FEMS Microbiology Ecology 32 (2000) 249^260
www.fems-microbiology.org
High incidence of halotolerant bacteria in Paci¢c hydrothermal-vent
and pelagic environments
Jonathan Z. Kaye *, John A. Baross
University of Washington, School of Oceanography, Box 357940, Seattle, WA 98195-7940, USA
Received 15 November 1999 ; received in revised form 10 April 2000; accepted 10 April 2000
Abstract
The abundance of halotolerant microorganisms in hydrothermal-vent and pelagic waters in the North and South Pacific was estimated by
the most probable number (MPN) technique using a heterotrophic 16% NaCl medium incubated at 20^24³C. Based on these MPNs and
direct counts with epifluorescence microscopy to enumerate the total microbial population, salt-tolerant microbes comprised from 6 0.01
to s 28% of the total microbial community. Fourteen isolates from these MPN enrichments were identified by sequencing a portion of the
16S rRNA gene, and all were found to belong to the genera Halomonas and Marinobacter. The response to salt of mesophilic hydrothermalvent microbial isolates obtained without selecting for salt tolerance was also examined. Forty-one of 65 strains cultured from hydrothermal
plume waters, low-temperature hydrothermal fluids, sulfide rock and an animal specimen at V2000^2200 m depth from the Endeavour
Segment of the Juan de Fuca Ridge were subjected to increasing concentrations of NaCl, and over half grew at a NaCl concentration that is
lethal to many commonly isolated marine bacteria. At least 36 of the 65 isolates (v 55%) grew in the enrichment medium supplemented with
10% NaCl; at least 30 of 65 (v 46%) grew with 16% NaCl; at least 20 of 65 (v 31%) tolerated 22% NaCl. Based on phylogenetic analysis of
the 16S rRNA gene in nine of these 65 isolates, four belonged to the genus Halomonas. These Halomonas strains tolerated 22^27% NaCl. It
is possible that a majority of the other 16 isolates which grew with 22% NaCl are also Halomonas based on their degree of halotolerance,
morphology, and apparent abundance as revealed by MPN enrichments. The four Halomonas strains obtained without selecting for
halotolerance were further characterized physiologically and metabolically. Overall, they grew between 31³C and 40³C, were facultative
aerobes, oxidized between 49 and 70 organic compounds according to Biolog plate substrate utilization matrices, grew with oligotrophic
quantities of carbon (0.002% yeast extract) in liquid media, reduced nitrate to nitrite, and tolerated up to 0.05^3 mM Cd2‡ . Halomonas is
one of the most abundant culturable organisms in the ocean, and its success may be attributed to its metabolic and physiological
versatility. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Culturability ; Halotolerance; Hydrothermal vent; Halomonas; Marinobacter
1. Introduction
Molecular phylogenetic analyses of prokaryotic communities in pelagic marine environments reveal that they
consist predominantly of clusters of uncultured bacterial
and archaeal species [1^3]. For example, the small-subunit
rRNA sequence databases show that as yet uncultured
bacteria belonging to the K-subclass of the Proteobacteria
(e.g. SAR clusters) comprise up to 25% of the total microbial community [3] and are abundant in both Paci¢c and
Atlantic waters [4]. The role that these numerically dominant microorganisms play in oceanic ecosystems remains
elusive however, because nothing is known about their
* Corresponding author. Tel. : +1 (206) 616-9041;
Fax: +1 (206) 543-0275; E-mail: [email protected]
metabolism or physiology [3,5] and because the phylogenetic distance is large between these organisms and nearly
all characterized bacteria, rendering phenotypic inferences
unreliable [5].
An understanding of the metabolic and other relevant
ecological functions of these numerically dominant microbes generally necessitates their isolation and characterization using pure-culture techniques. Media designed to
enrich for uncultured pelagic microorganisms, which are
hypothesized to be dominated by oligotrophs, attempt to
mimic in situ conditions by using low concentrations of
organic carbon [5]. E¡orts to culture such numerically
dominant microbes have made slow progress, and still
less than 0.1% of the total microbial community can be
cultured on an array of heterotrophic solid media [5]. This
low incidence of culturability among heterotrophs is attributed to imbalances in or inappropriate mixtures of
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 3 5 - 0
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J.Z. Kaye, J.A. Baross / FEMS Microbiology Ecology 32 (2000) 249^260
organic compounds in enrichment media, inhibition by
other organisms, and viral lysis due to the improved nutritional conditions provided to the bacteria [5]. However,
the marine oligotroph Sphingomonas sp. strain RB2256
was ¢rst isolated by dilution culture [6], and it may be a
metabolically active and numerically abundant species in
some pelagic environments [5,7]. Culturing very high percentages of the total microbial count has been achieved by
enumerating aggregate-forming units on solid media with
epi£uorescence microscopy [8], but this approach has limitations and does not allow for the isolation and subsequent characterization of microbial strains. Overall, attempts to isolate numerically dominant microorganisms
most often revolve around creating in situ conditions by
adjusting media compositions, culturing surfaces (e.g. agar
plates, membrane ¢lters), or dilutions with unamended
¢ltered seawater [5,6,8].
