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Applied and Environmental Microbiology, April 1999, p. 1710-1720, Vol. 65, No. 4
Center for Marine Biotechnology and
Biomedicine, Scripps Institution of Oceanography, University of
California, San Diego, La Jolla, California
92093-0202,1 and Calgene LLC Monsanto,
Davis, California 956162
Received 28 October 1998/Accepted 3 February 1999
There is considerable evidence correlating the production of
increased proportions of membrane unsaturated fatty acids (UFAs) with
bacterial growth at low temperatures or high pressures. In order to
assess the importance of UFAs to microbial growth under these
conditions, the effects of conditions altering UFA levels in the
psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by
P. profundum SS9 grown at various temperatures and
pressures were characterized, and differences in fatty acid composition
as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced
proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs)
fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis
and led to a decreased growth rate and yield at low temperature and
high pressure. In addition, oleic acid-auxotrophic mutants were
isolated. One of these mutants, strain EA3, was deficient in the
production of MUFAs and was both low-temperature sensitive and
high-pressure sensitive in the absence of exogenous 18:1 fatty acid.
Another mutant, strain EA2, produced little MUFA but elevated levels of
the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or
high-pressure sensitive. Finally, reverse genetics was employed to
construct a mutant unable to produce EPA. This mutant, strain EA10, was
also not low-temperature sensitive or high-pressure sensitive. The
significance of these results to the understanding of the role of UFAs
in growth under low-temperature or high-pressure conditions is discussed.
One of the characteristics believed
critical for life at low temperatures or high pressures is the
maintenance of appropriate membrane fluidity or phase. Reduced
temperature and increased hydrostatic pressure exert profound physical
influences on biological membranes, resulting in supraoptimal membrane
viscosity or phase transition caused primarily by the tighter packing
of the fatty acyl chains (9, 17, 26). Thus, at elevated
pressures and/or low temperatures, acyl chains assume a closely packed,
ordered array in which molecular motion is highly restricted. Such
membrane gelling effects are predicted to provide a strong inducement
for adaptive membrane restructuring in order to circumvent the
deleterious consequences of altered membrane function. The maintenance
of biological membranes in a narrow range of viscosity (homeoviscous response) (49) or within a liquid-crystalline phase
(homeophasic response) (28) may be key to an organism's
growth ability and survival. Membrane transport, intracellular
signaling and gene regulation, membrane protein dispersion and
protein-protein interactions within the lipid bilayer, and metabolic
electron transport are reliant on an appropriate membrane physical
structure (20).
Perhaps the most pervasive cellular response to a temperature change
entails the retailoring of the membrane's fatty acid composition. It
is well documented that the biological response of numerous bacteria to
decreases in temperature results in substantial increases in the
proportion of unsaturated fatty acids (UFAs) within membrane
phospholipids (30). Likewise, many deep-sea bacteria display
a high ratio of UFAs to saturated fatty acids (SFAs) in their membrane
phospholipids, which in many instances increases with increasing growth
pressure (11, 12, 21, 54). Fatty acyl chains containing one
or more double bonds adopt a more expanded conformation, pack less
compactly, and possess lower melting temperatures than their saturated
counterparts, allowing for their less orderly alignment within the
membrane phospholipids (17). Consequently, increases in
membrane unsaturation may be important for the homeostatic maintenance
of an appropriate physical structure of the membrane in response to
environmental variables which elicit membrane gelling effects, such as
reduced temperature and elevated pressure.
One particularly remarkable characteristic shared by many
low-temperature-adapted (psychrophilic or psychrotolerant) and
high-pressure-adapted (piezophilic or piezotolerant, previously
termed barophilic and barotolerant [56])
bacteria is the production of the omega-3 polyunsaturated
fatty acids (PUFAs) eicosapentaenoic acid (EPA; 20:5) and
docosahexanenoic acid (DHA; 22:6). There is a preponderance of
PUFA-producing bacteria associated with cold (or permanently cold) and
high-pressure environments compared to tropical and shallow-water
environments (12, 37, 60, 61). By virtue of their extremely
low melting temperatures, such PUFAs would be expected to exert
disproportionately large effects on membrane structure, effectively
reducing the melting temperature and increasing the melting pressure of
a lipid bilayer. Consequently, PUFA production and their temperature-
and pressure-dependent regulation by deep-sea bacteria have been
considered potentially important adaptations to the low temperatures
and high hydrostatic pressures of the deep sea (12, 15, 38, 54,
55). The deep sea is characterized by a temperature of 2°C in
most habitats and by hydrostatic pressures ranging from 10 MPa at a
depth of 1,000 m to approximately 110 MPa in the Challenger Deep of the
Mariana Trench at 10,898 m.
Whereas correlations have been found to exist between the degree of
membrane unsaturation and growth under low-temperature and
high-pressure conditions, confirmatory evidence that UFAs are indeed
required for growth under these conditions is lacking. Prior studies
have remained nearly exclusively descriptive and phenomenological,
reporting the phenotypic changes which ensue in response to temperature
and pressure variables. In the present study, we have addressed the
relative importance of UFAs for growth of the psychrotolerant
piezophilic deep-sea bacterium Photobacterium profundum SS9
at low temperatures and high pressures. P. profundum SS9 is
a genetically tractable model system for studies of low-temperature and
high-pressure adaptation (1). Isolated from an amphipod homogenate enrichment in the Sulu Sea at a depth of 2,551 m and an
ambient temperature of approximately 9°C, P. profundum SS9 is capable of growth at temperatures of less than 2°C to greater than
20°C (optimal temperature, 15°C) and from 0.1 MPa (0.101 MPa = 1 atm = 1.01 bar) to nearly 70 MPa (optimal pressure, 28 MPa)
(10). In addition, P. profundum SS9 produces the
PUFA EPA (39). To begin assessment of the importance of
fatty acid composition in vivo, the effects of altered UFA levels on
the growth properties of P. profundum SS9 have been analyzed
through UFA inhibitor analyses, the generation of mutants exhibiting
altered fatty acid profiles, and the engineering of a P. profundum SS9 mutant defective in EPA production.
