Content uploaded by Heinrich Lünsdorf
Author content
All content in this area was uploaded by Heinrich Lünsdorf
Content may be subject to copyright.
Thalassolituus oleivorans gen. nov., sp. nov.,
a novel marine bacterium that obligately utilizes
hydrocarbons
Michail M. Yakimov,
1
Laura Giuliano,
1
Renata Denaro,
1
Ermanno Crisafi,
1
Tatiana N. Chernikova,
2
Wolf-Rainer Abraham,
2
Heinrich Luensdorf,
2
Kenneth N. Timmis
2
and Peter N. Golyshin
2
Correspondence
Michail M. Yakimov
iakimov@ist.me.cnr.it
1
Istituto per l’Ambiente Marino Costiero, CNR Consiglio Nazionale Ricerche, Spianata San
Raineri 86, 98122 Messina, Italy
2
Division of Microbiology, GBF National Research Centre for Biotechnology, Mascheroder
Weg 1, 38124 Braunschweig, Germany
An aerobic, heterotrophic, Gram-negative, curved bacterial strain, designated MIL-1
T
, was isolated
by extinction dilution from an n-tetradecane enrichment culture that was established from sea
water/sediment samples collected in the harbour of Milazzo, Italy. In the primary enrichment, the
isolate formed creamy-white, medium-sized colonies on the surface of the agar. The isolate did not
grow in the absence of NaCl; growth was optimal at 2?7 % NaCl. Only a narrow spectrum of
organic compounds, including aliphatic hydrocarbons (C
7
–C
20
), their oxidized derivatives and
acetate, were used as growth substrates. The isolate was not able to grow under denitrifying
conditions. The DNA G+C content and genome size of strain MIL-1
T
were estimated to be
53?2 mol% and 2?2 Mbp, respectively. The major cellular and phospholipid fatty acids were
palmitoleic, palmitic and oleic acids (33?5, 29?5 and 11?0 % and 18, 32 and 31 %, respectively).
3-Hydroxy lauric acid was the only hydroxy fatty acid detected. Thirteen different compounds that
belonged to two types of phospholipid (phosphatidylethylamine and phosphatidylglycerol) were
identified. 16S rRNA gene sequence analysis revealed that this isolate represents a distinct
phyletic lineage within the c-Proteobacteria and has about 94?4 % sequence similarity to
Oceanobacter kriegii (the closest bacterial species with a validly published name). The deduced
protein sequence of the putative alkane hydrolase, AlkB, of strain MIL-1
T
is related to the
corresponding enzymes of Alcanivorax borkumensis and Pseudomonas oleovorans (81 and
80 % similarity, respectively). On the basis of the analyses performed, Thalassolituus oleivorans
gen. nov., sp. nov. is described. Strain MIL-1
T
(=DSM 14913
T
=LMG 21420
T
) is the type
and only strain of T. oleivorans.
INTRODUCTION
Many marine bacteria that are capable of degrading petro-
leum hydrocarbons have recently been isolated from sites all
over the world (Dyksterhouse et al., 1995; Button et al.,
1998; Yakimov et al., 1998; Hedlund et al., 1999; Syutsubo
et al., 2001; Golyshin et al., 2002). An alysis of 16S rRNA gene
sequences of these marine hydrocarbonoclastic bacteria
revealed that they all belong to the
c-subclass of the Proteo-
bacteria; however, they are separate and distinct from other
bacteria of this group and represent the genera Alcanivorax
(Yakimov et al., 1998), Cycloclasticus (Dyksterhouse et al.,
1995), Marinobacter (Gauthier et al., 1992), Neptunomonas
(Hedlund et al., 1999), Oleiphilus (Golyshin et al., 2002) and
Oleispira (Yakimov et al., 2003). The genera Marinobacter
and, especially, Alcanivorax, seem to play a major role in the
first step of crude oil biodegradation in marine environ-
ments (Harayama et al., 1999; Kasai et al., 2001). These
marine hydrocarbonoclastic bacteria appear to be novel in a
number of respects. They are obligate for hydrocarbon sub-
strates and additionally use only a small number of low-
molecular-mass organic acids, such as acetate and pyruvate .
Only a few rRNA operons (one to three) and cytoplasmic
proteins (not more than 300) and small genome sizes
(2?0–3?0 Mbp) are characteristic for these micro-organisms
Abbreviations: CID, collision-induced dissociations; GLFA, glycolipid fatty
acids; PE, phosphatidylethylamine; PG, phosphatidylglycerol; PLFA,
phospholipid fatty acids; TLFA, major cellular fatty acids.
Published online ahead of print on 11 July 2003 as DOI 10.1099/
ijs.0.02424-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and
putative alkane hydroxylase gene sequences of strain MIL-1
T
are
AJ431699 and AJ431700, respectively.
