Indibacter alkaliphilus gen. nov., sp. nov., an alkaliphilic bacterium isolated from a haloalkaline lake.

Abstract

A novel Gram-stain-negative, rod-shaped, non-motile bacterium, strain LW1(T), was isolated from a water sample collected at a depth of 3.5 m from Lonar Lake, Buldhana district, Maharashtra, India. The cell suspension was reddish-orange due to the presence of carotenoids. Strain LW1(T) was positive for catalase, oxidase, ornithine decarboxylase and lysine decarboxylase and negative for gelatinase, urease and lipase. Fatty acids were dominated by branched-chain fatty acids (>76 %), with a high abundance of iso-C(15 : 0) (48 %), anteiso-C(15 : 0) (7 %) and iso-C(17 : 0) 3-OH (11 %). Strain LW1(T) contained MK-4 and MK-7 as the major respiratory quinones and phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine as the major phospholipids. A blast sequence similarity search based on 16S rRNA gene sequences indicated that members of the genera Belliella and Aquiflexum were the nearest phylogenetic neighbours with similarities of 91.8-92.3 %. Phylogenetic analyses indicated that strain LW1(T) formed a deep-rooted lineage distinct from the clades represented by the genera Belliella, Aquiflexum, Cyclobacterium, Echinicola and Algoriphagus. Based on the above-mentioned phenotypic and phylogenetic characteristics, it is proposed that strain LW1(T) represents a novel species in a new genus, Indibacter alkaliphilus gen. nov., sp. nov. (type strain LW1(T)=KCTC 22604(T)=CCUG 57479(T)). The genomic DNA G+C content of strain LW1(T) is 42.7+/-1 mol%.

Full text

Page 1

Indibacter alkaliphilus gen. nov., sp. nov., an
alkaliphilic bacterium isolated from a haloalkaline
lake
P. Anil Kumar, T. N. R. Srinivas, S. Madhu, R. Ruth Manorama and S. Shivaji
Correspondence
S. Shivaji
shivas@ccmb.res.in
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
A novel Gram-stain-negative, rod-shaped, non-motile bacterium, strain LW1T, was isolated from a
water sample collected at a depth of 3.5 m from Lonar Lake, Buldhana district, Maharashtra, India.
The cell suspension was reddish-orange due to the presence of carotenoids. Strain LW1Twas
positive for catalase, oxidase, ornithine decarboxylase and lysine decarboxylase and negative for
gelatinase, urease and lipase. Fatty acids were dominated by branched-chain fatty acids (.76%),
with a high abundance of iso-C15:0(48%), anteiso-C15:0(7%) and iso-C17:03-OH (11%).
Strain LW1Tcontained MK-4 and MK-7 as the major respiratory quinones and
phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine as the major
phospholipids. A BLAST sequence similarity search based on 16S rRNA gene sequences
indicated that members of the genera Belliella and Aquiflexum were the nearest phylogenetic
neighbours with similarities of 91.8–92.3%. Phylogenetic analyses indicated that strain LW1T
formed a deep-rooted lineage distinct from the clades represented by the genera Belliella,
Aquiflexum, Cyclobacterium, Echinicola and Algoriphagus. Based on the above-mentioned
phenotypic and phylogenetic characteristics, it is proposed that strain LW1Trepresents a novel
species in a new genus, Indibacter alkaliphilus gen. nov., sp. nov. (type strain LW1T5KCTC
22604T5CCUG 57479T). The genomic DNA G+C content of strain LW1Tis 42.7±1 mol%.
Soda lakes are naturally occurring alkaline habitats with pH
values reaching 12 due to the presence of sodium carbonate
(Na2CO3), in the water (Joshi et al., 2008). Several novel
haloalkaliphilic bacterial species (close to 55 species) re-
presenting 41 genera have been described to date. These
genera belong to the phyla ‘Proteobacteria’ (19 genera, 26
species), ‘Firmicutes’ (15 genera, 21 species), ‘Actinobacteria’
(five genera, five species), ‘Bacteroidetes’ (one genus, one
species) and ‘Spirochaetes’ (one genus, two species). The
majority of the alkaliphiles have been isolated from soda
lakes (Milford et al., 2000; Borsodi et al., 2003; Doronina
et al., 2003; Sorokin et al., 2003; Zavarzina et al., 2006;
Carrasco et al., 2007; Delgado et al., 2006; Zhilina et al.,
2004; Hoover et al., 2003). Six novel archaeal species
representing five genera and belonging to the phylum
‘Euryarchaeota’ have also been isolated from soda lakes
(Kanal et al., 1995; Feng et al., 2005).
Lonar Lake is a haloalkaline water body in India. Although
several isolates representing 16 genera and belonging to
the phyla ‘Firmicutes’ (seven genera), ‘Proteobacteria’ (five
genera) and ‘Actinobacteria’ (four genera) have been iso-
lated from Lonar Lake (Joshi et al., 2008), no novel species
have been reported so far. In the present study, a novel
bacterium isolated from Lonar Lake and assigned to a new
genus in the family ‘Cyclobacteriaceae’, is described.
Strain LW1Twas isolated from a water sample (pH 10)
collected from Lonar Lake, located in the town of Lonar,
Buldhana district, Maharashtra, a state in India (19u 589 N
76u 309 E) on 25 August 2008. For isolation of bacteria, a
water sample (100 ml) was plated on Zobell marine agar
medium (Zobell, 1941) adjusted to pH 10.0 with Na2CO3
solution (20%, w/v) and incubated at room temperature
for 15 days. The viable bacterial cell counts obtained were
approximately 4.0–6.06105c.f.u. Based on colony mor-
phology, a reddish-orange coloured colony was selected
and characterized in the present study.
Cell morphology was studied using light microscopy.
Motility was assessed by light microscopy and on TSA
medium containing (g l21): pancreatic digest of casein
(17); papaic digest of soybean meal (3); NaCl (5); K2HPO4 ;
(2.5); glucose (2.5); and agar (0.4). Growth at different
temperatures, salt tolerance, biochemical characteristics,
carbon assimilation, H2S production and antibiotic sensi-
tivity were determined by previously described methods
(La ´nyı ´, 1987; Smibert & Krieg, 1994). Biochemical charac-
teristics were also double-checked with a Hi25 Entero-
%paper no. ije014076charlesworth ref: ije014076&
New Taxa - Bacteroidetes
Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Indibacter alkaliphilus sp. nov. LW1Tis FM883672.
International Journal of Systematic and Evolutionary Microbiology (2010), 60, 000–000
DOI 10.1099/ijs.0.014076
014076G2010 IUMSPrinted in Great Britain1