Little is known about the microbial community structure in the deep-sea, mid-ocean ridge setting. The hydrothermal-vent environments associated with mid-ocean
ridges include positively buoyant and neutrally buoyant
(lateral) hydrothermal plumes (V2³C), low-temperature
hydrothermal £uids (V5^100³C), high-temperature hydrothermal £uids (V150^400³C), sul¢de rock, basalt,
and pelagic and metalliferous sediments [9^12]. Vent
waters and bottom seawater in close proximity to hydrothermal vents are typically enriched in inorganic metabolic
energy sources, including H2 , CH4 and Mn [9,13]. The
concentration of organic carbon in hydrothermal plumes
is low, similar to the non-vent, deep-sea water column (D.
Butter¢eld, unpublished data). Community phylogenetic
analyses have focused on microbial mats and high-temperature hydrothermal £uids [14,15], and culturing work has
been biased towards hyperthermophiles, thermophiles, and
mesophilic sulfur- and metal-metabolizing chemoautotrophs [10,12,16].
We isolated numerous bacterial strains from hydrothermal plumes, low-temperature hydrothermal £uids, and
other vent samples using an oligotrophic medium
amended with enriched levels of reduced transition metals
and H2 or CH4 in some instances. A signi¢cant portion of
these isolates were found to tolerate high levels of salt and
heavy metals [17]. Here we report on the abundance and
characterization of these and other halotolerant microorganisms in both hydrothermal-vent and pelagic environments.
2. Materials and methods
2.1. Enrichment medium
The isolation medium contained synthetic seawater (Sea
Salts B), a trace elements solution (Trace Elements F) and
additional nutrients. Sea Salts B has (per liter of deionized
water) 19.6 g NaCl, 3.3 g Na2 SO4 , 0.5 g KCl, 0.05 g KBr,
0.02 g H3 BO3 and 8.8 g MgCl2 W6H2 O [18]. The medium
was supplemented with 10 ml of the trace elements solution (per liter of Sea Salts B). Trace Elements F consists of
(per liter of deionized water) 0.05 g Al2 (SO4 )3 , 0.1 g
H3 BO3 , 0.05 g LiCl, 0.1 g Na2 MoO4 W2H2 O, 0.05 g KBr,
0.05 g KI, 0.05 g NaF, 0.1 g ZnSO4 W7H2 O, 0.005 g BaCl2 ,
0.005 g CoCl2 W6H2 O, 0.01 g CuSO4 W5H2 O, 0.2 g
MnCl2 W4H2 O, 0.01 g NiCl2 W6H2 O, 0.005 g Na2 SeO4 ,
0.005 g SrCl2 W6H2 O, 0.005 g H2 WO4 and 0.005 g
VOSO4 WxH2 O [18,19]. For enrichments in 1991, the trace
elements solution contained 0.015 g NiCl2 W6H2 O but no
NaF. Lastly, the additional components of the isolation
medium include (per liter of Sea Salts B) 1.605 g NaNO3 ,
5.0 g Na2 S2 O3 W5H2 O, 0.02 g yeast extract, 1.0 g PIPES
bu¡er (piperazine-N,NP-bis[2-ethanesulfonic acid] disodium salt), 0.002 g FeSO4 W7H2 O, 0.15 g MnSO4 WH2 O, 0.1
g CaCl2 , 0.430 g (NH4 )2 SO4 and 0.036 g KH2 PO4 . Trace
Elements F, FeSO4 W7H2 O, MnSO4 WH2 O, CaCl2 ,
(NH4 )2 SO4 , 0.605 g of the NaNO3 and KH2 PO4 were
added after autoclaving via ¢lter sterilization. When necessary, the medium was solidi¢ed with 13 g (per liter)
puri¢ed agar (BBL). The pH was adjusted to 7 or 9 with
¢lter-sterilized 1 M HCl or 1 M KOH if needed. Some
agar slants were prepared in stoppered serum bottles with
a H2 /CO2 (80:20 vol.%) or CH4 headspace achieved by
purging and ¢lling the bottles with gas four times [20];
others simply contained an air headspace.
The medium used for most probable number (MPN)
enrichments contained (per liter of isolation medium)
156.8 g NaCl and 1.0 g sodium citrate as well.
2.2. Quantitative enrichments for halotolerant
microorganisms
To quantify halotolerant microorganisms in the vent
environment and overlying water column, the three-tube
MPN technique [21] was used with the growth medium at
an elevated NaCl concentration of 16% and with 0.1%
sodium citrate. Forty-three samples of low-temperature
hydrothermal £uid, buoyant hydrothermal plume, lateral
hydrothermal plume, bottom seawater in close proximity
to hydrothermal vents, and non-vent seawater from 10 to
2800 m depth were collected with the following samplers :
(1) a Niskin bottle rosette with a conductivity^temperature^depth^transmissometry (CTDT) package to detect
hydrothermal plume temperature and particle anomalies;
(2) Niskin bottles attached to the Deep-Submergence Vehicle Alvin basket; (3) the Remote-Operated Vehicle ROPOS suction sampler; (4) the Hot Fluid Sampler (NOAAPMEL VENTS Program, Seattle, WA, USA) mounted on
ROPOS; or (5) titanium syringe samplers [22] triggered by
Alvin. These samples were procured in 1998 from within or
above the Mothra vent ¢eld on the Endeavour Segment
(48³N, Juan de Fuca Ridge (JdFR)), Axial Seamount
(46³N, JdFR), and 17.5^21.5³S along the Southern East
Paci¢c Rise (SEPR). Seawater or vent water was diluted
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with autoclaved Sea Salts B in sterile Falcon tubes. Between 0.02 Wl and 200 Wl of sample water was then inoculated in triplicate onto agar plates or into broth and
incubated at room temperature (20^24³C) onboard ship.
