Psychromonas ingrahamii sp. nov., a novel gas vacuolate, psychrophilic bacterium isolated from Arctic polar sea ice

Abstract

A gas vacuolate bacterium, designated strain 37T, was isolated from a sea ice core collected from Point Barrow, Alaska, USA. Cells of strain 37T were large (6-14 microm in length), rod-shaped, contained gas vacuoles of two distinct morphologies, and grew well at NaCl concentrations of 1-10 % and at temperatures of -12 to 10 degrees C. The DNA G+C content was 40 mol%. Whole-cell fatty acid analysis showed that 16 : 1omega7c comprised 67 % of the total fatty acid content. Phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium was closely related to members of the genus Psychromonas, with highest sequence similarity (96.8 %) to Psychromonas antarctica. Phenotypic analysis differentiated strain 37T from P. antarctica on the basis of several characteristics, including cell morphology, growth temperature range and the ability to hydrolyse polymers. DNA-DNA hybridization experiments revealed a level of relatedness of 37 % between strain 37T and P. antarctica, providing further support that it represents a distinct species. The name Psychromonas ingrahamii sp. nov. is proposed for this novel species. The type strain is 37T (=CCUG 51855T=CIP 108865T).

Full text

Psychromonas ingrahamii sp. nov., a novel gas
vacuolate, psychrophilic bacterium isolated from
Arctic polar sea ice
Ann J. Auman,
1
Jennifer L. Breezee,
2
3 John J. Gosink,
2
4 Peter Ka¨mpfer
3
and James T. Staley
2
Correspondence
James T. Staley
jtstaley@u.washington.edu
1
Department of Biology, Pacific Lutheran University, Tacoma, WA 98447, USA
2
Department of Microbiology, University of Washington, Seattle, WA 98195, USA
3
Institut fu
¨
r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, Heinrich-Buff-Ring
26–32, D-35392 Giessen, Germany
A gas vacuolate bacterium, designated strain 37
T
, was isolated from a sea ice core collected from
Point Barrow, Alaska, USA. Cells of strain 37
T
were large (6–14 mm in length), rod-shaped,
contained gas vacuoles of two distinct morphologies, and grew well at NaCl concentrations of
1–10 % and at temperatures of ”12 to 10 6C. The DNA G+C content was 40 mol%. Whole-cell
fatty acid analysis showed that 16 : 1v7c comprised 67 % of the total fatty acid content.
Phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium was closely
related to members of the genus Psychromonas, with highest sequence similarity (96?8%) to
Psychromonas antarctica. Phenotypic analysis differentiated strain 37
T
from P. antarctica on the
basis of several characteristics, including cell morphology, growth temperature range and the
ability to hydrolyse polymers. DNA–DNA hybridization experiments revealed a level of relatedness of
37 % between strain 37
T
and P. antarctica, providing further support that it represents a distinct
species. The name Psychromonas ingrahamii sp. nov. is proposed for this novel species. The
type strain is 37
T
(=CCUG 51855
T
=CIP 108865
T
).
Most of the Earth’s biosphere never reaches tempera-
tures above 5
u
C and is home to a diverse group of micro-
organisms termed psychrophiles, having minimum,
optimum and maximum growth temperatures at or below
0, 15 and 20
u
C, respectively (Morita, 1975). One psychro-
philic ecosystem, polar sea ice, comprises 7–13 % of the
Earth’s surface at its maximum (Maykut, 1985; Parkinson &
Gloersen, 1993; Weeks & Ackley, 1982). Polar sea ice is
seasonably variable and its formation begins during polar
winter as the ocean surface waters freeze, forming a surface
slush termed ‘frazil ice’. This ice consolidates into circular
sheets of ‘pancake ice’, which become colonized by microbes
that eventually establish the sea ice microbial community
(SIMCO) (Nichol & Allison, 1997; Staley & Gosink, 1999;
Garrison et al., 1983). Polar sea ice is semisolid, contain-
ing channels of brine formed during ice crystallization.
Brine pockets may reach salinity levels of 150 % (Maykut,
1985), providing a liquid-phase environment at subzero
temperatures.