Page 2

bacteriaceae identification kit and HiCarbohydrate kit
parts A, B and C (both from HiMedia) according to the
manufacturer’s protocol. Growth of strain LW1Twas
checked at different pH values on TSA medium buffered
with citric acid/NaOH (for pH 5 and 6), phosphate (for
pH 7 and 8), glycine/NaOH (for pH 9 and 10) or Tris (for
pH 11 and 12) buffers.
Fatty acid methyl esters were prepared and analysed by the
Sherlock Microbial Identification System (MIDI) accord-
ing to the protocol described by Agilent Technologies. For
this purpose, strain LW1Twas grown on TSA medium at
30uC for 2 days. Polar lipids were extracted and analysed
according to the method described by Komagata & Suzuki
(1987). Menaquinones and polar lipids were determined
in freeze-dried cells. Menaquinones were extracted as
described by Collins et al. (1977) and analysed by HPLC
(Groth et al., 1997). DNA was isolated according to the
procedure of Marmur (1961) and the G+C content was
determined from melting point (Tm) curves (Sly et al.,
1986) obtained by using a Lambda 2 UV-Vis spectropho-
tometer (Perkin Elmer) equipped with the Templab 2.0
software package (Perkin Elmer). Escherichia coli was used
as a standard in determining DNA G+C content. For 16S
rRNA gene sequencing, DNA was prepared using the
Mo Bio microbial DNA isolation kit and sequenced as
described previously (Lane, 1991). The resultant almost
complete 16S rRNA gene sequence contained 1502 nt and a
BLAST sequence similarity search (Altschul et al., 1990) and
EzTaxon (Chun et al., 2007) were used to identify the
nearest taxa. All the 16S rRNA gene sequences belonging to
the family ‘Cyclobacteriaceae’ were downloaded from the
database (http://www.ncbi.nlm.nih.gov) and aligned using
the CLUSTAL_X program (Thompson et al., 1997); the alig-
nment was corrected manually. Phylogenetic trees were
constructed using two tree-making algorithms, maximum-
likelihood (ML) using the program PHYML (Guindon &
Gascuel, 2003) and neighbour-joining (NJ) (Saitou & Nei,
1987) using the PHYLIP package, version 3.5 (Felsenstein,
1993), and the resultant tree topologies were evaluated by
bootstrap analysis based on 1000 resamplings using the
SEQBOOT and CONSENSE programs in the PHYLIP package.
Pairwise evolutionary distances were computed using
DNADIST program with the Kimura two-parameter model
as developed by Kimura (1980).
Cells of strain LW1Twere rods, 0.5–0.7 mm wide and 2.0–
3.0 mm long, and multiplied by binary fission. Cells stained
Gram-negative and were non-motile. Colonies were cir-
cular, 2–3 mm in diameter, smooth, reddish-orange in
colour, opaque, convex and entire on half-strength marine
agar. The isolate was positive for catalase and oxidase and
negative for gelatinase and urease. Strain LW1Twas able to
reduce nitrate to nitrite and hydrolyse aesculin and starch.
It was negative for indole production, cellulose degrada-
tion, DNase and caseinase activity, and did not grow
on medium containing only 0.5% yeast extract. Growth
was observed at 15–40 uC; optimum growth was exhibited
at 30–37 uC. Growth was observed in 0–8% (w/v) NaCl,
with optimum growth in 0.5–6.0% (w/v) NaCl. Growth
occurred at pH 7.5–12.0, with optimum growth at pH
10.0. The absorption spectrum of an ethanol extract of
strain LW1Tshowed a broad peak with a maximum around
479 nm and a shoulder at 505 nm, peaks that are typical
for carotenoids. Alkalinization did not show a bath-
ochromatic
shift of the peaks. Therefore, strain LW1T
contains carotenoids, but no flexirubins, as often observed
for marine Cytophaga–Flavobacterium–Bacteroides bacteria
(Brettar et al., 2004b).
<
The cellular fatty acid composition of strain LW1Tshowed
a spectrum of 29 fatty acids with a pronounced dominance
of branched-chain C15:0and C17:0fatty acids (Table 1).
The branched-chain fatty acids were predominant and
constituted 76.86% of the total fatty acids, with the iso-
fatty acids forming the major fraction (67.59%). When
compared with Belliella baltica and Aquiflexum balticum,
the number of fatty acids detected in strain LW1T
was greater and the composition differed considerably
(Table 1). The menaquinones present in LW1Twere MK-4
(26%) and MK-7 (74%) and the phospholipids were
phosphatidylglycerol, phosphatidylcholine, phosphatidy-
lethanolamine and one unidentified lipid. Other character-
istics are listed in the species description. The DNA G+C
content of strain LW1Twas 42.7±1 mol%.
The phylogenetic relationship of the novel strain was
ascertained based on the 16S rRNA gene sequence
similarity of LW1Twith strains of other reported species
using a BLAST sequence similarity search (NCBI-BLAST/
EzTaxon). The results indicated that, at the 16S rRNA
gene sequence level, strain LW1Twas similar to various
cultivable bacterial strains of the genus Belliella that do not
have validly published names and uncultured bacterial
clone sequences with similarities of 90–93%. The phylo-
genetic neighbours of strain LW1Twith validly published
names included the type strains of B. baltica (Brettar et al.,
2004a), Aquiflexum balticum (Brettar et al., 2004b),
Cyclobacterium marinum (Raj & Maloy, 1990), Echinicola
vietnamensis (Nedashkovskaya et al., 2007a), Algoriphagus
ornithinivorans (Nedashkovskaya et al., 2007b), Echinicola
pacifica (Nedashkovskaya et al., 2006), Cyclobacterium
lianum (Ying et al., 2006), Cyclobacterium amurskyense
(Nedashkovskaya et al., 2005), Algoriphagus winogradskyi
(Nedashkovskaya et al., 2004), Algoriphagus hitonicola
(Copa-Patin ˜o et al., 2008), Algoriphagus terrigena (Yoon
et al., 2006) and Algoriphagus ratkowskyi (Bowman et al.,
2003), with similarities of 88.6–92.3%.
indicated that strain LW1Tprobably belongs to a new
genus. Phylogenetic analyses based on ML and NJ trees
further indicated that strain LW1Tformed a deep-rooted
lineage that was distinct from the clades represented by the
genera Algoriphagus, Aquiflexum, Belliella, Cyclobacterium
and Echinicola (Fig. 1).
BLAST results
Strain LW1Tcould also be differentiated phenotypically
from the closely related species B. baltica and Aquiflexum
balticum (Brettar et al., 2004a, b) (Table 2). For instance,
%paper no. ije014076charlesworth ref: ije014076&
P. Anil Kumar and others
2International Journal of Systematic and Evolutionary Microbiology 60