Growth of colonies or turbidity in tubes indicated preliminary positive results. A dissecting microscope was used to
verify the presence of colonies if necessary, and visualization with phase-contrast microscopy (Zeiss) con¢rmed all
positive and negative results for the MPNs in broth.
Thirteen cultures were isolated from the most dilute
positive MPN enrichment tube of a given sample by transferring 100 Wl from that MPN tube onto an agar plate of
the same composition, and subsequently isolating colonies
by triplicate streak transfers. One strain (A-sw1) was isolated from the second-most dilute MPN enrichment tube
in the same manner.
2.3. Counts of total microorganisms
Each water sample (36^37 ml) was preserved with either
glutaraldehyde (type II, 2% ¢nal concentration) or formalin (4% ¢nal concentration) and then stored at 2³C for 9^14
months before counting. Aliquots of ¢xed sample were
¢ltered onto 0.22-Wm black polycarbonate ¢lters (Osmonics, Inc.) and stained with 4P,6-diamidino-2-phenylindole. Cells were enumerated by epi£uorescence microscopy
(Zeiss) [23].
2.4. Isolation of oligotrophs without selecting for
halotolerance
Oligotrophic microbes were initially enriched from hydrothermal-vent samples in 1991 and 1995, all from
V2000^2200 m depth from the Main Endeavour Field
251
on the Endeavour Segment of the JdFR. Inocula included
hydrothermal plume water, low-temperature hydrothermal
£uids, sul¢de rock and a vestimentiferan (Ridgeia piscesae)
trophosome. Hydrothermal plume temperature and particle anomalies were detected with a CTDT package and
sampled with a rosette of Niskin bottles. Rocks and animals were procured with Alvin manipulators.
Solid samples were placed directly into the enrichment
medium. Water samples (1, 10 and 50 ml) were ¢rst ¢ltered onto 0.2-Wm polycarbonate ¢lters and then placed
onto agar slants in 55-ml serum bottles. Samples were
incubated at either 2 or V20³C aboard ship.
2.5. Phenotypic characterization of vent isolates
Nine oligotrophic isolates were characterized physiologically and metabolically. After isolation, they were grown
on agar plates and in broth composed of a medium
slightly modi¢ed from the enrichment medium. This
growth medium always had an air headspace, was made
at pH 7, and contained 0.1% sodium citrate to achieve
higher cell densities. Unless otherwise noted, all experiments occurred at 23³C, room temperature. Halomonas
paci¢ca and Halomonas aquamarina were obtained from
the American Type Culture Collection.
The Gram stain was performed according to a standard
procedure [24].
The ability to grow at elevated salt concentrations was
assessed with the growth medium augmented with NaCl.
Growth was monitored by phase-contrast microscopy
(Zeiss) and scored as positive if the concentration of cells
reached v 107 ml31 from an initial concentration of 9 105
ml31 .
To determine which organic carbon compounds could
Table 1
Range in the number of culturable halotolerant microorganisms (ml31 ) based on three-tube MPN estimates using a 16% NaCl heterotrophic medium,
range in the total number of cells (ml31 ) based on epi£uorescence microscopy, and range in the percentage of total microorganisms which are halotolerant, in seawater and hydrothermal-vent samples
Sample type
Depth
(m)
Halotolerant microbes
(ml31 )
Total microbes U104
(ml31 )a
Halotolerant percentage
(%)
Isolateb
Surface seawater
Mid-water
10^30
500^1 400
45^4 700
45^12 000
26 þ 6.5 to 62 þ 12
2.0 þ 0.57 to 7.4 þ 1.0
0.01^1.4
0.06^26
Deep water
Lateral plume
2 000^2 780
1 530^2 610
230^550
12^550
0.72 þ 0.20 to 1.3 þ 0.35
3.4 þ 1.0 to 18 þ 4.3
1.9^7.6
0.01^1.6
Buoyant plume
2 590^2 830
5 500^ s 12 000
4.1 þ 1.2 to 17 þ 3.1
3.3^ s 28
A-sw2, Mar.
A-sw1, Mar.
M-sw1, Hlm.
S-sw2, Hlm.
S-sw1, Hlm.
A-plume1, Mar.
S-plume1, Mar.
S-plume2, Mar.
S-plume3, Mar.
S-plume4, Hlm.
S-bplume1,
Hlm.
Bottom seawater
Low-temperature £uid
1 560^2 700
1 520^2 820
6 15^4 700
6 15^ s 12 000
3.2 þ 0.66 to 13 þ 1.8
1.6 þ 0.25 to 35 þ 5.4
6 0.01^12
6 0.02^ s 3.5
a
b
Mean of one subsample þ 95% con¢dence limits calculated from n = 32^64 ¢elds of a microscopic grid.
Isolate name followed by generic designation, Marinobacter or Halomonas.
FEMSEC 1135 9-6-00
A-lthf1, Hlm.
S-lthf1, Hlm.
S-lthf2, Hlm.