02424
G
2004 IUMS Printed in Great Britain 141
International Journal of Systematic and Evolutionary Microbiology (2004), 54, 141–148 DOI 10.1099/ijs.0.02424-0
(Button et al., 1998; Golyshin et al., 2002; Yakimov et al.,
2002). However, information about the genetics and bio-
chemistry of hydrocarbonoclastic bacteria is still very
limited (Wang et al., 1996; Hedlund et al., 1999; Dutta &
Harayama, 2001 ). Further studies on the nutrient require-
ments of hydrocarbonoclastic bacteria, coupled with inves-
tigations into the degradation pathways and genomic
analysis, need to be performed to achieve a better under-
standing of the superior metabolic capacities of these
bacteria, which would contribute significantly to the
management of oil pollution in marine environments.
During studies on the diversity of hydrocarbonoclastic
marine bacteria that occur naturally in coastal ecosystems
that have been chronically exposed to oil hydrocarbon pollu-
tion, a heterotrophic
c-proteobacterium that was obligate
for hydrocarbon utilization was isolated. In this work, the
phenotypic characterization of strain MIL-1
T
, its phylo-
genetic assignment and DNA base and lipid compositions
are pres ented. The isolate constitutes a species within a novel
genus for which, by considering its origin, morphology and
metabolism, the name Thalassolituus oleivorans gen. nov.,
sp. nov. is proposed. Strain MIL-1
T
(=DSM 14913
T
=LMG
21420
T
) is designated as the type strain.
METHODS
Bacterial strain isolation. Strain MIL-1
T
was isolated from sea
water/sediment samples that were collected in the harbour of
Milazzo, Sicily, Italy, from a depth of about 5 m, by using an enrich-
ment culture with 0?2 % (v/v) n-tetradecane as the sole carbon
source in ONR7a mineral medium (Dyksterhouse et al., 1995).
Replicate tenfold dilutions of the primary enrichment were made in
10 ml ONR7a mineral medium that was supplemented with sterile
n-tetradecane (0?2 %, v/v). Tubes were incubated in the dark at
20 uC until turbidity changes due to bacterial growth ceased
(approx. 2 weeks). Positive tubes that represented the highest dilu-
tion (10
25
) were plated further onto solid ONR7a mineral medium
that was supplemented with n-tetradecane; single colonies were
observed after 7 days incubation.
Growth conditions and phenotypic analysis. The isolate was
cultivated aerobically in ONR7a medium that was supplemented
with 0?2 % (v/v) n-tetradecane. Bacto agar (Difco) (15 g l
21
) was
added for the preparation of solid medium. For all phenotypic tests,
cultures were pre-grown in ONR7a medium that was supplemented
with n-hexadecane. Growth under anaerobic conditions and utiliza-
tion of carbon sources were determined and routine tests were
carried out as described previously (Golyshin et al., 2002).
The effects of hydrocarbons, salinity and temperature on growth were
also examined. The capacity of various aliphatic hydrocarbons to serve
as the sole source of carbon and energy was determined at 20 uCin
liquid ONR7a medium. Substrates were sterilized separately and added
aseptically at 0 ?2 % (v/v). To determine the salinity range for growth,
ONR7a medium that contained no sodium ions and was supplemented
with n-tetradecane was prepared by adjustment with appropriate
concentrations of NaCl [0?01–2?0 M, i.e. 0?06–12 % (w/v)]. Tem-
perature range for growth was determined by incubation of cultures
in the same medium at 4, 10, 15, 20, 25 and 30 uC. In all experiments,
growth was scored by measuring OD
600
. Five replicates of test cul-
tures of each strain were analysed after three serial transfers under
identical conditions.
Electron microscopy. The isolate was cultivated aerobically in
ONR7a medium that was supplemented with 0?2 % (v/v) n-tetradecane;
cells in mid-exponential growth phase were sedimented and fixed in
5 % glutaraldehyde that was buffered with 50 mM PBS, pH 7?1.
Negative-staining, shadow-casting, embedding and ultra-thin sec-
tioning were done according to methods described previously
(Yakimov et al., 1998; Golyshina et al., 2000).
Cellular fatty acid analysis. Lipids were extracted from mid-
exponential cells that had been grown in ONR7a/tetradecane
medium by using a modified Bligh–Dyer procedure (Bligh & Dyer,
1959). Later on, fatty acid methyl esters were generated and analysed
by GC as described previously (Vancanneyt et al ., 1996).
Phospholipid analysis. Lipids of cells that had been harvested in
the mid-exponential phase were extracted and polar lipids were
separated by flash chromatography, as described previously
(Abraham et al., 1997). The polar lipid fraction was analysed by
using electrospray ionization in the negative mode in a quadrupol-
time-of-flight mass spectrometer. Abundant molecular ions were
separated and the parent ions then underwent collision-induced dis-
sociations (CID); resulting fragments were finally detected in the
time-of-flight part of the instrument.