Page 3

strain LW1Tdiffered from both with respect to colony
colour, cell size, optimal salt concentration for growth,
salt tolerance, growth at pH 12 and DNA G+C content
(Table 2). In addition, specific differences were observed
between strain LW1Tand B. baltica and Aquiflexum
balticum with respect to acid production and utilization
of various carbon sources (Table 2). Thus, the cumula-
tive differences that strain LW1Texhibits with the above
two closely related species unambiguously support the
creation of a novel species within a new genus, Indibacter
alkaliphilus gen. nov., sp. nov., to accommodate this
isolate.
Description of Indibacter gen. nov.
Indibacter (In.di.bac9ter. L. n. India India; N.L. masc. n.
bacter a rod; N.L. masc. n. Indibacter a rod from India,
referring to the isolation of the bacterial strain from India).
Cells are Gram-stain-negative, aerobic rods that are
positive for catalase and oxidase and negative for gela-
tinase, urease and lipase. Major fatty acids are iso-C15:0,
anteiso-C15:0and iso-C17:03-OH. MK-4 and MK-7 are
the predominant respiratory quinones. Phospholipids
include phosphatidylglycerol, phosphatidylcholine and
phosphatidylethanolamine and an unknown lipid. The
genus is affiliated to the family ‘Cyclobacteriaceae’, order
‘Sphingobacteriales’, class ‘Sphingobacteria’.
Description of Indibacter alkaliphilus sp. nov.
Indibacter alkaliphilus (al.ka.li9phi.lus. N.L. n. alkali alkali;
Gr. adj. philos loving; N.L. adj. alkaliphilus alkali-loving).
Cells are non-motile rods (0.5–0.7 mm in width and 2.0–
3.0 mm in length) that occur singly and multiply by binary
fission. Colonies on half-strength marine agar are circular,
2–3 mm in diameter, smooth, reddish-orange in colour,
opaque, convex and entire. Cells grow at 15–40 uC, with
optimum growth at 30–37 uC, and tolerate up to 8.0%
%paper no. ije014076charlesworth ref: ije014076&
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences
showing the relationship between strain LW1Tand members of the
family ‘Cyclobacteriaceae’. Phylogenetic trees were constructed
using both the NJ and ML methods. The topology of the two trees
was similar. Filled circles indicate a node common to both trees
marked in the ML tree; open circles signify a node that differs.
Numbers at nodes are bootstrap values (ML/NJ; XX, no bootstrap
value in NJ tree where nodes differ in both dendrograms; ”, value
,50%). Bar, 0.02 substitutions per alignment position.
Table 1. Fatty acid composition of strain LW1Tand related
strains
Strains: 1, LW1T; 2, Belliella baltica BA134T(data from Brettar et al.,
2004a); 3, Aquiflexum balticum BA160T(Brettar et al., 2004b). Results
are percentages of the total fatty acids. Fatty acids representing more
than 5% are in bold. ND, Not detected. Fatty acid C13:1AT 12–13 was
not detected in any of the strains studied.
Fatty acid123
C12:0
iso-C13:0
anteiso-C13:0
C12:0
iso-C14:0
C15:0
iso-C15:0
anteiso-C15:0
C15:02-OH
iso-C15:03-OH
iso-C15:1F
iso-C15:1G
C15:1v5c
C15:1v6c
C16:0
C16:0N alcohol
iso-C16:0
anteiso-C16:0
C16:03-OH
iso-C16:03-OH
iso-C16:1H
iso-C16:13-OH
C16:1v5c
C16:1v7c/iso-C15:02-OH*
C17:0
C17:02-OH
C17:03-OH
iso-C17:0
anteiso-C17:0
iso-C17:03-OH
anteiso-C17:1B
C17:1v6c
C17:1v8c
iso-C17:1v9c
anteiso-C17:1v9c
C18:1v9c
iso-C19:1I
Unidentified
0.24
0.29
0.36
0.28
NDND
NDND
NDND
ND
1.78
3.86
18.93
4.81
0.90
2.29
ND
4.77
ND
NDND
22.59
18.53
47.91
6.75
0.34
1.26
1.59
ND
1.59
NDND
9.40
ND
0.41
1.46
1.60
0.36
2.12
0.60
0.78
0.58
0.76
10.07
ND
2.51
ND
ND
NDND
ND
2.78
ND
4.23
ND ND
NDND
ND
3.22
1.81
2.02
7.05
ND
9.45
2.00
1.98
6.10
ND
0.86
ND
0.35
1.82
0.31
2.41
0.51
10.68
NDND
NDND
ND
0.50
ND
ND
ND
3.04
3.95
9.81
1.49
10.15
ND
1.44
2.55
2.99
ND
1.20
0.38
ND
5.22
1.10
ND
1.05
0.26
ND
ND
0.77
8.30
ND
1.46
4.60
ND
12.48
*Not differentiated by the MIDI system.
Indibacter alkaliphilus gen. nov., sp. nov.
http://ijs.sgmjournals.org3

