Download Thiorhodococcus fuscus sp. nov., isolated from a lagoon

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3938–3943
DOI 10.1099/ijsem.0.000517
Thiorhodococcus fuscus sp. nov., isolated from a
lagoon
K. V. N. S. Lakshmi,1 B. Divyasree,2 K. Sucharita,3 Ch. Sasikala2 and
Ch. V. Ramana1
Correspondence
Ch. Sasikala
[email protected]
Ch. V. Ramana
[email protected]
1
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central
University, Hyderabad 500046, India
2
Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology,
Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085, India
3
Government Women’s college, Railpet, Guntur, Andhra Pradesh 522001, India
A brown, moderately halophilic, photoautotrophic bacterium designated strain JA363T was
purified from a photoheterotrophic enrichment obtained from sediment from Chilika lagoon,
Odisha, India. Cells of the isolate were coccoid, motile by means of single polar flagellum and
Gram-stain-negative. Strain JA363T had an obligate requirement for NaCl and could tolerate up
to 7 % (w/v) NaCl. Strain JA363T had complex growth factor requirements. Internal
photosynthetic membranes were present as vesicles. Strain JA363T contained
bacteriochlorophyll a and spirilloxanthin series carotenoids with rhodopin as a major (.85 %)
component. C16 : 1v7c/C16 : 1v6c, C18 : 1v7c and C16 : 0 were the major fatty acids and
phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. Q8 was the
predominant quinone system of strain JA363T. The DNA G+C content was 64 mol%. The
highest 16S rRNA gene sequence similarity of strain JA363T was found with the type strains of
Thiorhodococcus kakinadensis (98.7 %), Thiohalobacter thiocyanaticus (98.2 %),
Thiophaeococcus fuscus (97.4 %) and Thiorhodococcus bheemlicus (96.3 %). However,
the phylogenetic trees generated firmly placed strain JA363T in the genus Thiorhodococcus,
which was further supported by phenotypic and chemotaxonomic evidence. Consequently,
strain JA363T is described as representing a novel species of the genus Thiorhodococcus
as Thiorhodococcus fuscus sp. nov. The type strain is JA363T (5KCTC 5701T5NBRC
104959T).
Purple sulfur bacteria are often found in anoxic sediments
and in shallow waters where there is the presence of both
reduced sulfur compounds and solar light (Rabold et al.,
2006). During our survey of one such water body at Chilika
lagoon, a marine habitat with neutral pH, an unexpectedly
rich diversity of cultivable purple anoxygenic phototrophs
was revealed. Among the several bacteria isolated, a moderately halophilic bacterium affiliated to the genus Thiorhodococcus was isolated from the sediment of Chilika lake at
Satpada, Odisha, India (GPS position: 198 399 E
858 229 N). The sample had a pH of 7.0 and salinity of
Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ,
neighbour-joining.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain JA363T is AM993157.
Five supplementary figures and a supplementary table are available
with the online Supplementary Material.
3938
3.5 %. Members of the genus Thiorhodococcus are spherical
to slightly ovoid, motile, multiply by binary fission and
show obligate phototrophy. These strains are morphologically similar to species of the genera Thiocystis and Thiophaeococcus, but differ from them in their 16S rRNA gene
sequences and several other aspects (Imhoff et al., 1998;
Anil Kumar et al., 2008). This truly marine genus is currently represented by five species with validly published
names; Thiorhodococcus minor (Guyoneaud et al., 1997),
Thiorhodococcus mannitoliphagus (Rabold et al., 2006),
Thiorhodococcus bheemlicus, Thiorhodococcus kakinadensis
(Anil Kumar et al., 2007) and Thiorhodococcus modestalkaliphilus (Sucharita et al., 2010). The name of a sixth
member ‘Thiorhodococcus drewsii’ (Zaar et al., 2003) has
not been validly published.
Strain JA363T was recovered from a photoheterotrophic
enrichment in a medium described previously (Shivali
et al., 2011; without supplementing sodium bicarbonate),
incubated at 2400 lx, 30 8C for 7 days in 45 ml fully
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A novel species of Thiorhodococcus
filled screw-capped bottles. Purification was done by
repeated streaking on agar slants (Lakshmi et al., 2011).
Purified cultures were grown in completely filled screw
cap test tubes (106100 mm) under the phototrophic conditions described above. Cultures were maintained on agar
slants or as lyophilized cultures preserved at 4 8C.