G+C content and genome format. The DNA G+C content of
strain MIL-1
T
was determined by using an HPLC method that was
described previously (Mesbah et al., 1989; Tamaoka & Komagata,
1984). Purified non-methylated
l-phage DNA (Sigma) was used as a
control. PFGE separation of the DNA digests by endonucleases AscI,
PacI, PmeI, SwaI and SfiI (New England Biolabs) was performed by
using a Gene Navigator Electrophoresis device (Pharmacia) with
switch times that ramped between 2 and 64 s at 6 V cm
21
. In order
to obtain plasmids, cells of MIL-1
T
were extracted with a Large
Construct kit (Qiagen). The extracted DNA was later analysed by
gel electrophoresis.
16S rRNA gene sequence analysis. To investigate the phylo-
genetic relationships of strain MIL-1
T
, isolation of genomic DNA,
PCR amplification, determination of the sequence of the 16S rRNA
gene and its subsequent phylogenetic affiliation were performed
according to previously described protocols (Golyshin et al., 2002).
Cloning of the putative alkB gene. Chromosomal DNA of strain
MIL-1
T
was amplified by using oligonucleotides and conditions
described by Smits et al. (1999) and the deduced putative AlkB pro-
tein sequence from MIL-1
T
was aligned manually by using the Se-Al
sequence alignment editor, version 1.0
a1 (Rambaut, 1996). Maximum-
likelihood evolutionary distances of the proteins were calculated by
using the
PROTDIST program and a dendrogram depicting phylo-
genetic relationships was derived by using the Fitch–Margoliash
method (
FITCH version 3.572c) with random-order input of se-
quences and using the global rearrangement option (Felsenstein, 1993).
RESULTS AND DISCUSSION
Phenotypic and ultrastructural characteristics
Strain MIL-1
T
was isolated after serial dilutions from an
enrichment culture that was established from sea water/
sediment samples collected in the harbour of Milazzo, Italy,
by addition of n-tetradecane as the sole carbon source.
Exponentially growing cells were subjected to ultra-thin
sectioning after embedding in epoxy resin and were analysed
with an energy-filtered transmission electron microscope.
Characteristically, the bacteria showed a curved, vibrioid,
occasionally screw-like morphology (Fig. 1a) and distinctly
142 International Journal of Systematic and Evolutionary Microbiology 54
M. M. Yakimov and others
presented a Gram-negative cell wall architecture with an
outer membrane. However, under the fixation protocol
used, the murein sacculus could be not be recognized as a
typical central periplasmic layer (Fig. 1a, inset). Cells were
of various lengths in the range 1?2–3?1
mm and measured
0?32–0?77 mm in diameter (mean value: 0?566±0?108 mm;
n=37) and, under the growth conditions used, the cyto-
plasm contained electron-translucent inclusions, possibly of
hydrocarbon polymers (Fig. 1a, asterisk). From shadow-
casted samples, inclusions were located mainly at cell poles
(Fig. 1b, asterisk). The bacteria characteristically showed
monopolar, monotrichous flagellation (Fig. 1b, fl), whereas
a monopolar tuft of four flagella was also detected.
The new isolate required NaCl for growth; growth was
observed at NaCl concentrations of 0?5–5?7 % (w/v).
Optimum growth occurred at 2?7 % NaCl. The isolate
grew at 4–30
u
C, w ith an optimum growth temperature of
20–25
u
C. The pH range for growth was 7?5–9?0, with
optimum growth at pH 8?0.
Physiology and biochemic al characteristics
Consistent with its phylogenetic placement, strain MIL-1
T
shares many phenotypic properties with Oceanospirillum
and related genera. However, there are some crucial pheno-
typic differences that suggest that the new strain does not
belong to any previously described genus. Isolate MIL-1
T
was ox idase-positive and did not catabolize any substrate
tested except for acetate, aliphatic hydrocarbons with a
carbon chain length between C
7
and C
20
and their oxidized
derivatives. Poor growth was observed in ONR7a medium
that was sup plemented with
L-arabinose and psicose.
During growth on Tweens 20, 40 and 80, production of
extracellular lipase was detected. Neither nitrate reduction
nor denitrifying activity was detected. The reaction for
catalase was positive. Biochemical and physiological charac-
teristics that differentiate isolate MIL-1
T
from related genera
are summarized in Table 1. In contrast with the genera
Marinobacter, Marinomonas and Oceanobacter, which are
characterized by nutritional versatility, uptake by isolate
MIL-1
T
is almost restricted to aliphatic hydrocarbons. Such
a narrow spectrum of substrates that support growth of
MIL-1
T
is a typical physiological feature for marine,
obligately alkane-degrading c-proteobacteria that belong
to the recently described genera Alcanivorax, Oleiphilus and
Oleispira (Yakimov et al., 1998, 2002; Golyshin et al., 2002).