Page 4

(w/v) NaCl. Growth occurs at pH 7.5–12.0. Catalase,
oxidase, ornithine decarboxylase and lysine decarboxylase
are positive, but urease, b-galactosidase, arginine dihydro-
lase and tryptophan deaminase are negative. Methyl red
and Voges–Proskauer reactions are negative. Starch and
aesculin are hydrolysed, but Tween 60, Tween 85 and
gelatin are not hydrolysed. Nitrate is reduced to nitrite and
H2S gas is not produced. Cells produce acid from xylose,
melezitose, maltose,
D-fructose,
cellobiose, D-glucose, lactose and L-rhamnose, but not from
D-arabinose, melibiose,
inulin, aesculin, xylitol, salicin, erythritol, adonitol, dulcitol,
D-galactose, inositol, mannitol, D-mannose, raffinose, suc-
rose, D-sorbitol or trehalose, even after 1 week of incubation
at optimum temperature and pH. Assimilates lactose, xylose,
maltose, fructose, glucose, galactose, raffinose, trehalose,
melibiose, sucrose, L-arabinose, mannose, glucosamine, ado-
nitol, rhamnose, aesculin and D-arabinose, but not inulin,
sodium gluconate, glycerol, salicin, dulcitol, inositol, sorbi-
tol, mannitol, methyl a-D-glucoside, methyl a-D-mannoside,
ribose, melezitose, xylitol, ONPG, citrate, malonate, sorbose
%paper no. ije014076charlesworth ref: ije014076&
Table 2. Differential features of strain LW1Tand related strains
Strains: 1, LW1T; 2, Belliella baltica BA134T(data from Brettar et al., 2004a); 3, Aquiflexum balticum BA160T(Brettar et al., 2004b). All strains are
positive for nitrate reduction to nitrite, hydrolysis of starch and acid production from maltose. All strains are negative for utilization of malate,
citric acid, gluconic acid, malonic acid and succinic acid. +, Positive; W, weak; 2, negative.
Characteristic123
Cell morphology
Cell size (mm)
Colour
DNA G+C content (mol%)
Salinity growth range (%)
Salinity optimum (%)
Temperature growth range (uC)
Temperature optimum (uC)
pH growth range
Growth at pH 12
Hydrolysis of gelatin
Acid production from:
N-Acetylglucosamine
Amygdalin
L-Arabinose
Cellobiose
5-Ketogluconate
D-Mannose
Melezitose
Methyl a-D-glucoside
Methyl a-D-mannoside
Rhamnose
Xylitol
D-Xylose
Utilization of:
N-Acetylglucosamine
L-Alanine
L-Alanyl glycine
Gluconate
L-Glutamic acid
a-Ketobutyric acid
a-Ketoglutaric acid
DL-Lactic acid
Mannose
D-Mannose
Methyl b-D-glucoside
L-Proline
Propionic acid
L-Serine
D-Sorbitol
L-Threonine
Rods, straight
0.5–0.762.0–3.0
Reddish-orange
42.7
0–8
0–6
15–40
30–37
7.5–12.0
+
2
Rods, straight
0.3–0.461.5–3.0
Pink/orange
35.3
0–6
0–3.4
4–37
25–30
6–10
2
2
Long rods, straight
0.3–0.661.1–4.8
Red
38.4
0–6
1–2
4–40
30–35
7–9
2
+
2
2
+
2
2
+
+
2
2
2
2
2
2
2
+
W
W
2
+
W
2
W
W
+
2
2
W
W
W
W
+
W
2
+
W
2
2
2
2
2
2
2
2
2
+
+
2
2
2
2
2
2
2
+
2
2
+
2
2
W
W
2
+
+
WW
+
+
W
2
2
2
W
W
2
W
+
W
2
W
2
W
2
W
P. Anil Kumar and others
4International Journal of Systematic and Evolutionary Microbiology 60