Genomic DNA was extracted and purified according to the
method of Marmur (1961) and the G+C content of the
DNA of strain JA363T, as determined by reverse-phase
HPLC, (Mesbah et al., 1989) was 64 mol%. Well isolated
colonies were used for 16S rRNA gene amplification by
using PCR master mix (GeNei) as described previously
(Subhash et al., 2013a). 16S rRNA gene sequencing was performed on a 3130xl ABI prism automated DNA sequencer
(Applied Biosystems) as described previously (Subhash
et al., 2013b). The 16S rRNA gene sequence of the strain
was identified by BLAST search analysis on the EzTaxon-e
server (Kim et al., 2012). The BLAST search analysis
(1438 nt) indicated that JA363T shared highest 16S rRNA
gene sequence similarity with the type strains of Thiorhodococcus kakinadensis (98.79 %), Thiohalobacter thiocyanaticus
(98.26 %), Thiophaeococcus fuscus (97.43 %) and Thiorhodococcus bheemlicus (96.32 %). The CLUSTAL W algorithm of
MEGA -5 (Tamura et al., 2011) software was used for phylogenetic analyses. Distances were calculated by using the
Kimura two-parameter method (Kimura, 1980) in a pair
wise deletion procedure. The neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP)
methods in the MEGA -5 software were used to reconstruct
phylogenetic trees and the combined phylogenetic tree
(NJ, ML, MP; Fig. 1) revealed that strain JA363T clustered
with the members of the genus Thiorhodococcus and was
not specifically affiliated with lineages formed by members
of the genera Thiophaeococcus and Thiohalobacter of the
family Chromatiaceae, as otherwise indicated by BLAST
search analysis. A more detailed analysis of the phylogenetic
tree (Fig. S1 available in the online Supplementary Material)
using some of the cloned sequences from NCBI further confirmed the affiliation of JA363T with the members of the
genus Thiorhodococcus.
Further characteristics of strain JA363T were studied in
detail as per recommended minimal standards (Imhoff &
Caumette, 2004) together with those of the closely related
strains Thiorhodococcus kakinadensis JCM 14150T
(5 JA130T) and Thiorhodococcus bheemlicus JCM 14149T
(5 JA132T). The taxonomic relationship between strain
JA363T and the closely related type strains was examined
using DNA–DNA hybridization which was performed
using a membrane filter technique (Tourova & Antonov,
1987), using a nick translation kit supplied by BRIT,
Jonaki, CCMB campus, Hyderabad. When strain JA363T
was radioactively labelled, the levels of DNA–DNA
reassociation with type strains of Thiorhodococcus kakinadensis and Thiorhodococcus bheemlicus were 45.5 % and
42.2 %, respectively. However, when the type strains of
Thiorhodococcus kakinadensis and Thiorhodococcus bheemlicus were labelled and used for DNA–DNA hybridization
with JA363T in the reciprocal reaction, the reassociation
values were 43.3 % and 41.7 %, respectively. Based on the
hybridization results, strain JA363T thus represents a
novel species according to recommendations for delineating a bacterial species (Stackebrandt & Goebel, 1994).
Morphological properties (cell shape, cell division, cell size,
motility) were observed under a phase-contrast microscope
(BH-2, Olympus). Flagellar position was determined using
a transmission electron microscope (H-7500, Hitachi). The
internal membrane structures were also viewed with a
transmission electron microscope, after the cells had been
processed as described by Hanada et al. (2002). Cells of
Escherichia coli ATCC 11775T (X80725)
Thiohalobacter thiocyanaticus DSM 21152T (FJ482231)
Phaeobacterium nitratireducens AK40T (HF968497)
98/100/100
99/100/100/96/-
95/99/87/85/52
54/-/65/59/61/63/61
0.02
78/70/75
‘Thiorhodococcus drewsii’ DSM 15006 (AF525306)
Thiorhodococcus kakinadensis JA130T (AM282561)
Thiorhodococcus fuscus JA363T (AM993157)
Thiophaeococcus mangrovi JA304T (AM748925)
Thiophaeococcus fuscus A633T (FN824810)
Thiorhodococcus bheemlicus JA132T (AM282559)
Thiorhodococcus mannitoliphagus WST (AJ971090)
Thiorhodococcus minor CE2203T (Y11316)
Thiorhodococcus modestalkaliphilus JA395T (AM993156)
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing phylogenetic relationships of strain JA363T
(1438 nt) with a few members of the family Chromatiaceae. The tree was computed with MEGA 5.2 software and rooted by
using Escherichia coli as the outgroup. GenBank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bootstrap percentages refer to NJ/MP/ML analysis. Bar, 0.02 nucleotide substitutions per position.