252
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be metabolized, Gram-negative Biolog plates (Biolog, Inc.,
Hayward, CA, USA) containing 95 wells pre-¢lled with
di¡erent organic substrates and a color-sensitive metabolic
indicator were used; each plate also has a control well pre¢lled only with the metabolic indicator [25]. Cells were
grown to the exponential phase in Falcon tubes to a density of V108 cells ml31 and pelleted by spinning in a
clinical centrifuge at maximum speed at room temperature
for 20^40 min. The supernatant was decanted, and the
pellet was resuspended in 15 ml of the growth medium
modi¢ed by removing the yeast extract and sodium citrate.
150 Wl of this suspension was inoculated into each of the
96 wells of the Biolog plate, which were then incubated at
23³C for at least 4 days in a moisture chamber and scored
every 8^24 h. All control wells remained negative, indicating that the residual organic carbon (from the growth
medium) retained by the pellets and subsequently inoculated into the wells had a negligible impact on results.
Tolerance to divalent cadmium was assayed by adding
Cd to the growth medium from a stock of ¢lter-sterilized
1.52% CdCl2 W2.5H2 O in Sea Salts B.
For anaerobic growth, the growth medium was dispensed into Balch tubes which were sealed with rubber
stoppers and aluminum crimps. The headspace was purged
of air and ¢lled with argon four times [20]. The medium
was reduced with ¢lter-sterilized Na2 SW9H2 O (0.05%), and
resazurin (0.0002%) indicated oxygen removal. Anaerobic
nitrate reduction was assayed colorimetrically via nitrite
production [26].
tionally bases 450^483, E. coli numbering, in Fig. 2). Phylogenetic trees were created with PAUP* (D.L. Swo¡ord,
Smithsonian Inst.) as found in the Genetics Computer
Group (GCG) (Wisconsin Package version 9.1, GCG,
Madison, WI, USA) using a neighbor-joining distance algorithm [29,30] with negative branch lengths prohibited
and with bootstrap analysis (100 replicates). Trees were
visualized with TreeView [31]. GenBank accession numbers are E-plume1 AF212202; E-plume2 AF212201; Eplume3 AF212203; E-sul¢de1 AF212204; M-sw1
AF212205; A-lthf1 AF212206; A-plume1 AF212207; Asw1 AF212208; A-sw2 AF212209; S-plume1 AF212210;
S-plume2 AF212211; S-sw1 AF212212; S-plume3
AF212213; S-bplume1 AF212214; S-sw2 AF212215; Splume4
AF212216;
S-lthf1
AF212217;
S-lthf2
AF212218; E-limpetgut1 AF251770; E-twt2 AF251771;
E-twt1 AF251772; E-lthf1 AF251773; and E-plume4
AF251774.
3. Results
The MPNs revealed that microorganisms capable of
growing with 16% NaCl in hydrothermal-vent and pelagic
waters ranged from 6 15 to s 12 000 ml31 (95% con¢dence limits : 6 2.5 to 65 000 ml31 ) (Table 1). The total
number of microbes in these samples varied from
2.6. Phylogenetic analyses
Twenty-three strains were identi¢ed phylogenetically : 14
isolated from MPN enrichments and nine oligotrophs isolated without selection for halotolerance from various hydrothermal-vent samples. Pure cultures of cells were
grown in 500-ml £asks and pelleted by centrifuging at
10 000Ug for 20^40 min at 4³C. Genomic DNA was extracted using the IsoQuick kit (Orca Research, Bothell,
WA, USA). A portion of the 16S rRNA gene was ampli¢ed by polymerase chain reaction (PCR) using the bacterial primers 8F (5P-AGA GTT TGA TCC TGG CTC AG)
and 519R (5P-GWA TTA CCG CGG CKG CTG) [27].
After cleaning the PCR products, they were re-ampli¢ed
with the same primers and £uorescently tagged dideoxyribonucleotides and then sequenced with an Applied Biosystems sequencer (ABI) Model 373A at the University of
Washington Marine Molecular Biotechnology Laboratory
or an ABI100 at the Molecular Genetics Instrumentation
Facility at the University of Georgia, Athens, GA, USA.
Sequences of approximately 400 bp length (bases 100^493
and 101^484, Escherichia coli numbering, in Figs. 2 and 3,
respectively) were aligned with other sequences acquired
from GenBank using the Ribosomal Database Project
website [28] after excising a variable stem-loop (bases
198^219, E. coli numbering, in Figs. 2 and 3, and addi-
Fig. 1. Halotolerant percentage of the total microbial community. Percentages are derived from dividing three-tube MPN estimates of the
number of halotolerant microorganisms which grew on a 16% NaCl,
0.1% sodium citrate medium incubated at 20^24³C by enumerations of
the total microbial community obtained with epi£uorescence microscopy. Samples include non-vent ocean water, buoyant and lateral hydrothermal plumes, bottom seawater in close proximity to hydrothermal
vents, and low-temperature hydrothermal £uids from the North and
South Paci¢c. MPN values which provide percentages that are less than
a given number (e.g. 6 0.01%) are not included; values which provide
percentages that are greater than a given value (e.g. s 28%) are included.
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253
Fig. 2. Phylogenetic tree of nine hydrothermal-vent isolates obtained without selecting for tolerance to salt. The tree was constructed by aligning V400
bp of the 16S rRNA gene with known organisms. Scale bar indicates 10% sequence change. Bootstrap values (100 replicates) given at branch points.