Sea ice is an active environment with large gradients in
light, temperature, nutrient availability and salinity, all of
which change seasonally (Eicken, 1992). The SIMCOs are
typically concentrated in the lower 10–20 cm of a sea ice
column, at the ice–water interface, where both sufficient
nutrients from the water column and sufficient surface light
are present (Staley & Gosink, 1999). The SIMCOs are
stratified, containing large varieties of both eukaryotes and
prokaryotes. Recent attempts to characterize the bacterial
component of SIMCOs have revealed great diversity. To
our knowledge, six new genera of the phylum Bacteroidetes
(Gosink et al., 1998; Bowman et al., 1998a, 1997, 2003;
Bowman & Nichols, 2002) and three new genera of Proteo-
bacteria (Gosink et al., 1997; Irgens et al., 1996; Bowman
et al., 1998b) have been identified within or near the SIMCO,
along with known Gram-positive genera (Junge et al., 1998).
Among the SIMCOs, gas vacuolate heterotrophs have been
discovered in high numbers from both the Arctic and the
Antarctic (Gosink et al., 1993; Staley et al., 1989), located
either in the water column below or in the ice above the
3Present address: Department of Special Bacteriology, Washington
State Department of Health, Shoreline, WA 98155, USA.
4Present address: Department of Bioinformatics, Amgen, Inc., Seattle,
WA 98119, USA.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain 37
T
is U73721.
Abbreviations: PUFA, polyunsaturated fatty acid; SIMCO, sea ice
microbial community.
64068
G
2006 IUMS Printed in Great Britain 1001
International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1001–1007 DOI 10.1099/ijs.0.64068-0

nutrient-rich SIMCO band (Gosink et al., 1993). Gas
vacuoles contain small, rigid, proteinaceous subunit vesicles
that are gas-permeable, reducing cell density as compared
with the cytoplasm, and thereby providing buoyancy
(Walsby, 1972). Gas vesicles act as organelles of motility,
regulating the vertical movement of cells via their synthesis
and degradation (Staley, 1980). Although gas vacuolate bac-
teria in polar sea ice are prevalent and phylogenetically
diverse, belonging to the Alpha-, Beta- and Gammaproteo-
bacteria and to the Bacteroidetes (Gosink & Staley, 1995), the
function of gas vacuoles in this environment remains
unknown.
Recently, we reported that a bacterial isolate, designated
strain 37
T
, isolated from a sea ice core from Point Barrow,
Alaska, USA, could grow at subfreezing temperatures, with a
generation time of 240 h at 212
u
C, the lowest recorded
growth temperature of any organism verified by a growth
curve (Breezee et al., 2004). Strain 37
T
was considered to
represent a novel species, which was provisionally named
Psychromonas ingrahamii sp. nov. It is most similar to
Psychromonas antarctica and other members of the genus
Psychromonas, a group of psychrophiles having a wide vari-
ety of physiological characteristics including piezophily,
facultative psychrophily and halophily (Breezee et al., 2004;
Mountfort et al., 1998; Kawasaki et al., 2002; Nogi et al.,
2002; Xu et al., 2003; Groudieva et al., 2003; Ivanova et al.,
2004). Here we report additional information for this sea
ice isolate to justify recognition of this novel species.
Strain 37
T
was isolated in May 1991 from Elson Lagoon
(Point Barrow, Alaska) about 130 cm from the ice–water
interface of a 1?8 m ice core (Gosink et al., 1993). Ordal’s sea
water cytophaga medium (SWC
m
) prepared in full-strength
artificial sea water (ASW) was used for the isolation and
routine growth of strain 37
T
(Irgens et al., 1989). Colonies
on plates were white, circular, smooth and convex, with an
entire margin. Phenotypic characteristics of strain 37
T
are
summarized in Table 1.