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3939
K. V. N. S. Lakshmi and others
strain JA363T were coccoid, 2–4 mm in diameter (Fig. S2),
multiplied by binary fission and were motile by means of
single polar flagella (Fig. S3A). Transmission electron
microphotographs of ultrathin sections of the strain
revealed the presence of a vesicular type of internal membrane structures (Fig. S3B).
Utilization of organic carbon compounds like formate,
propionate, butyrate, caproate, valerate, lactate, glycerol,
methanol and ethanol as carbon sources/electron donors
was tested at 0.1 % (v/v) along with NaHCO3 (0.1 %, w/v),
while for other substrates it was tested at 0.3 % (w/v) without
NaHCO3. Growth was measured turbidometrically at
660 nm. Good growth was observed only on limited substrates such as acetate, benzoate, caprylate, pyruvate, formate,
fructose, lactate, malate, succinate, tartrate and valerate.
Those which could not be utilized included ascorbate, aspartate, butanol, butyrate, caproate, citrate, crotonate, cysteine,
ethanol, fumarate, glutamate, glucose, glycerol, gluconate,
glycolate, 2-oxoglutarate, mannitol, methionine, methanol,
peptone, propanol, propionate, sorbitol, sucrose, starch and
yeast extract (Table 1).
For testing the utilization of sulfur sources, MgSO4 . 7H2O
was replaced with MgCl2 . 6H2O (0.2 %) and various sulfur
sources (sodium sulfide, sodium thiosulfate, thioglycolate,
cysteine, magnesium sulfate, sodium sulfate, sodium sulfite
all at 0.5 mM concentration) were added to the medium.
While JA363T utilized sulfite, sulfide, thioglycolate and
thiosulfate as sulfur sources, methionine, cysteine, elemental sulfur and sulfate did not support growth. Nitrogen
source requirements for growth were tested by replacing
ammonium chloride with different nitrogen sources at a
concentration of 7 mM. JA363T grew well with ammonium
chloride, glutamine and glutamate as nitrogen sources
while aspartate, nitrite, urea and nitrate did not support
growth. Vitamin requirement was tested by replacing
yeast extract with single vitamins and combinations of vitamins as growth factors, and JA363T showed a complex
growth requirement.
Table 1. Characteristics differentiating strain JA363T from the closely related species of the genus Thiorhodococcus
Strains: 1, JA363T; 2, Thiorhodococcus kakinadensis JCM 14150T; 3, Thiorhodococcus bheemlicus JCM 14149T. All data were obtained by the authors.
All the strains are coccoid–spherical, motile by polar flagella, divide by binary fission, have vesicles as internal cytoplasmic membrane structures, Q8
quinone system and polar lipids phosphatidylglycerol, phosphatidylethanolamine and aminophospholipid. Organic substrate utilization was tested
during photoheterotrophic growth. Pyruvate was utilized by all the strains. Crotonate, butyrate, propanol, ethanol, caproate and methionine were
not utilized by any of the strains. n, Niacin; b, biotin; p, pantothenate; y.e., yeast extract; +, good growth; 2, no growth; (+), weakly positive
growth; Rp, rhodopin; Ly, lycopene; UL1–6, unknown polar lipids.
Characteristic
1
Cell diameter (mm)
2–3
Source of isolation
Sediment of lagoon
Colour of cell suspensions
Brown
Growth factors
y.e.
pH optimum (range)
7.5 (6.0–8.5)
Temperature optimum (range) (8C)
30 (25–35)
NaCl optimum (range) (w/v, %)
2–4 (0.5–7)
Photolithoautotrophy
+
Major carotenoid
Rp
Polar lipids
UL1,2,3,4
Substrates used as carbon/electron donors for growth
Acetate
+
Benzoate
+
Casamino acids
+
Caprylate
+
Formate
+
Fructose
+
Glucose
2
Glycerol
2
Glycolate
2
Lactate
+
Malate
+
Propionate
2
Succinate
+
Tartrate
+
Valerate
+
DNA G+C content (HPLC) (mol%)
64
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2
3
3–5
Water of marine aquaculture pond
Purple–violet
n, b, p
7.5 (6.5–8.0)
25–30 (25–35)
1–2 (0.5–5)
2
Ly
UL1,3,4
4–6
Marine tidal waters
Purple–violet
–
7.0–7.5 (6.5–8.0)
25–30 (25–35)
1–3 (0.5–6)
2
Ly
UL1,2,3,4,5,6
(+)
2
2
2
2
2
2
2
2
(+)
+
+
+
2
2
60
+
2
+
2
2
2
+
+
+
+
2
2
2
2
(+)
65
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A novel species of Thiorhodococcus
Chemotrophic growth was determined by growing the
cultures in Erlenmeyer flasks placed in an orbital shaker
(in the dark) at 30 8C. It was observed that the JA363T
was able to grow photoorganoheterotrophically [anaerobic,
light (2400 lx) with pyruvate (0.03 %, w/v) as a carbon
source/electron donor] and photolithoautotrophically
[anaerobic, light (2400 lx), Na2S . 9H2O/Na2S2O3 . 5H2O
(1 mM/5 mM) and NaHCO3 (0.1 %,w/v)]. Chemolithoautotrophy [aerobic, dark, Na2S2O3 . 5H2O (5 mM) and
NaHCO3 (0.1 %, w/v)] and chemoorganoheterotrophy
[aerobic (respiration)/anaerobic (fermentation), dark and
pyruvate (0.3 %, w/v)] could not be demonstrated in
strain JA363T. JA363T had an obligate requirement of
NaCl for growth, where optimum growth occurred at 2–
4 %, but it could tolerate up to 7 % NaCl (range 0.5–7 %,
w/v). The pH range for growth of strain JA363T was 6.0–
8.5 with the optimum being pH 7.5. JA363T was a mesophile, growing optimally at 30 8C (range 25–35 8C).