(7.3 þ 2.0)U103 ml31 to (6.2 þ 1.2)U105 ml31 . Overall,
these culturable halotolerant microbes comprised between
6 0.01 and s 28% of the total population. Combining the
95% con¢dence limits of the MPNs and total counts shows
that the absolute minimum percentage is 6 0.00% and
absolute maximum percentage is s 100%. There is no apparent relationship between sample type or geographic
location and percentage of halotolerant microbes (Fig.
1). Thirteen bacterial strains were isolated from the greatest dilution (and one from the second greatest dilution, Asw1) of di¡erent MPN enrichments and analyzed phylogenetically. Eight belonged to the genus Halomonas and
six belonged to the genus Marinobacter.
In 1991 and 1995, 17 samples of hydrothermal plume
water, low-temperature hydrothermal £uid, sul¢de rock,
and a tubeworm trophosome were inoculated onto the
enrichment media and incubated at 2³C or V20³C (Table
2). Sixty-¢ve microbial strains were then puri¢ed from
these primary enrichments. Forty-one of these 65 strains
remained viable until 1997 and were screened for tolerance
to increasing levels of NaCl (Table 2). At least 36 (v 55%
of the original 65) grew in the enrichment medium with
10% NaCl ; at least 30 (v 46% of 65) grew with 16% NaCl ;
and at least 20 (v 31% of 65) grew with 22% NaCl. A
minimum of ¢ve (v 8% of 65) could not grow at a NaCl
concentration at or above 10%. The 20 strains which grew
with 22% NaCl were each rods, 1 Wm by 1.5^4 Wm in
dimension.
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Table 2
Sample source, incubation conditions and degree of halotolerance among 41 isolates
Sample
Sample source
Incubation
Sample type
T (³C)
Year
Headspace
T (³C)
pH
A
Lateral plume
2
1991
CH4
2
7
B
Lateral plume
2
1991
CH4
2
7
C
D
Lateral plume
Lateral plume
2
2
1991
1991
H2 /CO2
H2 /CO2
2
2
7
7
E
Lateral plume
2
1991
H2 /CO2
2
7
G
Lateral plume
2
1991
H2 /CO2
2
9
H
Lateral plume
2
1991
H2 /CO2
2
9
I
Lateral plume
2
1991
H2 /CO2
2
7
J
Lateral plume
2
1991
H2 /CO2
2
7
K
Lateral plume
2
1991
H2 /CO2
2
7
L
Low-temperature £uid
V10
1995
air
V20
7
M
Ridgeia trophosome
V10
1995
air
V20
7
N
O
P
Q
Sul¢de rock
Lateral plume
Lateral plume
Lateral plume
V10
2
2
2
1995
1991
1991
1991
air
CH4
CH4
CH4
V20
2
2
2
7
9
9
9
R
Limpet gut
V10
1995
air
V20
7
a
Isolatea
Salt tolerance
(% NaCl)
E-plume1, Hlm.
E-plume5
E-plume2, Hlm.
E-plume6
E-plume7
E-plume8
E-plume3, Hlm.
E-plume9
E-plume10
E-plume11
E-plume12
E-plume13
E-plume14
E-plume15
E-plume16
E-plume17
E-plume18
E-plume4, new genus?
E-plume19
E-plume20
E-plume21
E-plume22
E-plume23
E-plume24
E-lthf2
E-lthf1, new genus?
E-twt3
E-twt4
E-twt5
E-twt6
E-twt7
E-twt1, Pseudoalt.
E-twt2, Vibrio
E-sul¢de1, Hlm.
E-plume25
E-plume26
E-plume27
E-plume28
E-plume29
E-limpetgut1, Vibrio
E-limpetgut2
25
22
22
2
22
22
27
22
22
22
22
10
22
10
22
22
22
14
16
16
16
16
16
22
16
16
22
16
10
10
16
16
8
22
22
2
22
22
22
8
10
Isolate name followed by generic designation, if determined : Halomonas and Pseudoalteromonas abbreviated.
A portion of the 16S rRNA gene was sequenced for
nine of the 65 isolates (Fig. 2). These nine organisms
were chosen based on di¡erences in morphology, colony
appearance, Biolog plate assays and sample source, but
not based on salt tolerance. Four of the nine strains (Eplume1, E-plume2, E-plume3 and E-sul¢de1) belonged to
the genus Halomonas. The remaining ¢ve belonged to
Pseudoalteromonas or Vibrio, or were distantly related to
named genera ; isolate E-plume4 was closely related to an
unnamed Mn-oxidizing bacterium, however [32].
A phylogenetic tree was constructed with V400-bp 16S
rRNA sequences from the 18 Halomonas and Marinobacter strains (14 from MPN enrichments and four from
oligotrophic enrichments without selecting for halotoler-
ance) and previously characterized Halomonas and Marinobacter species (Fig. 3). Isolates A-plume1, S-plume1, Asw1, A-sw2, S-plume2 and S-plume3 formed a cluster
within the genus Marinobacter. Strains A-lthf1, S-sw1, Ssw2, S-lthf1 and E-plume2 clustered with H. aquamarina
and Halomonas meridiana. Halomonas venusta fell out with
S-bplume1, S-plume4 and S-lthf2. Strains E-plume1 and
E-plume3 formed their own branch within the Halomonas
genus, as did Halomonas variabilis and isolate E-sul¢de1.
