylan (0.4 %), for hydrolysis of allantoin, nitrate reduction,
urea, ability of growth at L-arabinose, D-cellobiose, D-fructose,
D-galactose, D-mannose, D-melezitose, L-rhamnose, maltose,
inulin, dextran, L-glutamic acid, L-sorbose, xylose and succinic
acid (0.1 %) as sole carbon sources (1.0 %), ability of growth
at alpha-isoleucine, glycine, L-alanine, L-cysteine, L-histidine,
L-hydroxyproline, L-methionine, L-phenylalanine, L-proline, L-
serine, L-threonine and L-valine as sole nitrogen sources
(0.1 %), and growth at pH 4, 5, 6 and 12, temperature 4, 10,
20 and 50 °C, 4, 5, 6, 7, 8, 9 and 10 % NaCl concentrations.
All data were obtain in this study
Strains 1 S2704
T
,2 S. specialis DSM 41924
T
.?positive,
-negative
Antonie van Leeuwenhoek
123
41-1564
T
[MK-10(H
4
) and MK-10(H
6
)]. In addition,
the isolate exhibited quantitative differences in the
fatty acid profile, notably in the amount of anteiso-
C
17:0
.
It can be concluded that strain S2704
T
represents a
novel species of the genus Streptomyces. Therefore it
is proposed that the organism be classified in the genus
as Streptomyces klenkii sp. nov.
Description of Streptomyces klenkii sp. nov
Streptomyces klenkii (klenk’i.i. N.L. gen. masc. n.
klenkii of Klenk, named in honour of Hans-Peter
Klenk for his contributions to bacterial systematics).
Aerobic, Gram-stain positive, non-motile, non-acid
alcohol-fast actinomycete which forms branched sub-
strate hyphae and aerial mycelium that differentiates
S. endus NRRL 2339T(AY999911)
S. demainii NRRL B-1478T(DQ334782)
S. sporocinereus NBRC 100766T(AB249933)
S. hygroscopicus subsp. hygroscopicus NRRL 2387T (AB231803)
S. iranensis HM 35T(FJ472862)
S. albiflaviniger NRRL B-1356T(AJ391812)
S. yogyakartensis NBRC 100779T(AB249942)
S. javensis NBRC 100777T(AB249940)
S. violaceusniger NBRC 13459T(AB184420)
S. rhizosphaericus NBRC 100778T(AB249941)
S. samsunensis M1463T(EU077190)
S. melanosporofaciens NBRC 13061T(AB184283)
S. castelarensis DSM 40830T(AY508511)
S. sporoclivatus NBRC 100767T(AB249934)
S. antimycoticus NBRC 12839T(AB184185)
S. cacaoi subsp. cacaoi NBRC 12748T(AB184115)
S. qinglanensis 172205T(HQ660227)
S. flocculus NBRC 13041T(AB184272)
S. rangoonensis LMG 20295T(AJ781366)
S. gibsonii NBRC 15415T(AB184663)
S. almquistii NBRC 13015T(AB184258)
S. pharmamarensis PM267T(FR693804)
S. aidingensis TRM46012T(HQ286045)
Streptomyces klenkii S2704T(KF656772)
S. burgazadensis Z1R7T(KF793919)
S. sedi YIM 65188T(EU925562)
S. zhaozhouensis NEAU-LZS-5T(KC304791)
S. specialis GW 41-1564T(AM934703)
S. hoynatensis S1412T(JQ582693)
S. avicenniae MCCC 1A01535T(EU399234)
S. hainanensis YIM 47672T(AM398645)
S. mayteni YIM 60475T(EU200683)
Micrococcus lylae DSM 20315T(X80750)
100*
100*
56
69
100
81*
84* 80*
87*
64
98
71*
99*
99 84
89
98*
58*
98
0.01
*
*
Fig. 2 Neighbour-joining tree (Saitou & Nei 1987) based on
almost complete 16S rRNA gene sequences showing the
position of strain S2704
T
amongst its phylogenetic neighbours.
Asterisks indicate branches of the tree that were also recovered
using the maximum-likelihood (Felsenstein 1981) and maxi-
mum-parsimony (Kluge & Farris 1969) tree-making algorithms.
Micrococcus lylae DSM 20315
T
(X80750) was used as an
outgroup. Numbers at the nodes indicate the levels of bootstrap
support (%); only values C50 % are shown. GenBank accession
numbers are given in parentheses. Bar 0.01 substitutions per site
Antonie van Leeuwenhoek
123
into spiral or retinaculum-apertum spore chains with
smooth-surfaced spores. Growth occurs at pH 7.0–11
and at 28–45 °C but not at pH 4.0, 5.0, 6.0 and 12 and
at temperatures of 4, 10, 20 and 50 °C. Optimum
growth occurs on N-Z-amine medium (DSM medium
554) at 28–37 °C and pH 8.0. NaCl is not required for
optimum growth. Positive for anaerobic growth.
Arbutin, allantoin and urea are not hydrolysed. Nitrate
reduction is negative. Starch, tween 40 and 80 are
degraded but adenine, casein, elastin, guanine, hypo-
xanthine, xanthine and xylan are not. Utilizes adonitol,
D-mannitol and sucrose as sole carbon sources but not
myo-inositol, D-arabinose, L-arabinose, D-cellobiose,
D-fructose, D-sorbitol, D-galactose, D-mannose,
D-melezitose, D-ribose, dextrin, dextran, xylitol,
xylose, L-rhamnose, lactose, L-glutamic acid, maltose,
succinic acid, inulin and L-sorbose. Utilizes L-arginine
as sole nitrogen source but not alpha-isoleucine,
L-cysteine, glycine, L-alanine, L-phenylalanine, L-ser-
ine, L-valine, L-tyrosine, L-histidine, L-proline,
L-hydroxyproline, L-methionine, and L-threonine.
The predominant menaquinones are MK-9(H
8
) and
MK-10(H
6
) with minor amounts of MK-9(H
6
) and
MK-11. The polar lipid profile contains diphosphat-
idylglycerol, phosphatidylethanolamine, phosphati-
dylmethylethanolamine, phosphatidylinositol, glyco-
phospholipid, a phospholipid and two unknown glycolipids.
The major fatty acids are anteiso-C
17:0
,iso-C
16:0
and
anteiso-C
15:0
. The G?C content of the genomic DNA
of the type strain is 72.2 mol%.
The type strain, S2704
T
(=DSM 42104
T
=KCTC
29202
T
) was isolated from a sediment from the
southern Black Sea coast, Turkey. The GenBank
accession number for the 16S rRNA gene sequence of
Streptomyces klenkii S2704
T
is KF656772.
Acknowledgments This research was supported by Ondokuz
Mayis University (OMU), project no. PYO. FEN. 1901.12.014.
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