Phototrophically grown cells were brown, while all the
other five members of the genus Thiorhodococcus are redshaded. The in vivo absorption spectrum of intact cells of
strain JA363T as measured using a Genesys2 spectrophotometer (Spectronic) in sucrose solution (Trüper & Pfennig, 1981) exhibited maxima at 374, 491, 530, 590, 803
and 857 nm (Fig. S4) indicating the presence of bacteriochlorophyll a. Carotenoid composition of strain JA363T
as determined by C18-HPLC (eluted with acetonitrile/
methanol/ethyl acetate; 5 : 4 : 1, v/v; flow rate 1 ml min21;
absorption at 450 nm) using a photodiode array detector
indicated the presence of rhodopin (.80 %) as the major
carotenoid while the type strains of Thiorhodococcus kakinadensis and Thiorhodococcus bheemlicus contained lycopene (.85 %); both the carotenoids belong to the
spirilloxanthin series.
For fatty acid analysis, cells were harvested by centrifugation (10 000 g for 15 min at 4 8C) on reaching a cell density of 70 % of the maximum optical density (100 %50.9;
OD660) and the pellet was used for analysis. Cellular fatty
acids were methylated, separated and identified according
to the instructions for the Microbial Identification
System (Microbial ID; MIDI 6.0 version; method,
RTSBA6) (Sasser, 1990) (www.midi-inc.com), which was
outsourced to Royal Research Laboratories, Secunderabad,
India. The fatty acid profile of JA363T was found to be in
congruence with those of the type strains of species of
the genus Thiorhodococcus with major proportions of
C16 : 1v7c/C16 : 1v6c (48.2 %), C18 : 1v7c/C18 : 1v6c (19.1 %)
and C16 : 0 (18.0 %) and minor amounts of C12 : 0 (5 %),
C14 : 0 (3.7 %), iso-C17 : 1v9c (1.4 %) and C18 : 1v5c (1.1 %;
Table S1). However, strain JA363T differed from the
other closely related type strains of the genus in the presence of iso-C17 : 1v9c and absence of C18 : 1v9c, anteisoC15 : 0, C17 : 0 and anteiso-C17 : 0. Members of the genera
Thiophaeococcus, Chromatium, Thiocapsa and Thiocystis,
which belong to the family Chromatiaceae, have also been
reported to have similar fatty acid profiles with C18 : 1,
C16 : 1 and C16 : 0 as major fatty acids (Divyasree et al.,
http://ijs.microbiologyresearch.org
2014; Imhoff & Bias-Imhoff, 1995); however, the position
of bonds was not indicated for the latter three genera.