Lastly, isolate M-sw1 and Halomonas marina are most
closely related to each other. High bootstrap values at
the Halomonas and Marinobacter generic branch points
(99 and 88, respectively) highlight the robustness of the
genus-level groupings.
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255
Fig. 3. Phylogenetic tree of 18 Halomonas and Marinobacter strains, constructed as in Fig. 2. Fourteen strains were isolated from the most dilute MPN
enrichments (second-most dilute for A-sw1), and four were isolated without selecting for halotolerance. Scale bar indicates 10% sequence change. Bootstrap values (100 replicates) given at branch points.
Halomonas isolates E-plume1, E-plume2, E-plume3 and
E-sul¢de1, along with type cultures of H. paci¢ca and H.
aquamarina, were further characterized phenotypically
(Table 3). The four novel strains were motile, Gram-negative, and grew with 22^27% NaCl. Colonies were tinted
orange and had an encrusted appearance. The minimum
temperature that permitted growth was 31³C for Eplume1, E-plume3 and E-sul¢de1, and 2³C for E-plume2,
H. aquamarina and H. paci¢ca. While H. aquamarina and
H. paci¢ca grew at 45³C, these four new Halomonas
strains grew only up to 40³C. Addition of Cd2‡ retarded
growth, but only at extremely high (mM) levels for three
of the Halomonas strains. Strain E-plume2 grew in the
presence of 3.0 mM Cd2‡ , and strains E-plume1 and Eplume3 tolerated 2.0 mM Cd2‡ . H. paci¢ca survived with
0.5 mM Cd2‡ while isolate E-sul¢de1 and H. aquamarina
only tolerated 0.05 mM Cd2‡ .
These four Halomonas isolates were heterotrophic facultative aerobes, and each reduced nitrate to nitrite. According to Biolog plates, strains E-plume1, E-plume2, Eplume3 and E-sul¢de1 overall oxidized 52^74% of 95 provided organic compounds. All four strains utilized a vari-
FEMSEC 1135 9-6-00
256
J.Z. Kaye, J.A. Baross / FEMS Microbiology Ecology 32 (2000) 249^260
Table 3
Temperature range, metabolism and cadmium tolerance of four novel Halomonas strains isolated without selecting for halotolerance, H. aquamarina
and H. paci¢ca
Organism
E-plume1, Halomonas
E-plume2, Halomonas
E-plume3, Halomonas
E-sul¢de1, Halomonas
H. aquamarina
H. paci¢ca
a
Temperature range (³C)
Minimum
Maximum
Carbon compounds
oxidized (Biolog plate)
(% of 95 tested)
31
2
31
31
2
2
40
40
40
40
45
45
54
52
62
74
^a
48
Anaerobic nitrate
reduction
Cd2‡ tolerance
(mM)
+
+
+
+
+
^
2.0
3.0
2.0
0.05
0.05
0.5
Not determined.
4. Discussion
ety of 42 sugars, starches, amino acids, organic acids and
other organic compounds. None of the four oxidized 20
other organic compounds that fall into those same categories. However, only some of the four Halomonas strains
could oxidize the 33 remaining organic compounds assayed (Table 4).
Halomonas is a cosmopolitan microbial genus, its range
encompassing the oceans, sediments, lakes and subsurface
environments [33,34]. It not only has penetrated most
every environment on Earth, but the results presented
Table 4
Organic compounds oxidized by the four Halomonas strains isolated without selecting for halotolerance according to Biolog plate substrate utilization
matrices
Compound
Organism
E-plume1
Glycogen
Tween 80
N-Acetyl-D-glucosamine
Cellobiose
D-Mannose
D-Psicose
L-Rhamnose
Methyl pyruvate
Mono-methyl succinate
Acetic acid
cis-Aconitic acid
Formic acid
D-Galactonic acid lactone
D-Galacturonic acid
D-Glucosaminic acid
K-Keto glutaric acid
Propionic acid
Sebacic acid
Succinamic acid
Glucuronamide
D-Alanine
L-Alanyl-glycine
L-Aspartic acid
Glycyl-L-glutamic acid
Hydroxy L-proline
L-Leucine
L-Ornithine
L-Phenylalanine
L-Pyroglutamic acid
L-Serine
Inosine
Uridine
Glucose-1-phosphate
E-plume2
E-plume3
E-sul¢de1
H. paci¢ca
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Data from H. paci¢ca shown for comparison.
FEMSEC 1135 9-6-00
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
J.Z. Kaye, J.A. Baross / FEMS Microbiology Ecology 32 (2000) 249^260
here indicate that it is also an abundant group in North
and South Paci¢c pelagic and deep-sea, hydrothermal-vent
waters. Less is known about the distribution of Marinobacter, though it is probably ubiquitous given that strains
have been cultured from French Mediterranean coast sediments, from deep-sea sediments in the Western Paci¢c,
from oil wells o¡ of the coasts of Vietnam and California,
and from the tropical Paci¢c Ocean o¡ of the Hawaiian
coast [33,35^39]. All of the 14 microbial strains cultured
from di¡erent MPN dilution-series enrichments were phylogenetically identi¢ed as Halomonas or Marinobacter, and
therefore these halotolerant genera were likely well represented in the majority if not all of the remaining 29 MPNs.