Lipid analys is
After a whole-cell methanolysis procedure and saponifica -
tion of phospho- and glycolipids, three different fatty acid
profiles were detected in strain MIL-1
T
(Table 2). The
fraction of the saturated fatty acids C
12
–C
18
represented
>92 % of total extracted glycolipid fatty acids (GLFA), with
lauric acid as a major component. The major cellular and
phospholipid fatty acid (TLFA and PLFA, resp ectively)
profiles were characterized by an almost equal presence of
saturated and monounsaturated fatty acids, with a strong
predominance of C
14 : 0
,C
16 : 1
,C
16 : 0
and C
18 : 1
. These
profiles were different from that of Oceanobacter kriegii,
which is characterized by the strong abundance of monoun-
saturated fatty acids (63 %) (Gonzalez & Whitman, 2001).
Analysis of hydroxy fatty acids in strain MIL-1
T
TLFA
revealed the presence of a single hydroxy fatty acid, C
12 : 0
3-OH, whereas three different 3-hydroxy fatty acids are
Fig. 1. Electron micrograph of (a) ultrathin-sectioned and (b) shadow-casted exponentially growing cells of Thalassolituus
oleivorans. The nucleoplasm (ch) occupies most of the cell lumen and electron-translucent inclusions (asterisks) are found
mainly at the cell poles. Inset shows the cytoplasmic (CM) and outer (OM) membranes. A single flagellum (fl) is inserted at
one cell pole of a dividing cell, which shows the start of septation (S). Direction of shadow-casting is marked by an arrow.
Bars, 1?1 mm (a); 100 nm (inset); 600 nm (b).
http://ijs.sgmjournals.org 143
Thalassolituus oleivorans gen. nov., sp. nov.
present in Oceanobacter kriegii:C
10 : 0
(19 %), C
12 : 0
(54 %)
and C
16 : 0
(27 %).
Analysis of intact phospholipids
Analysis of CID-MS spectra revealed the presence of two
different types of phospholipid: the phosphatidylethylamine
(PE) and phosphatidylglycerol (PG) types. Thirteen differ-
ent compounds could be identified and are listed in Table 3.
The position of the two fatty acids at the glycerol moiety
could be deduced because for the fatty acid positioned at sn-
2, the neutral loss as free fatty acid, as well as substituted
ketene, is more frequent than for that positioned at sn-1
(Murphy & Harrison, 1994). From the struc ture of the
lipids, it was evident that all lipids possessed an unsaturated
fatty acid at sn-2 of the glycerol moiety, whereas the sn-1
position was mainly occupied by saturated fatty acids. Such
a preference for having longer and saturated fatty acids at
sn-1 was described previously as a general feature of bac terial
phospholipids (Lechevalier, 1977), with only a few excep-
tions (Fang et al., 2000). As the distribution of fatty acids in
the molecule has some influence on the rigidity of the cell
wall, the finding that the proportion of saturated fatty acids
at the sn-1 position is higher for PG than for PE may have
consequences for the stability of cell-wall contact with
hydrocarbons.
DNA G+C content and genome format
The G+C content of the genomic DNA of strain MIL-1
T
is
53?2 mol%, which is comparable with the DNA G+C
contents of Marinobacterium and Oceanobacter (Table 1).
The G+C content of the amplified 16S rRNA gene sequence
of strain MIL-1
T
is 53?37 mol%. As revealed by PFGE
Table 1. Selected phenotypic properties that distinguish the genus Thalassolituus from other
related genera of marine c-Proteobacteria
Genera: 1, Marinobacterium;2,Marinomonas;3,Oceanobacter;4,Oleispira;5,Thalassolituus. Data from
Bowditch et al. (1984), Gonzalez & Whitman (2001), Satomi et al. (2002), Yakimov et al. (2002) and
this study.
Trait 1 2 3 4 5
Cell shape* St St St Cr Cr, Cb
FlagellationD 1 1–2 1 1 1–4
Accumulation of PHBd + 2 + 22
Lipase 22++ +
Reduction of nitrate ++++2
Utilization of:
D-Glucose ++22 2
D-Fructose V§ ++22
Ethanol +++22
n-Propanol +++22
n-Butanol +++22
Mannitol
ND|| ++22
Sorbitol +++22
Citrate +++22
p-Hydroxybenzoate +++22
DL-b-Hydroxybutyrate + 2 + 22
2-Oxoglutarate +
V + 22
DL-Lactate +++22
DL-Malate V ++22
Quinate +++22
Propionate +++22
DL-Alanine +++22
Aliphatic hydrocarbons 22
ND ++
Major 3-OH fatty acid (%) C
10 : 0
(100) C
10 : 0
(60) C
12 : 0
(54) None C
12 : 0
(100)
DNA G+C content (mol%) 55 44–48 55 41–42 53?2
*Cb, Coccoid bodies; Cr, curved rods; St, straight rods.