Cell size, shape and the presence of gas vacuoles were deter-
mined for cells grown in Difco marine broth 2216 (Becton
Dickinson) via phase contrast microscopy using a Zeiss
Photomicroscope I. Electron micrographs were obtained of
unstained whole cells by using a Zeiss EM900 transmission
electron microscope at 50 kV. Cells of strain 37
T
were
unusually large, ranging from 6 to 14
mm long by 1?25 to
1?5
mm wide (Fig. 1; Breezee et al., 2004), and were arranged
singly, in pairs or in short chains. Motility was examined by
incubation of strain 37
T
in liquid SWC
m
for 12 days with
periodic examination by phase contrast microscopy, and
cells were found to be non-motile. Gas vacuoles were also
visible, appearing as bright, refractive areas inside cells
(Fig. 1). Electron microscopy revealed two distinct mor-
phologies of gas vacuoles: numerous short, wide cylinders
with conical tips; and rare, longer but narrower cylinders
with conical ends (Fig. 1b). The presence of two gas vacuole
types is unusual, having been reported before only in the
halophilic archaeon Halobacterium halobium (Walsby, 1994).
The range of temperatures allowing growth of strain 37
T
was determined by observation of growth on SWC
m
plates at
5, 10, 15 and 20
u
C for 8 days. Growth at subzero tem-
peratures was also tested using liquid SWC
m
. Strain 37
T
was
psychrophilic, growing at temperatures from 212 to 10
u
C
with a generation time of 240 h at 212
u
C (Breezee et al.,
2004). No growth was observed at 15
u
C. Attempts to grow
strain 37
T
at 215
u
C were unsuccessful because the culture
medium routinely froze. The true minimum growth tem-
perature may in fact be lower than 212
u
C.
The pH range for growth was tested using SWC
m
buffered to
various pH values with 25 mM solutions of the following
buffers: MES, pH 5?7; ACES, pH 6?6; TAPSO, pH 7?4;
TAPS, pH 8?3; CHES, pH 9?0) (Dyksterhouse et al., 1995).
Growth at each pH was determined turbidometrically using
a Bausch and Lomb 20-D spectrophotometer at 600 nm.
Growth was observed at near neutral pH values (pH 6?5, 6?8
and 7?4), but not at moderately acidic (pH 5?0) or basic
(pH 8?3, 9?0) values.
Requirement for and tolerance to NaCl were determined by
observing growth on CLED agar (Difco) supplemented with
0–22 % NaCl. Strain 37
T
required NaCl for growth, showing
no growth at 0 % NaCl. It grew well at 1–12 % NaCl, and
weak growth was observed at NaCl concentrations as high as
20 %.
The ability of strain 37
T
to use a particular substrate as its
sole carbon source was tested at substrate concentrations
of 0?2 % in SWC
m
in microtitre plate wells. Strain 37
T
was
inoculated in triplicate and incubated for 21 days at 5
u
C.
Growth was determined by measuring the absorbance at
600 nm using a DeltaSoft II microplate reader. Strain 37
T
was able to use a wide variety of carbon sources, as detailed
in the species description later. Sugar fermentation was
tested using the Hugh–Leifson method (Gerhardt et al.,
1981). BBL brand OF basal medium was dissolved in ONR-
7a salt solution (Dyksterhouse et al., 1995). Each carbon
source was diluted to a concentration of 1 %. Vibrio splendi-
dus and inoculated medium without added carbon source
were used as positive and negative controls, respectively. Gas
production from glucose metabolism was detected by grow-
ing strain 37
T
in liquid SWC
m
supplemented with glucose
into which Durham tubes were placed for gas detection.
Strain 37
T
was facultatively anaerobic and fermented several
carbon sources, including lactose, sucrose,
D-mannitol,
salicin, maltose, trehalose, cellobiose,
D-galactose, melibiose
and
D-glucose (without gas production), but not dulcitol,
myo-inositol,
D-sorbitol, L-arabinose or D-xylose.
Biochemical tests were performed using standard methodo-
logy (Gerhardt et al., 1981). For these tests, cultures of strain
37
T
were grown in SWC
m
supplemented with the appro-
priate substrates. For nitrate reduction, strains were supple-
mented with 0? 1or0?01 % NaNO
3
and 0?17 % agar. Cells
of strain 37
T
were Gram-negative, oxidase-positive, weakly
catalase-positive and positive for nitrate reduction, all traits
characteristic of members of the genus Psychromonas.