Regardless, halotolerant microorganisms, Halomonas,
Marinobacter and otherwise, comprised 0.01^10% (order
of magnitude) of the microbial community in the Paci¢c
Ocean and its sea£oor hydrothermal-vent environments.
The percentages that halotolerant microorganisms constituted of the total microbial community may be in£ated
due to cell loss while the ¢xed samples were stored at 2³C
for 9^14 months. The maximum estimated cell loss could
approach one order of magnitude [40]. However, nearly all
counts are in agreement with literature values for marine
waters. In the hydrothermal-vent environment, the concentration of microorganisms in lateral hydrothermal
plumes and buoyant hydrothermal plumes 2^20 m above
a black smoker has been estimated at (11^130)U104 ml31 ,
and the concentration in bottom water in close proximity
to vents was found to be (2^130)U104 ml31 [12,41]. Lowtemperature hydrothermal-vent £uids contain (1^11)U104
ml31 [42]. Of all enumerations reported here, only three
buoyant hydrothermal plume counts fall below these
ranges.
Halomonas and Marinobacter appear to be numerically
signi¢cant and ubiquitous in marine waters. Typically
6 0.01^0.1% of the heterotrophic microbial population in
a given seawater sample is culturable, and such percentages are achieved by employing numerous types of media
incubated under a variety of conditions [5]. It is therefore
striking that Halomonas spp. and Marinobacter spp. are so
prevalent and so readily cultured, implying that media
formulated to mimic the environment may not always be
the only approach to culture numerically signi¢cant, metabolically active microorganisms.
Our enrichment medium di¡ers from traditional oligotrophic and copiotrophic heterotrophic media in several
ways. For the 65 oligotrophs isolated without selecting
for halotolerance, there were elevated levels of reduced
Fe and Mn, frequently a reduced gas headspace (CH4 or
H2 /CO2 ), and trace elements provided in concentrations
modeled on 350³C hydrothermal £uid [11], frequently
10^10 000 times higher than measured in seawater. In addition to these factors, abundant halotolerant microbes
from MPN enrichments were isolated of course with elevated levels of NaCl, roughly ¢ve times higher than seawater. It is unclear which of these factors increased success
257
in culturability, or the mechanism(s) by which these media
components operate.
Four Halomonas strains were also obtained when enriching for oligotrophs without selecting for halotolerance
from hydrothermal plumes and sul¢de rock. While these
four strains were identi¢ed phylogenetically, additional
Halomonas strains may be present among the other 37
oligotrophs obtained without selecting for tolerance to
salt. In particular, it is possible that some of the 16 other
strains (for a total of 20, 31% of the 65 original isolates)
belong to Halomonas because they could also tolerate at
least 22% NaCl and had a rod morphology. While highorganic, high-salt media are typically used to select for
Halomonas spp. [34], these four strains (and perhaps
some of the 16 others) are another example of the infrequent instance when Halomonas spp. are isolated with seawater salinity media. It is the ¢rst instance, however, when
Halomonas spp. have been isolated with an oligotrophic
medium. Again, it is not known whether the elevated levels of Fe, Mn and trace elements or the reduced gas headspace in the enrichment medium enabled Halomonas to
out-compete other heterotrophs.
The 16S rRNA phylogenetic tree of Halomonas and
Marinobacter environmental isolates reveals clusters of
closely related organisms. Mirroring the SAR groups
[3,4], strains of nearly identical Halomonas and Marinobacter according to 16S rRNA gene sequence were found
thousands of kilometers apart. For example, while nearly
identical in 16S rRNA sequence, strains S-sw1, S-sw2 and
S-lthf1 were found hundreds of kilometers apart at 21.5³S
(2000 m depth), 18.5³S (1000 m depth) and 17.5³S (2570 m
depth, low-temperature hydrothermal £uid), respectively,
in the Paci¢c Ocean. Conversely, Halomonas strains with
di¡erent 16S rRNA sequences were found in close proximity to each other. Strains S-lthf1 and S-lthf2 were isolated from water samples roughly 1 m apart at a lowtemperature hydrothermal £uid site at 17.5³S on the
SEPR. The geographic distribution of a single cluster is
phenomenal. The Marinobacter cluster containing isolates
A-sw1, A-sw2, S-plume1, S-plume2 and S-plume3 came
from 21.5³S and 46³N, from 10^2600 m depth, and from
hydrothermal plume and non-plume environments.
Another important aspect of these data is that cultured
organisms do indeed form clusters, thereby allowing the
determination of the range of phenotypes within a cluster
in conjunction with DNA^DNA homologies. Interestingly, the Biolog plate data indicate that strains E-plume1
and E-plume2 had 92% identity in their ability to metabolize 95 organic compounds, whereas there is only 79%
and 74% identity between strain E-plume1 and the more
closely related strains E-plume3 and E-sul¢de1, respectively. In addition, strain E-plume1 had only 59% identity
with H. paci¢ca, similar to its 58% identity with strain Etwt2, a Vibrio isolate. Using Biolog plates as a metric,
there appears to be as much metabolic variation within
a cluster as between a cluster and other organisms. This
FEMSEC 1135 9-6-00
258
J.Z. Kaye, J.A. Baross / FEMS Microbiology Ecology 32 (2000) 249^260
metabolic diversity among closely related Halomonas spp.
corroborates the phenotypic variability found in the phylogenetically close-knit cluster of H. aquamarina, H. meridiana and H. variabilis when examining polar lipid patterns, respiratory lipoquinones and proportions of fatty
acids [43].