DNumber of flagella at one pole.
dPHB, Poly-
b-hydroxybutyrate.
§
V, Variable among strains
||
ND, No data available.
144 International Journal of Systematic and Evolutionary Microbiology 54
M. M. Yakimov and others
analysis of endonuclease digests of the genomic DNA of
isolate MIL-1
T
, the genome size was about 2?2 Mbp. No
plasmids were observed.
Molecular phylogenetic analysis
An almost-complete 16S rDNA sequence (1366 bp) was
determined for isolate MIL-1
T
. Preliminary sequence com-
parison against the 16S rRNA sequences held in GenBank
and the Ribosomal Database Project database (Altschul
et al., 1997; Maidak et al., 1997) indicated that the organism
belongs to the
c-subclass of the Proteobacteria. The sequence
was aligned manually against representatives of the
c-
Proteobacteria by using the secondary structure model of
bacterial 16S rRN A (Gutell, 1994). On the basis of 16S rDNA
similarity, strain MIL-1
T
showed an apparent relationship
with bacteria that belonged to the Marinomonas assemblage,
within a heterogeneous group that also contained the genus
Oceanospirillum. The closest relatives are Oceanobacter
kriegii ATCC 27133
T
(94?4 % 16S rDNA sequence similar-
ity), Oleispira antarctica LMG 21398
T
(92?5 %), Marino-
bacterium georgiense IAM 1419 (91?6 %), Oceanospirillum
multiglobuliferum NBRC 13614
T
(91?5 %), Marinomonas
mediterranea ATCC 700492
T
(91?2 %) and Oceanospirillum
linum ATCC 11336
T
(90?9 %). According to the method of
analysis (Satomi et al., 2002), strain MIL-1
T
formed a stable
phyletic group with Oceanobacter kriegii and Oleispira
antarctica and was evidently placed in the Marinomonas
assemblage. The branching point of MIL-1
T
was stable, as
the corresponding bootstrap values were very high (100, 71
and 78 %, respectively; Fig. 2). A very similar tree topology
was reconstructed by using the Jukes–Cantor treeing
algorithm (data not shown).
Alkane hydroxylase gene (alkB)
The putative gene for alkane hydroxylase, the key enzyme of
alkane catabolism, was cloned by using the approach of
Smits et al. (1999). Searches for coding areas revealed that
the sequenced DNA represented part of a larger ORF that
encoded a protein of 185 aa. Phylogenetic analysis of this
deduced polypeptide is shown in Fig. 3. The protein
sequence exhibited 80 % similarity to the corresponding
part of the 404 aa alkane hydroxylase of Alcanivorax borku-
mensis and clustered distinctly with the branch of pseudo-
monad alkane hydroxylases. Interestingly, we failed to
amplify the putative alkB gene from the most closely related
micro-organisms, Oceanobacter kriegii ATCC 27133
T
and
Oleispira antarctica DSM 14852
T
.
Polyphasic taxonomic treatment of strain MIL-1
T
unequivo-
cally indicates that the phylogenetic and phenotypic
differences between strain MIL-1
T
and its closest relatives
justify the description of a novel genus and species, Thalas-
solituus oleivorans gen. nov., sp. nov.
Description of Thalassolituus gen. nov.
Thalassolituus (Tha.las.so.li.tu9us. Gr. fem. n. thalassa the
sea; L. masc. n. lituus a curved rod, crook; N.L. masc. n.
Thalassolituus a marine, curve-shaped organism).
Gram-negative, vibrioid to spiral, motile cells, 1 ?2–3?5
mm
long by 0?6 mm wide. Strictly halophilic: Na
+
ions are
required for growth. Chemoorganoheterotrophic; strictly
aerobic; unable to grow under anaerobic conditions by
fermentation, nitrate reduction or phototrophically. Oxidase-
positive. Ammonia and nitrate may serve as nitrogen
sources. Indole-, arginine dihydrolase- and gelatinase-
negative. Acetate, C
7
–C
20
aliphatic hydrocarbons and their
oxidized derivatives are the only carbon sources that are
Table 3. Polar lipids identified by CID-MS in lipid extract of
T. oleivorans MIL-1
T
No. Mass Type sn-1 sn-2
1 687 PE C
16 : 1
C
16 : 1
2 687 PE C
14 : 1
C
18 : 1
3 687 PE C
14 : 0
C
18 : 2
4 689 PE C
16 : 0
C
16 : 1
5 689 PE C
14 : 0
C
18 : 1
6 715 PE C
16 : 1
C
18 : 1
7 717 PE C
16 : 0
C
18 : 1
8 717 PE C
18 : 0
C
16 : 1
9 720 PG C
16 : 0
C
16 : 1
10 720 PG C
14 : 0
C
18 : 1
11 746 PG C
16 : 1
C
18 : 1
12 748 PG C
16 : 0
C
18 : 1
13 776 PG C
18 : 0
C
18 : 1
Table 2. Fatty acid profiles of T. oleivorans MIL-1
T
and
another marine hydrocarbonoclastic c-proteobacterium,
Oleispira antarctica RB-8
T
Abbreviations: GL, glycolipids; PL, phospholipids; TL, total lipids.