1002 International Journal of Systematic and Evolutionary Microbiology 56
A. J. Auman and others

However, strain 37
T
could not hydrolyse starch or gelatin.
For determination of indole production, cultures were grown
in SWC
m
lacking succinate and were tested at three different
stages of growth; indole production was not observed.
The whole-cell fatty acid composition was determined using
fatty acid methyl ester analysis of cells grown on SWC
m
plates at 4
u
C. Cells were scraped into 136100 mm Teflon-
lined tubes, frozen at 280
u
C and lysed. The fatty acids were
Table 1. Comparison of characteristics of P. ingrahamii sp. nov. and other members of the genus Psychromonas
Taxa: 1, P. ingrahamii 37
T
;2,P. antarctica DSM 10704
T
;3,P. arctica Pull 5.3
T
;4,P. kaikoae JT7304
T
;5,P. marina 4-22
T
;6,P. profunda
2825
T
. All were Gram-negative, oxidase-positive, catalase-positive, and able to use D-glucose and D-fructose as sole carbon sources.
Characteristics are scored as: +, positive; 2, negative;
W, weakly positive after 3 weeks; (W), weakly positive after 6 weeks. NR, Not
reported;
ND, not determined. Data for other Psychromonas species were taken from Breezee et al. (2004), Mountfort et al. (1998), Kawasaki
et al. (2002), Nogi et al. (2002), Groudieva et al. (2003), Xu et al. (2003) and Brenner et al. (2005).
Characteristic 1 2 3 4 5 6
Cell morphology and arrangement Large rods;
singles, pairs
Ovoid rods;
singles, pairs
Rods; singles,
pairs
Ovoid rods Rods Rods
Cell length (
mm) 6–14 2?5–6 1?3–2?6 2–4 1?5–2?02?0–5?5
Production of gas vesicles + 22222
Colony colour White White White
NR Colourless Colourless
Motility 2 +++++
Carbon sources utilized:
D-Galactose ++ND +++
D-Mannose 22 ++22
D-Mannitol ++ +++W
D
-Sorbitol 22ND 222
N-Acetylglucosamine ++
ND ND + ND
Arabinose 2 ND ND 222
D-Xylose 22 22++
Cellobiose + 2
ND +++
Lactose 22 + 2 ++
Maltose
ND +++++
Sucrose ++ +++
W
Fumarate + 2 + ND ND +
DL-Lactate + 22ND ND (W)
DL-Malate 22 2ND ND ND
Glycerol + 2 + 2 + (W)
Carbon sources fermented:
D-Glucose (Gas) ++(G) + (G) +++
myo-Inositol 22
ND 22+
Lactose + 2
ND 2 ++
Trehalose ++
ND + 2 +
D-Xylose 22 22++
Polymers hydrolysed:
Starch 2 ++2 +
W
Gelatin 2 + 2 + 22
NaCl concentration allowing growth (%) 1–12* 0–4 1–7 >0 0–7 % >0
Growth temperature range (uC) 212 to 10D 2–17 0–25 4–15 0–25 2–14
pH range (optimum) 6?5–7?4(6?5) 6?5–9?8(8?8)
ND ND ND
O
2
requirement for growth Facultative
anaerobe
Aerotolerant
anaerobe
Aerobe Facultative
anaerobe
Facultative
anaerobe
Facultative
anaerobe
Growth at atmospheric pressure ++ +2 ++
Indole 22
ND 22+
Nitrate reduction + 22+++
DNA G+C content (mol%) 40 42?840?143?843?538?1
*Weak growth was seen at concentrations up to 20 %.
DAttempts to grow P. ingrahamii at temperatures below 212 uC were inconclusive because the culture medium froze.
http://ijs.sgmjournals.org 1003
Psychromonas ingrahamii sp. nov.

saponified with methanolic base, then converted to fatty
acid methyl esters with HCl using the MIDI protocol as pre-
viously described (MIDI, 1993). A Hewlett Packard model
5890 Series II gas chromatograph was used to identify and
quantify the fatty acid methyl esters. This analysis revealed
the principal constituents to be 16-carbon unsaturated and
saturated fatty acids 16 : 1
v7c and 16 : 0, making up 67 and
18?7 %, respectively, of the whole-cell fatty acid content.