The abundance and ubiquity of Halomonas in marine
waters and heavy metal-enriched, low-temperature hydrothermal environments may be attributed to its metabolic
and physiological versatility. The four characterized Halomonas strains oxidized 52^74% of 95 organic compounds,
grew with 0.002^0.1% organic carbon, grew between 31
and 40³C, utilized oxygen or nitrate, and tolerated 0.05^3
mM Cd2‡ . These traits are generally consistent with many
other characterized Halomonas spp. [34]. The temperature
range, ability to grow without oxygen, and metabolic versatility encompass the conditions encountered in the modern ocean; in addition, tolerance to heavy metals permits
growth in low-temperature hydrothermal £uids which usually derive from a mixture of hot, metal-laden hydrothermal £uid and seawater. Accordingly, Halomonas should be
able to grow under most oceanic and low-temperature,
hydrothermal-vent conditions.
Halomonas and Marinobacter have neither been previously cultured from the vent environment nor been seen as
numerically important in community phylogenetic studies
in the water column [3]. However, most numerically dominant microbes are currently unculturable by traditional
methods [7]. While the oligotroph Sphingomonas sp. strain
RB2256 was proposed to comprise 15^35% of the pelagic
microbial community in an Alaskan bay [44], its putative
high abundance was neither con¢rmed by £uorescence in
situ hybridization (perhaps due to low ribosome content)
nor by quantitative enrichments [5]. Ultimately, however,
Sphingomonas spp. were frequently isolated (V20% of
cellular clones) in a coastal seawater sample [7] though
its abundance in open-ocean and deep-sea waters remains
unknown. Di¡erent regions of the ocean may harbor different populations of numerically dominant microorganisms.
The observation that Halomonas and Marinobacter are
abundant members in the water column and in low-temperature hydrothermal £uids raises questions about the
role of tolerance to high levels of salt in microorganisms
that may experience little variation in salt concentration in
the environment. In addition to natural and arti¢cial highsalt environments found along coastlines and in bays (e.g.
hypersaline lagoons, solar salterns) and unique settings
like the deep Red Sea hot brines [45], there is hypothesized
to be an even more pervasive, high-salt environment:
brines beneath the global network of mid-ocean ridges.
Brines derive from two processes in deep-sea, mid-ocean
ridge hydrothermal systems. The ¢rst process, phase separation, occurs at temperatures and pressures above 407³C
and 298 bar whereby hydrothermal £uids are transformed
into immiscible volatile-rich vapors and droplets of metal-
rich brines [46,47]. The phases physically segregate as they
circulate within the fracture network of the oceanic crust
[48,49]. The Endeavour Segment of the JdFR and 9³N on
the East Paci¢c Rise host robust hydrothermal systems
that have been undergoing phase separation as long as
observations have been made [50^53]. The second process
by which brines may form at depth in submarine environments is by exsolution of magmatic £uids during the ¢nal
stages of melt crystallization, creating £uids of up to 70%
NaCl [47,54^58]. Fluid inclusion analyses of rocks from
the Troodos ophiolite and Mid-Atlantic Ridge indicate
that hydrothermal £uids with up to 20% NaCl circulate
within the lower crustal lithologies in open fracture networks at temperatures of 200^500³C [54,59]. Based on
vent £uid compositions and £uid inclusion data, a briny
subsurface environment within the gabbroic and lower
dike sequences of the oceanic crust was proposed [60].
Here, brines are generated by supercritical phase separation (and possibly degassing magmatic £uid) and subsequently accumulate and persist [61]. Supercritical phase
separation and segregation processes are strongly re£ected
in vent £uid chemistry [50,62,63] in which chlorinities of
one-tenth to greater than twice seawater (1^7% NaCl
equivalent) are commonly measured in mid-ocean ridge
hydrothermal vents [11]. Once cooled or mixed with seawater in a low-temperature hydrothermal £uid system,
these brines may create a salty, metal-rich subsea£oor microbial habitat. Halomonas, Marinobacter and other halotolerant genera have previously been isolated from coastal
oil well and terrestrial subsurface brines [36,37,64,65].
These data are the ¢rst report indicating that halotolerant bacteria comprise a signi¢cant component of the microbial community in hydrothermal-vent and pelagic marine environments. The ability to grow with high
concentrations of NaCl and Cd2‡ , anaerobically, on a
variety of organic compounds, and over a wide mesophilic
temperature range implies that the subsea£oor brine habitat associated with deep-sea, hydrothermal-vent systems
may be a globe-encircling biotope suitable for Halomonas,
Marinobacter and other halotolerant and halophilic bacteria and archaea.
Acknowledgements
This research required the aid of numerous fellow scientists, ships and submersibles. We would like to express
our gratitude to chief scientists John Delaney, Bob Embley
and Marv Lilley for enabling ample sample collection and
to the crews of the R/V Thomas G. Thompson, R/V Atlantis, DSV Alvin and ROV ROPOS. A special thank-you is
extended to Debbie Kelley, Dave Butter¢eld, Jim Holden,
Melanie Summit and Byron Crump for their helpful suggestions and advice in the laboratory. This research was
supported by Sea Grant (NA36RG0071) and the National
Science Foundation (BCS9320070) to J.A.B.
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