Values given are percentages of the total for each type of lipid.
Fatty acid T. oleivorans O. antarctica*
PL GL TL PL TL
C
12 : 0
0?037?22?01?41?2
C
12 : 0
3-OH 0?00?01?40?00?0
C
14 : 1
v90?00?01?22?40?2
C
14 : 0
8?128?710?25?31?2
C
16 : 1
v717?60?033?549?929?9
C
16 : 0
31?620?629?532?523?9
C
18 : 1
v6/v930?77?910?92?432?0
C
18 : 0
12?05?67?70?01?2
OtherD 0?00?03?66?110?4
Total 100?0 100?0 100?0 100?0 100?0
*Grown at 20 uC.
DOther mean content of unidentified fatty acids in TL of T.
oleivorans and different fatty acids detected only in O. antarctica.
http://ijs.sgmjournals.org 145
Thalassolituus oleivorans gen. nov., sp. nov.
used for growth. Principal cellular fatty acids are laurate,
palmitate and octadecenoate. According to 16S rRNA gene
sequence analysis, the genus belongs to the
c-subgroup of
the Proteobacteria, namely to the Oceanospirillum/Marino-
monas/Marinobact erium assemblage. The type and only
species (to date) of the genus is Thalassolituus oleivorans.
Description of Thalassolituus oleivorans sp. nov.
Thalassolituus oleivorans (o.le.i.vo9rans.L.n.oleum oil; L. part.
adj. vorans devouring; N.L. adj. oleivorans oil-devouring).
Polymorphic bacteria that ar e motile by means of one to
four polar flagella. Genome size is about 2?2 Mbp. Mari ne;
requires at least 25 % sea water salinity for growth. Na
+
ions
are required; growth occurs at NaCl concentration s of
0?5–5?7 % (w/v), with optimum growth at 2?3 % NaCl.
Growth occurs at 4–30
u
C, with optimum growth at
20–25
u
C. pH range for growth is 7?5–9?0, with optimum
growth at pH 8?0. Tweens 20, 40 and 80 are degraded,
whereas agarase, amylase, arginine dihydrolase, ornithine
decarboxylase, lysine decarboxylase, gelatinase and aesculi-
nase activities are not detected. Nitrate is not reduced to
nitrite. Acetate, aliphatic hydrocarbons with a chain-length
between C
7
and C
20
and their oxid ized derivatives are the
only substrates that support growth. The principal fatty
Fig. 3. Phylogenetic position of the deduced
protein sequence for the T. oleivorans
MIL-1
T
cloned putative alkane hydroxylase
AlkB, among relevant enzymes of the c-
Proteobacteria. Numbers at nodes are boot-
strap confidence values (percentage of 100
bootstrap replications). Tree was rooted with
the sequence of the alkane-1-monooxygenase
of Rhodococcus erythropolis (GenBank
accession no. AJ301871). Bar, 0?1 substitu-
tion per sequence position.
Fig. 2. Position of T. oleivorans MIL-1
T
among
related representatives of the Oceanospirillum
assemblage with validly published names,
based on 16S rRNA phylogenetic analysis. The
tree, based on 1360 nucleotide positions, was
constructed by using the neighbour-joining
method and nucleotide substitution rates were
computed by using Kimura’s two-parameter
model, as described previously (Satomi et al.,
2002; Yakimov et al., 2002). Numbers at
nodes are bootstrap values (percentage of 500
trees analysed; only values >60 % are shown).
Bar, 0?02 substitution per sequence position.
146 International Journal of Systematic and Evolutionary Microbiology 54
M. M. Yakimov and others
acids in total TLFA, PLFA and GLFA profiles are C
12 : 0
,C
16 : 0
and C
18 : 1
. The TLFA and PLFA profiles are characterized
by an almost equal presence of saturated and monoun-
saturated fatty acids, with a strong predominance of C
14 : 0
,
C
16 : 1
,C
16 : 0
and C
18 : 1
. Phospholipids are represented by
the PE and PG types. DNA G+C content is 53?2 mol%.
According to analysis of the 16S rRNA gene sequence, this
bacterium belongs to the
c-subclass of the Proteobacteria and
forms a stable phyletic group with Oceanobacter kriegii.