Other Psychromonas species also contain high concentra-
tions of 16 : 1, ranging between 39 % in Psychrom onas marina
4-22
T
and 58 % in P. antarctica DSM 10704
T
(Table 2).
Other fatty acids found in measurable quantities in strain
37
T
included 18 : 1 (3?6 %) and 12 : 0 (2?5 %). Our analysis
was unable to distinguish between the fatty acids 12 : 0 alde,
16 : 1 ISO and 14 : 0 3-OH, and 4?5 % of the fatty acids from
strain 37
T
were among this group. The fatty acid com-
position of strain 37
T
is summarized in Table 2.
Genomic DNA from strain 37
T
was isolated using a hexadecyl-
trimethylammonium bromide miniprep protocol (Ausubel
et al., 1989). The DNA G+ C content of strain 37
T
was deter-
mined by HPLC according to the method of Mesbah et al.
(1989) and found to be 40 mol%, within the range of
38?1–43?8 mol% reported for other members of the genus
Fig. 1. Phase contrast (a) and transmission electron (b) micrographs of cells of strain 37
T
. Bars, 5 and 0?6 mm, respectively.
Bright areas within the cells observed by phase contrast microscopy are gas vacuoles. The characteristic morphology of the
subunit gas vesicles, i.e. their cylindrical shape with conical polar caps, is shown in (b).
Table 2. Fatty acid content of P. ingrahamii sp. nov. and other members of the genus Psychromonas
Taxa: 1, P. ingrahamii 37
T
;2,P. antarctica DSM 10704
T
;3,P. arctica Pull 5.3
T
;4,P. kaikoae JT7304
T
;5,P. marina 4-22
T
;6,P. profunda
2825
T
. Values are percentages of total fatty acids. Isomers are shown in parentheses if known. Results below 1 % are not shown. Data for
other Psychromonas species were taken from Kawasaki et al. (2002), Nogi et al. (2002), Groudieva et al. (2003) and Xu et al. (2003).
Fatty acid 1 2 3 4 5 6
12 : 0 2?51 2?7–5?21
14 : 0 6
15 : 0 1
16 : 0 18?724 7?0–16?21543?631
14 : 1 8 (
v7t)2?7–5?2(v5t)10(v7t)3?215
16 : 1 67 (
v7c)58(v7c) ~50 (v7c), 7?0–16?2(v7t)52(v7c), 2 (v9c)39?444
18 : 1 3?63(
v7c)7?0–16?2(v7) 2 (v7c)3?1
20 : 5
v3 2
22 : 6 21?6
12 : 0 3-OH 2
12 : 0 alde, 16 : 1 ISO
or 14 : 0 3-OH
4?5 6 (14 : 0 3-OH) 4 (14 : 0 3-OH) 2?7 (16 : 1 ISO)
1004 International Journal of Systematic and Evolutionary Microbiology 56
A. J. Auman and others

Psychromonas (Mountfort et al., 1998; Kawasaki et al., 2002;
Nogi et al., 2002; Groudieva et al., 2003; Xu et al., 2003).
The 16S rRNA gene from strain 37
T
was sequenced as
described by Gosink & Staley (1995). The EMBL accession
numbers for additional 16S rRNA gene sequences used for
analysis are given in parentheses in Fig. 2. These sequences
were aligned using
CLUSTAL_X (Thompson et al., 1997).
Phylogenetic trees were constructed by determining dis-
tances (according to the Kimura two-parameter model) and
clustering (with the neighbour-joining method) by using the
MEGA (Molecular Evolutionary Genetics Analysis) version
2.1 software package (Kumar et al., 2001). Phylogenetic
analysis of the 16S rRNA gene sequence revealed that strain
37
T
was a member of the Gammaproteobacteria, was related
most closely to P. antarctica DSM 10704
T
, showing 96? 8%
similarity at the nucleotide level, and clustered with other
members of the genus Psychromonas (Fig. 2). Strain 37
T
was
also related closely (>98 % sequence similarity) to two other
polar sea ice taxa, strain 174 (EMBL accession no. U73722)
and strain 90Pgv1 (EMBL accession no. U14582), isolated
from the Arctic and Antarctic, respectively, that have not yet
been fully characterized (Staley & Gosink, 1999).