Page 5

or amygdalin. Susceptible to (mg per disc except where
indicated): cefazolin (30), colistin (10), co-trimoxazole (25),
lincomycin (2), nalidixic acid (30), polymyxin B (50 U),
amikacin (30), ampicillin (10), bacitracin (10), carbenicillin
(100), cefotaxime (30), chloramphenicol (30), ciprofloxacin
(5), erythromycin (15), gentamicin G (30), kanamycin (30),
lomefloxacin (30), nitrofurantoin (300), norfloxacin (10),
novobiocin (30), oleandomycin (15), penicillin G (10),
rifampicin (30), spectinomycin (100), tetracycline (30),
doxycycline (10), cefuroxime (30), cefoperazone (75),
roxithromycin (30), streptomycin (10) and vancomycin
(30). Polar lipids comprise phosphatidylglycerol, phospha-
tidylcholine, phosphatidylethanolamine and one uniden-
tified lipid. Isoprenoid quinones are MK-4 and MK-7.
Cellular fatty acid composition is given in Table 1.
The type strain, LW1T(5KCTC 22604T5CCUG 57479T),
was isolated from a water sample collected at a depth
of 3.5 m from Lonar Lake, Lonar, Buldhana district,
Maharashtra, India. The DNA G+C content of the type
strain is 42.7±1 mol%.
Acknowledgements
We would like to thank Dr S. W. A. Naqvi, Scientist, National
Institute of Oceanography, Goa, India, for providing the samples. S.S.
is grateful to National Centre for Antarctic and Ocean Research, Goa,
Department of Biotechnology, New Delhi, and the CSIR Network
Project on Biodiversity for funding.
References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.
(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.
Borsodi, A. K., Micsinai, A., Kova ´cs, G., To ´th, E., Schumann,
P., Kova ´cs, A. L., Bo ¨ddi, B. & Ma ´rialigeti, K. (2003). Pannonibacter
phragmitetus gen. nov., sp. nov., a novel alkalitolerant bacterium
isolated from decomposing reed rhizomes in a Hungarian soda lake.
Int J Syst Evol Microbiol 53, 555–561.
Bowman, J. P., Nichols, C. M. & Gibson, J. A. (2003). Algoriphagus
ratkowskyi gen. nov., sp. nov., Brumimicrobium glaciale gen. nov., sp.
nov., Cryomorpha ignava gen. nov., sp. nov. and Crocinitomix
catalasitica gen. nov., sp. nov., novel flavobacteria isolated from
various polar habitats. Int J Syst Evol Microbiol 53, 1343–1355.
Brettar, I., Christen, R. & Ho ¨fle, M. G. (2004a). Belliella baltica gen.
nov., sp. nov., a novel marine bacterium of the Cytophaga–
Flavobacterium–Bacteroides group isolated from surface water of the
central Baltic Sea. Int J Syst Evol Microbiol 54, 65–70.
Brettar, I., Christen, R. & Ho ¨fle, M. G. (2004b). Aquiflexum balticum
gen. nov., sp. nov., a novel marine bacterium of the Cytophaga–
Flavobacterium–Bacteroides group isolated from surface water of the
central Baltic Sea. Int J Syst Evol Microbiol 54, 2335–2341.
Carrasco, I. J., Ma ´rquez, M. C., Xue, Y., Ma, Y., Cowan, D. A., Jones,
B. E., Grant, W. D. & Ventosa, A. (2007). Bacillus chagannorensis sp.
nov., a moderate halophile from a soda lake in Inner Mongolia,
China. Int J Syst Evol Microbiol 57, 2084–2088.
Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W.
(2007). EzTaxon: a web-based tool for the identification of
prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst
Evol Microbiol 57, 2259–2261.
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977).
Distribution of menaquinones in actinomycetes and corynebacteria. J