The type and only strain to date, MIL-1
T
(=DSM 14913
T
=
LMG 21420
T
), was isolated after serial dilutions from an
enrichment culture that was established from sea water/
sediment samples collected in the harbour of Milazzo, Sicily,
Italy, by addition of n-tetradecane as the sole carbon source.
ACKNOWLEDGEMENTS
We are indebted to Rene Huppmann for assistance in fatty acid
analysis. This work was supported by grants from the Italian Ministry
for Education, University and Research (PEA 1999–2000, Research
Project 1.4 and CLUSTER 10, Project ‘SAM’). K. N. T. gratefully
acknowledges the generous support of the Fonds der Chemischen
Industrie. We thank Hans Tru
¨
per (University of Bonn) for advice and
corrections of the bacterial name.
REFERENCES
Abraham, W.-R., Meyer, H., Lindholst, S., Vancanneyt, M. & Smit, J.
(1997). Phospho- and sulfolipids as biomarkers of Caulobacter sensu
lato, Brevundimonas and Hyphomonas. Syst Appl Microbiol 20,
522–539.
Altschul, S. F., Madden, T. L., Scha
¨
ffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res 25,
3389–3402.
Bligh, E. G. & Dyer, W. J. (1959). A rapid method for total lipid
extraction and purification. Can J Biochem Physiol 37, 911–917.
Bowditch, R. D., Baumann, L. & Baumann, P. (1984). Description
of Oceanospirillum kriegii sp. nov. and O. jannaschii sp. nov.
and assignment of two species of Alteromonas to this genus as
O. commune comb. nov. and O. vagum comb. nov. Curr Microbiol
10, 221–230.
Button, D. K., Robertson, B. R., Lepp, P. W. & Schmidt, T. M. (1998).
A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by
extinction culture and having kinetic constants compatible with
growth at ambient concentrations of dissolved nutrients in seawater.
Appl Environ Microbiol 64, 4467–4476.
Dutta, T. K. & Harayama, S. (2001). Biodegradation of n-
alkylcycloalkanes and n-alkylbenzenes via new pathways in Alcanivorax
sp. strain MBIC 4326. Appl Environ Microbiol 67, 1970–1974.
Dyksterhouse, S. E., Gray, J. P., Herwig, R. P., Lara, J. C. & Staley,
J. T. (1995). Cycloclasticus pugetii gen. nov., sp. nov., an aromatic
hydrocarbon-degrading bacterium from marine sediments. Int J Syst
Bacteriol 45, 116–123.
Fang, J., Barcelona, M. J., Nogi, Y. & Kato, C. (2000). Biochemical
implications and geochemical significance of novel phospholipids of
the extremely barophilic bacteria from the Mariana Trench at
11,000 m. Deep-Sea Res Part I Oceanogr Res Pap 47, 1173–1182.
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version
3.5c. Department of Genetics, University of Washington, Seattle, USA.
Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M.,
Bonin, P. & Bertrand, J.-C. (1992).
Marinobacter hydrocarbonoclas-
ticus gen nov., sp. nov., a new, extremely halotolerant, hydrocarbon-
degrading marine bacterium. Int J Syst Bacteriol 42, 568–576.
Golyshin, P. N., Chernikova, T. N., Abraham, W.-R., Lu¨ nsdorf, H.,
Timmis, K. N. & Yakimov, M. M. (2002).
Oleiphilaceae fam. nov., to
include Oleiphilus messinensis gen. nov., sp. nov., a novel marine
bacterium that obligately utilizes hydrocarbons. Int J Syst Evol
Microbiol 52, 901–911.
Golyshina, O. V., Pivovarova, T. A., Karavaiko, G. I. & 7 other
authors (2000).
Ferroplasma acidiphilum gen. nov., sp. nov., an
acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking,
mesophilic member of the Ferroplasmaceae fam. nov., comprising
a distinct lineage of the Archaea. Int J Syst Evol Microbiol 50,
997–1006.
Gonza
´
lez, J. M. & Whitman, W. B. (2001). Oceanospirillum and
related genera. In The Prokaryotes (available at http://link.springer-ny.
com/link/service/books/10125/tocs.htm).
Gutell, R. R. (1994). Collection of small subunit (16S- and 16S-like)
ribosomal RNA structures: 1994. Nucleic Acids Res 22, 3502–3507.
Harayama, S., Kishira, H., Kasai, Y. & Shutsubo, K. (1999).
Petroleum biodegradation in marine environments. J Mol
Microbiol Biotechnol 1, 63–70.
Hedlund, B. P., Geiselbrecht, A. D., Bair, T. J. & Staley, J. T. (1999).
Polycyclic aromatic hydrocarbon degradation by a new marine
bacterium, Neptunomonas naphthovorans gen. nov., sp. nov. Appl
Environ Microbiol 65, 251–259.