Although strain 37
T
differed significantly at the phenotypic
level from P. antarctica DSM 10704
T
and other members of
the genus Psychromonas (see Table 1), the high degree of 16S
rRNA gene sequence similarity warranted further examina-
tion at the molecular level. DNA–DNA hybridization experi-
ments were performed using the method described by
Ziemke et al. (1998), except that for nick translation, 2
mg
DNA was labelled during 3 h incubation at 15
u
C using
genomic DNA isolated from strain 37
T
and P. antarctica
DSM 10704
T
. The reassociation value between these two
strains was 37?1 % (reciprocal 38?8 %), confirming that
strain 37
T
represents a novel species, according to accepted
criteria (Wayne et al., 1987).
Members of the genus Psychromonas have been isolated
from a variety of low-temperature environments, including
a high-salinity pond on the McMurdo ice-shelf (Mountfort
et al., 1998), deep-sea cold-seep sediments near Japan (Xu
et al., 2003; Nogi et al., 2002), Japanese cold-current coastal
sea water (Kawasaki et al., 2002), and northern European
Arctic sea water and sea ice (Groudieva et al., 2003). Members
of this genus display great phenotypic diversity, ranging in
degrees of piezophily and temperature range of growth.
Strain 37
T
, isolated from a sea ice core, represents a novel
species within this genus and is unique among this group in
its unusually large cell size, its ability to grow at subfreezing
temperatures, its tolerance to high salt concentrations and
its ability to produce gas vacuoles (see Table 1). Unlike other
Psychromonas strains, strain 37
T
cannot hydrolyse the poly-
mers starch or gelatin and appears to be non-flagellated.
The abilities of strain 37
T
to withstand both high salt con-
centrations and subfreezing temperatures are consistent
with the polar sea ice environment from which it was
isolated. The semisolid matrix of polar sea ice consists of ice
crystals around which extruded brine accumulates to high
concentrations. The high salt concentrations within these
brine pockets allow the water to remain liquid at tem-
peratures well below freezing. It is within these high-salt,
low-temperature microenvironments that members of the
SIMCOs persist.
Although the formation of gas vacuoles by strain 37
T
is
unique among members of the Psychromonas genus, it is not
unusual for a polar sea ice bacterium. Within the SIMCOs,
gas vacuolate bacteria are abundant and phylogenetically
diverse, with representatives in the Alpha-, Beta- and
Gammaproteobacteria, and within the phylum Bacteroidetes
(Gosink et al., 1993; Gosink & Staley, 1995; Irgens et al.,
1989). Strain 37
T
is unusual, however, in its ability to pro-
duce two distinct gas vacuole morphotypes within a single
Fig. 2. Phylogenetic analysis based on 16S
rRNA gene sequences available from the
European Molecular Biology Laboratory data-
base (accession numbers are given in paren-
theses), constructed after multiple alignment of
data by using
CLUSTAL_X (Thompson et al.,
1997). Distances (distance options accord-
ing to the Kimura two-parameter model) and
clustering with the neighbour-joining method
were determined by using the software pack-
age
MEGA (Molecular Evolutionary Genetics
Analysis) version 2.1 (Kumar et al., 2001).
Bootstrap values, based on 1000 replica-
tions, are given as percentages at branch
points. Bar, 0?02 substitutions per mean
nucleotide position.
http://ijs.sgmjournals.org 1005
Psychromonas ingrahamii sp. nov.

cell, previously reported only in a halophilic archaeon
(Walsby, 1994).
Unlike Ps ychromonas kaikoae JT7304
T
and P. marina 4-22
T
,
strain 37
T
cell membranes contain no measurable amounts
of polyunsaturated fatty acids (PUFAs) such as 20 : 5
(eicosapentaenoic acid) or 22 : 6 (docosahexaenoic acid).