Gen Microbiol 100, 221–230.
Copa-Patin ˜o, J. L., Arenas, M., Soliveri, J., Sa ´nchez-Porro, C. &
Ventosa, A. (2008). Algoriphagus hitonicola sp. nov., isolated from an
athalassohaline lagoon. Int J Syst Evol Microbiol 58, 424–428.
Delgado, O., Quillaguama ´n, J., Bakhtiar, S., Mattiasson, B.,
Gessesse, A. & Hatti-Kaul, R. (2006). Nesterenkonia aethiopica sp.
nov., an alkaliphilic, moderate halophile isolated from an Ethiopian
soda lake. Int J Syst Evol Microbiol 56, 1229–1232.
Doronina, N. V., Darmaeva, T. D. & Trotsenko, Y. A. (2003).
Methylophaga alcalica sp. nov., a novel alkaliphilic and moderately
halophilic, obligately methylotrophic bacterium from an East
Mongolian saline soda lake. Int J Syst Evol Microbiol 53, 223–229.
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version
3.5.1. Department of Genome Sciences, University of Washington,
Seattle, USA.
Feng, J., Zhou, P., Zhou, Y. G., Liu, S. J. & Warren-Rhodes, K. (2005).
Halorubrum alkaliphilum sp. nov., a novel haloalkaliphile isolated
from a soda lake in Xinjiang, China. Int J Syst Evol Microbiol 55, 149–
152.
Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. &
Augsten, K. (1997). Demetria terragena gen. nov., sp. nov., a new
genus of actinomycetes isolated from compost soil. Int J Syst Bacteriol
47, 1129–1133.
Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum likelihood. Syst
Biol 52, 696–704.
Hoover, R. B., Pikuta, E. V., Bej, A. K., Marsic, D., Whitman, W. B.,
Tang, J. & Krader, P. (2003). Spirochaeta americana sp. nov., a new
haloalkaliphilic, obligately anaerobic spirochaete isolated from soda
Mono Lake in California. Int J Syst Evol Microbiol 53, 815–821.
Joshi, A. A., Kanekar, P. P., Kelkar, A. S., Shouche, Y. S., Vani, A. A.,
Borgave, S. B. & Sarnaik, S. S. (2008). Cultivable bacterial diversity
of alkaline Lonar lake, India. Microb Ecol 55, 163–172.
Kanal, H., Kobayashi, T., Aono, R. & Kudo, T. (1995). Natronococcus
amylolyticus sp. nov., a haloalkaliphilic archaeon. Int J Syst Bacteriol
45, 762–766.
Kimura, M. (1980). A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16, 111–120.
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in
bacterial systematics. Methods Microbiol 19, 161–207.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
Techniques in Bacterial Systematics, pp 115–175. Edited by E.
Stackebrandt & M. Goodfellow. Chichester: Wiley.
La ´nyı ´, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
acid from microorganisms. J Mol Biol 3, 208–218.
Milford, A. D., Achenbach, L. A., Jung, D. O. & Madigan, M. T. (2000).
Rhodobaca bogoriensis gen. nov. and sp. nov., an alkaliphilic purple
nonsulfur bacterium from African Rift Valley soda lakes. Arch
Microbiol 174, 18–27.
Nedashkovskaya,
Vandemeulebroecke, K., Lysenko, A. M., Rohde, M., Falsen, E.,
Frolova, G. M., Mikhailov, V. V. & Swings, J. (2004). Description of
Algoriphagus aquimarinus sp. nov., Algoriphagus chordae sp. nov. and
Algoriphagus winogradskyi sp. nov., from sea water and algae, transfer
of Hongiella halophila Yi and Chun 2004 to the genus Algoriphagus as
Algoriphagus halophilus comb. nov. and emended descriptions of the
O.I.,Vancanneyt,M., VanTrappen,S.,
%paper no. ije014076 charlesworth ref: ije014076&
Indibacter alkaliphilus gen. nov., sp. nov.
http://ijs.sgmjournals.org5