Kasai, Y., Kishira, H., Syutsubo, K. & Harayama, S. (2001).
Molecular detection of marine bacterial populations on beaches
contaminated by the Nakhodka tanker oil-spill accident. Environ
Microbiol 3, 246–255.
Krieg, N. R. (1984). Genus Oceanospirillum Hylemon, Wells, Krieg
and Jannasch 1973, 361
AL
.InBergey’s Manual of Systematic
Bacteriology, vol. 1, pp. 104–110. Edited by N. R. Krieg & J. G.
Holt. Baltimore: Williams & Wilkins.
Lechevalier, M. P. (1977). Lipids in bacterial taxonomy – a
taxonomist’s view. Crit Rev Microbiol 5, 109–210.
Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey,
M. J. & Woese, C. R. (1997).
The RDP (Ribosomal Database Project).
Nucleic Acids Res 25, 109–111.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by
high-performance liquid chromatography. Int J Syst Bacteriol 39,
159–167.
Murphy, R. C. & Harrison, K. A. (1994). Fast atom bombardment
mass spectrometry of phospholipids. Mass Spectrom Rev 13, 57–75.
Pot, B., Gillis, M. & De Ley, J. (1992). The genus Oceanospirillum.
In The Prokaryotes. A Handbook on the Biology of Bacteria:
Ecophysiology, Isolation, Identification, Applications, 2nd edn,
vol. 4., pp. 3230–3236. Edited by A. Balows, H. G. Tru
¨
per,
M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Rambaut, A. (1996). Se-Al (Sequence Alignment Editor), version
1.0
a1. Distributed by the author and available via http://evolve.zoo.
ox.ac.uk/software.html?id=seal. Department of Zoology, University
of Oxford, UK.
Satomi, M., Kimura, B., Hayashi, M., Shouzen, Y., Okuzumi, M. &
Fujii, T. (1998).
Marinospirillum gen. nov., with descriptions of
Marinospirillum megaterium sp. nov., isolated from kusaya gravy,
and transfer of Oceanospirillum minutulum to Marinospirillum
minutulum comb. nov. Int J Syst Bacteriol 48, 1341–1348.
http://ijs.sgmjournals.org 147
Thalassolituus oleivorans gen. nov., sp. nov.
Satomi, M., Kimura, B., Hamada, T., Harayama, S. & Fujii, T. (2002).
Phylogenetic study of the genus Oceanospirillum based on 16S rRNA
and gyrB genes: emended description of the genus Oceanospirillum,
description of Pseudospirillum gen. nov., Oceanobacter gen. nov. and
Terasakiella gen. nov. and transfer of Oceanospirillum jannaschii and
Pseudomonas stanieri to Marinobacterium as Marinobacterium
jannaschii comb. nov. and Marinobacterium stanieri comb. nov.
Int J Syst Evol Microbiol 52, 739–747.
Smits, T. H. M., Ro
¨
thlisberger, M., Witholt, B. & van Beilen, J. B.
(1999). Molecular screening for alkane hydroxylase genes in Gram-
negative and Gram-positive strains. Environ Microbiol 1, 307–317.
Syutsubo, K., Kishira, H. & Harayama, S. (2001). Development of
specific oligonucleotide probes for the identification and in situ
detection of hydrocarbon-degrading Alcanivorax strains. Environ
Microbiol 3, 371–379.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reverse-phase high-performance liquid chromato-
graphy. FEMS Microbiol Lett 25, 125–128.
Vancanneyt, M., Witt, S., Abraham, W.-R., Kersters, K. &
Fredrickson, H. L. (1996).
Fatty acid content in whole-cell
hydrolysates and phospholipid fractions of pseudomonads: a
taxonomic evaluation. Syst Appl Microbiol 19, 528–540.
Wang, Y., Lau, P. C. K. & Button, D. K. (1996). A marine
oligobacterium harboring genes known to be part of aromatic
hydrocarbon degradation pathways of soil pseudomonads. Appl
Environ Microbiol 62, 2169–2173.
Yakimov, M. M., Golyshin, P. N., Lang, S., Moore, E. R. B.,
Abraham, W.-R., Lu¨ nsdorf, H. & Timmis, K. N. (1998). Alcanivorax
borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading
and surfactant-producing marine bacterium. Int J Syst Bacteriol 48,
339–348.
Yakimov, M. M., Giuliano, L., Gentile, G., Crisafi, E., Chernikova,
T. N., Abraham, W.-R., Lu¨ nsdorf, H., Timmis, K. N. & Golyshin, P. N.
(2003). Oleispira antarctica gen. nov., sp. nov., a novel hydro-
carbonoclastic marine bacterium isolated from Antarctic coastal sea
water. Int J Syst Evol Microbiol 53, 779–785.
148 International Journal of Systematic and Evolutionary Microbiology 54
M. M. Yakimov and others