As the concentration of PUFAs has been suggested to be
inversely proportional to optimum growth temperature, the
lack of PUFAs in strain 37
T
, which grows at subfreezing
temperatures lower than those of other Psychromonas
species, is inconsistent with this hypothesis (Bowman
et al., 1998c).
Phylogenetic analysis of 16S rRNA gene sequences indicated
that strain 37
T
, isolated from sea ice from Point Barrow,
Alaska, was most closely related to P. antarctica DSM
10704
T
, isolated from a high-salinity pond sediment (96?8%
sequence similarity). Strain 37
T
was also closely related to
two polar sea ice taxa, 174 and 90Pgv1, isolated from Arctic
and Antarctic sea ice, respectively (Staley & Gosink, 1999). It
is interesting that such closely related organisms have been
isolated from opposite polar regions. This phenomenon has
been shown previously for other sea ice genera, including
Polaribacter and Octadecabacter (Gosink et al., 1997, 1998),
and is supported by studies of Arctic and Antarctic sea ice
communities using culture-independent molecular techni-
ques (Brown & Bowman, 2001; Brinkmeyer et al., 2003).
This suggests that organismal dispersal was followed by
acquisition of traits required for adaptation to particular
microenvironments.
Description of Psychromonas ingrahamii
sp. nov.
Psychromonas ingrahamii (in.gra.ham9.i.i. N.L. gen. n. ingra-
hamii of Ingraham, in honour of John L. Ingraham for his
extensive research on psychrophilic bacteria).
Cells are Gram-negative, non-motile large rods, 6–14
mm
long and 1?25–1?5 mm wide, found either singly or in pairs,
and containing two gas vesicle morphotypes. On SWC
m
,
colonies are white, circular, smooth and convex, with an
entire margin. Moderately halophilic (growth at NaCl con-
centrations of 1–12 %, with weak growth up to 20 %, but
no growth without NaCl), and strictly psychrophilic. Tem-
perature range for growth is 212
u
C (with a generation time
of 240 h) or lower (not tested) to 10
u
C or higher (not tested
between 10 and 15
u
C, but no growth is observed at 15
u
C).
The pH range for growth is 6?5–7?4. Grows at atmospheric
pressure. Facultative anaerobe, capable of both respiratory
and fermentative metabolism. Catalase- and cytochrome
oxidase-positive. Reduces inorganic nitrate. Indole test is
negative. Predominant cellular fatty acids are 16 : 1
v7c and
16 : 0. Utilizes as sole carbon sources D-glucose, D-ribose,
D-fructose, sucrose, L-glutamate, L-cysteine, DL-aspartate,
fumarate, succinate, pyruvate, propionate, acetate, glycerol,
N-acetylglucosamine, glucosamine, cellobiose,
DL-lactate,
D-mannitol, salicin, trehalose and D-glucuronate, but not
lactose,
L-leucine, L-proline, a-ketoglutarate, citrate, benzo-
ate, glycolate, methanol, arabinose, caproate,
D-gluconate,
myo-inositol, DL-malate, D-mannose, D-sorbitol or D-
xylose. Can ferment lactose, sucrose,
D-mannitol, salicin,
maltose, trehalose, cellobiose,
D-galactose, melibiose and
D-glucose (without gas production), but not dulcitol, myo-
inositol, D-sorbitol, L-arabinose or D-xylose. No starch or
gelatin hydrolysis. The DNA G+C content is 40 mol%.
The type and only strain, 37
T
(=CCUG 51855
T
=CIP
108865
T
), was isolated from Elson Lagoon (Point Barrow,
Alaska, USA) about 130 cm from the ice–water interface
from a 1? 8 m ice core.
Acknowledgements
This work was supported in part by NSF grant BSR 9006788, the
University of Washington (UW) NSF IGERT DGE-9870713 as well as
the UW NASA NAI programs. We thank Margaret L. Hudson at Seattle
University (Seattle, WA) for assisting with transmission electron
microscopy.
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http://ijs.sgmjournals.org 1007
Psychromonas ingrahamii sp. nov.