Page 6

genera Algoriphagus Bowman et al. 2003 and Hongiella Yi and Chun
2004. Int J Syst Evol Microbiol 54, 1757–1764.
Nedashkovskaya, O. I., Kim, S. B., Lee, M. S., Park, M. S., Lee, K. H.,
Lysenko, A. M., Oh, H. W., Mikhailov, V. V. & Bae, K. S. (2005).
Cyclobacterium amurskyense sp. nov., a novel marine bacterium
isolated from sea water. Int J Syst Evol Microbiol 55, 2391–2394.
Nedashkovskaya, O. I., Kim, S. B., Vancanneyt, M., Lysenko, A. M.,
Shin, D. S., Park, M. S., Lee, K. H., Jung, W. J., Kalinovskaya, N. I. &
other authors (2006). Echinicola pacifica gen. nov., sp. nov., a novel
flexibacterium isolated from the sea urchin Strongylocentrotus
intermedius. Int J Syst Evol Microbiol 56, 953–958.
Nedashkovskaya, O. I., Kim, S. B., Hoste, B., Shin, D. S., Beleneva,
I. A., Vancanneyt, M. & Mikhailov, V. V. (2007a). Echinicola viet-
namensis sp. nov., a member of the phylum Bacteroidetes isolated
from seawater. Int J Syst Evol Microbiol 57, 761–763.
Nedashkovskaya, O. I., Kim, S. B., Kwon, K. K., Shin, D. S., Luo,
X., Kim, S. J. & Mikhailov, V. V. (2007b). Proposal of Algoriphagus
vanfongensis sp. nov., transfer of members of the genera Hongiella
Yi and Chun 2004 emend. Nedashkovskaya et al. 2004 and
Chimaereicella Tiago et al. 2006 to the genus Algoriphagus, and
emended description of the genus Algoriphagus Bowman et al. 2003
emend. Nedashkovskaya et al. 2004. Int J Syst Evol Microbiol 57, 1988–
1994.
Raj, H. D. & Maloy, S. R. (1990). Proposal of Cyclobacterium marinus
gen. nov., comb. nov., for a marine bacterium previously assigned to
the genus Flectobacillus. Int J Syst Bacteriol 40, 337–347.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–
425.
Sly, L. I., Blackall, L. L., Kraat, P. C., Tian-Shen, T. & Sangkhobol,
V. (1986). The use of second derivative plots for the determination of
mol% guanine plus cytosine of DNA by the thermal denaturation
method. J Microbiol Methods 5, 139–156.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
Methods for General and Molecular Bacteriology, pp. 607–654. Edited
by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg.
Washington, DC: American Society for Microbiology.
Sorokin, D. Y., Tourova, T. P., Sjollema, K. A. & Kuenen, J. G. (2003).
Thialkalivibrio nitratireducens sp. nov., a nitrate-reducing member of
an autotrophic denitrifying consortium from a soda lake. Int J Syst
Evol Microbiol 53, 1779–1783.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
Ying, J. Y., Wang, B. J., Yang, S. S. & Liu, S. J. (2006). Cyclobacterium
lianum sp. nov., a marine bacterium isolated from sediment of an
oilfield in the South China Sea, and emended description of the genus
Cyclobacterium. Int J Syst Evol Microbiol 56, 2927–2930.
Yoon, J. H., Lee, M. H., Kang, S. J. & Oh, T. K. (2006). Algoriphagus
terrigenasp.nov.,isolatedfromsoil.IntJSystEvolMicrobiol56,777–780.
Zavarzina, D. G., Kolganova, T. V., Bulygina, E. S., Kostrikina, N. A.,
Turova, T. P. & Zavarzin, G. A. (2006). Geoalkalibacter ferrihydriticus
gen. nov., sp. nov., the first alkaliphilic representative of the family
Geobacteraceae, isolated from a soda lake. Mikrobiologiia 75, 775–785
(in Russian).
Zhilina, T. N., Appel, R., Probian, C., Brossa, E. L., Harder, J., Widdel,
F. & Zavarzin, G. A. (2004). Alkaliflexus imshenetskii gen. nov. sp. nov.,
a new alkaliphilic gliding carbohydrate-fermenting bacterium with
propionate formation from a soda lake. Arch Microbiol 182, 244–253.
Zobell, C. E. (1941). Studies on marine bacteria. I. The cultural
requirements of heterotrophic aerobes. J Mar Res 4, 42–75.
%paper no. ije014076 charlesworth ref: ije014076&
P. Anil Kumar and others
6International Journal of Systematic and Evolutionary Microbiology 60