Polar lipids were extracted from 1 g freeze-dried cells
with methanol/chloroform/saline (2 : 1 : 0.8, by vol.) as
described by Kates (1986). Lipids were separated using
silica gel TLC (Kieselgel 60 F254, Merck) by two-dimensional chromatography using chloroform/methanol/
water (65 : 25 : 4, by vol.) in the first dimension and
chloroform/methanol/acetic acid/water (80 : 12 : 15 : 4, by
vol.) in the second dimension (Tindall, 1990a, b; Oren
et al., 1996). The total lipid profile was visualized by spraying with 5 % ethanolic molybdophosphoric acid and was
further characterized by spraying with ninhydrin (specific
for amino groups), molybdenum blue (specific for phosphates), Dragendorff (quaternary nitrogen) or a-naphthol
(specific for sugars) (Kates, 1972; Oren et al., 1996). The
polar lipid profile of strain JA363T was observed to be in
consonance with those of closely related type strains of
species of the genus Thiorhodococcus, which contained
phosphatidylglycerol and phosphatidylethanolamine as
the major lipids with minor amounts of an aminophospholipid and four unidentified lipids that, based on
their staining behaviour, did not contain either a sugar
moiety or a phosphate or quaternary ammonium or an
amino group (Fig. S5). The type strain of Thiorhodococcus
bheemlicus additionally contained minor amounts of two
other unidentified lipids. The lipid profile of species of
the genus Thiorhodococcus (strain JA363T, Thiorhodococcus
kakinadensis JCM 14150T and Thiorhodococcus bheemlicus
JCM 14149T) differed from those of the species of the
genus Thiophaeococcus (Divyasree et al., 2014) by the
absence of diphosphatidylglycerol, phosphotidylcholine
and phospholipids. Members of the genus Thiorhodococcus
analysed in the present study additionally lacked glycolipids and cardiolipins which are otherwise reported to
occur in the members of Chromatiaceae such as members
of the genera Chromatium, Thiocapsa and Thiocystis
(Imhoff et al., 1982). Quinones were extracted with a
chloroform/methanol (2 : 1, v/v) mixture, purified by
TLC and analysed by HPLC (Imhoff, 1984; Hiraishi &
Hoshino, 1984; Hiraishi et al., 1984) and ubiquinone 8
(.90 %) was observed to be the predominant quinone
system in strain JA363T and the type strains of Thiorhodococcus kakinadensis and Thiorhodococcus bheemlicus. This
matched the quinone system found in the members of
the genus Thiophaeococcus (Divyasree et al., 2014) and
other phototrophic members of the family Chromatiaceae
(Imhoff & Bias-Imhoff, 1995).
The phylogenetic distance of strain JA363T from type
strains of closely related species, which is also reflected in
a number of distinctive phenotypic (colour, growth
factor requirement, organic substrate utilization, photolithoautotrophic growth; Table 1) and chemotaxonomic
traits (polar lipids, carotenoids; Fig. S5, Table 1), justifies
the description of strain JA363T as representing a novel
species of the genus Thiorhodococcus for which the name
Thiorhodococcus fuscus sp. nov. is proposed.
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3941
K. V. N. S. Lakshmi and others
Description of Thiorhodococcus fuscus sp. nov.
Thiorhodococcus fuscus (fus9cus. L. masc. adj. fuscus tawny).
Individual cells are coccoid, 2–4 mm in diameter, Gramstain-negative, motile by means of a single polar flagellum,
multiply by binary fission and have internal photosynthetic
membranes of the vesicular type. Photoorganoheterotrophy and photolithoautotrophy are the growth modes. Chemolithoautotrophy,
chemoorganoheterotrophy
and
fermentative growth are absent. Phototrophically grown
cultures are brown. The in vivo absorption spectrum of
intact cells in sucrose exhibits maxima at 374, 491, 530,
590, 803 and 857 nm. Bacteriochlorophyll-a and rhodopin
of the spirilloxanthin series are present as major pigments.
NaCl is obligate for growth; optimal growth is observed at
2–4% NaCl (range 0.5–7%, w/v). Optimum pH and temperature for growth are pH 7.5 (range pH 6.0–8.5) and
30 uC (range 25–35 uC), respectively. Acetate, benzoate,
caprylate, pyruvate, formate, fructose, lactate, malate, succinate, tartrate and valerate are good carbon sources/electron donors for growth. Assimilates sulfite, sulfide,
thioglycolate and thiosulfate, while ammonium chloride,
glutamine and glutamate are used as nitrogen sources.
Has complex growth requirement. Major fatty acids are
C16:1v7c/C16:1v6c, C18:1v7c/C18:1v6c and C16:0 with
minor amounts of C12:0, C14:0, iso-C17:1v9c and
C18:1v5c. Phosphatidylglycerol, phosphatidylethanolamine, an aminophospholipid and four unknown lipids
are the polar lipids. Q8 is the predominant quinone.
The type strain, JA363T (5KCTC 5701T5NBRC 104959T),
was isolated from sediment of Satapada, Chilika lagoon,
Odisha, India. The DNA G+C content of the type strain
is 64 mol% (determined by HPLC).
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Acknowledgements
K. V. N. S. L. thanks University Grants Commission for a D. S. Kothari
Postdoctoral fellowship and B. D. thanks Department of Science and
Technology for an Inspire fellowship.
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