Page 7

Dear Authors,
Please find enclosed a proof of your article for checking.
When reading through your proof, please check carefully authors’ names, scientific data, data in tables, any mathematics and
the accuracy of references. Please do not make any unnecessary changes at this stage. All necessary corrections should be
marked on the proof at the place where the correction is to be made; please write the correction clearly in the margin (if in the
text they may be overlooked).
Any queries that have arisen during preparation of your paper for publication are listed below and indicated on the proof.
Please provide your answers when returning your proof.
Please return your proof by Fax (+44 (0)118 988 1834) within 2 days of receipt.
Query no.Query
1
Author: Please check that this change is correct.
2
Author: Please check that this change is correct.

Page 8

IJSEM
International
Journal of
Systematic and
Evolutionary
Microbiology
Offprint Order Form
PAPER
ije014076
Please quote this number in any correspondence
Authors
P. Anil Kumar and others
Date _____________________
I would like 25 free offprints, plusadditional offprints, giving a total ofoffprints
Dispatch address for offprints (BLOCK CAPITALS please)
Please complete this form even if you do not want extra offprints. Do not delay returning your proofs by waiting for a
purchase order for your offprints: the offprint order form can be sent separately.
Please pay by credit card or cheque with your order if possible. Alternatively, we can invoice you. All remittances
should be made payable to ‘Society for General Microbiology’ and crossed ‘A/C Payee only’.
Tick one
% Charge my credit card account (give card details below)
% I enclose a cheque/draft payable to Society for General Microbiology
% Purchase order enclosed
Return this form to: IJSEM Editorial Office, Marlborough House, Basingstoke Road, Spencers Wood,
Reading RG7 1AG, UK.
CHARGES FOR ADDITIONAL OFFPRINTS
50 75100 Copies
No. of pages
1-2
3-4
5-8
9-16
17-24
each 8pp extra
25 125150 175 200 Per 25 extra
OFFICE USE ONLY
Issue:
Vol/part:
Page nos:
Extent:
Price:
Invoice: IR/
£23
£35
£46
£58
£70
£18
£40
£58
£76
£92
£110
£23
£58
£81
£104
£128
£151
£29
£76
£104
£133
£162
£191
£35
£92
£128
£162
£196
£231
£40
£110
£150
£191
£231
£272
£46
£128
£173
£219
£267
£312
£53
£145
£191
£249
£301
£353
£58
£23
£29
£35
£40
£46
PAYMENT BY CREDIT CARD (Note: we cannot accept American Express)
Please charge the sum of £____________ to my credit card account.
My Mastercard/Visa number is (circle appropriate card; no others acceptable):
Expiry
date
Security
Number
Signature: _________________________Date: _______________
Cardholder’s name and address*:
*Address to which your credit card statement is sent. Your offprints will be sent to the address shown at the top of the form.
May 2006