Strains and growth conditions.
All bacterial strains and
plasmids used in this study are listed in Table
1. P. profundum strains were
routinely cultured at 15°C in 2216 marine medium (28 g/liter; Difco
Laboratories, Detroit, Mich.). All temperature experiments (15, 9, and
4°C) were conducted aerobically in 2216 medium. For solid media, agar (Difco Laboratories) was added at 17 g/liter. The antibiotics kanamycin
(50 µg/ml for Escherichia coli; 200 µg/ml for P. profundum strains), rifampin (100 µg/ml), and ampicillin (100 µg/ml) were added to the medium when required. The antibiotic
cerulenin (2,3-epoxy-4-oxo-7,10-dodecadienamide) was added at 12 µg/ml when used in inhibition studies. All antibiotics were obtained
from Sigma Chemical Co. (St. Louis, Mo.). When required, media were
supplemented with oleic acid (18:1) in the form of Tween 80 (polyoxyethylenesorbitan-monooleate) at a final concentration of
0.025%. Tween 20 (polyoxyethylenesorbitan-monolaurate), Tween 40 (polyoxyethylenesorbitan-monopalmitate), and Tween 80 were obtained
from Sigma Chemical Co.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Monounsaturated but Not Polyunsaturated Fatty
Acids Are Required for Growth of the Deep-Sea Bacterium
Photobacterium profundum SS9 at High Pressure and
Low Temperature
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
High-pressure growth studies. High-pressure cultivation of P. profundum strains for growth studies or fatty acid analysis was conducted as previously described (6). Each culture was grown to stationary phase in 2216 marine medium at 1 atm (1 atm = 0.101 MPa) and 15°C. Stationary-phase cultures were diluted 1/200 into 2216 medium buffered with HEPES (100 mM, pH 7.5; Sigma Chemical Co.) containing 22 mM glucose (Sigma Chemical Co.). The diluted culture was used to fill 4.5- or 15-ml polyethylene transfer pipettes (Samco, San Fernando, Calif.). The pipettes were filled completely and then heat sealed with a hand-held heat sealing clamp (Nalgene, Rochester, N.Y.). Cells were incubated at a hydrostatic pressure of 0.1, 28, or 50 MPa (1, 280, or 500 atm, respectively) at 9°C (unless otherwise stated) in stainless steel vessels which could be pressurized by using water and a hydraulic pump and which were equipped with quick-connect fittings for rapid decompression and recompression as described by Yayanos and Van Boxtel (57).
Chemical mutagenesis.
P. profundum DB110, a
Lac
, rifampin-resistant derivative of wild-type P. profundum SS9 (6), served as the parental strain for
mutagenesis experiments.
N-Methyl-N'-nitro-N-nitrosoguanidine (NG) (Sigma Chemical Co.) mutagenesis experiments were performed according to the protocols of Miller (31). Streptozotocin
(Sigma Chemical Co.) enrichment and selection were performed similarly to the procedures of Jacobson et al. (18). Prior to
mutagenesis, kill curves were obtained for strain DB110, using various
concentrations of the mutagen NG and the antibiotic streptozotocin in
order to determine the effectiveness of killing. The conditions used
here represent concentrations and times of exposure that resulted in 50% killing. Strain DB110 was grown in 25-ml batches in 2216 marine medium supplemented with 0.4% N-acetylglucosamine (NAG)
(Sigma Chemical Co.) at 15°C in an environmental shaker to an optical density at 600 nm of 1.0, corresponding to a density of approximately 108 cells ml1. NAG supplementation was
performed to preinduce cells for uptake of the antibiotic
streptozotocin for later antibiotic selection. Streptozotocin is an
analogue of NAG which is transported into the cell by the same
phosphotransferase system proteins (23). Hence, preinduction
with NAG facilitates uptake of streptozotocin. The 25-ml batches were
divided into 5 equal volumes and centrifuged at 5,000 × g for 5 min. The cell pellets were washed once in 0.1 M
citrate buffer (pH 7.2) supplemented with sea salts (32 g/liter; Sigma
Chemical Co.) and resuspended in an equal volume of citrate buffer. NG
was added to the cultures to a final concentration of 100 µg/ml. The
cultures were allowed to incubate at 15°C for 4 h without
shaking. After 4 h, the cultures were washed twice in 0.1 M
phosphate buffer to remove the NG and resuspended in an equal volume of
2216 marine medium. The cultures were then allowed a 6-h outgrowth
recovery period at 15°C. To enrich for fatty acid-requiring
auxotrophs, the antibiotic streptozotocin was then added to each
culture to a final concentration of 50 µg/ml and the cultures were
allowed to incubate at 15°C with shaking for an additional 2 h.
At 30-min intervals, 1 ml from each culture was removed and 100 µl
was plated onto each of five antibiotic-free 2216 agar plates
containing 0.005% oleic acid-Na+ salt and 0.05% Tween 40 (Sigma Chemical Co.) as a solubilizing agent for the oleic acid. The
plates were incubated at 15°C in the dark for approximately 5 to 7 days to allow growth of mutagenized cells. Colonies were then replica
plated onto 2216 agar supplemented with oleic acid, as well as onto
unsupplemented 2216 agar. Of the 6,345 colonies screened, 5 displayed
an auxotrophic requirement for oleic acid, showing no growth on
unsupplemented 2216 agar. These oleic acid auxotrophs were designated
P. profundum EA1 to EA5.
Gene disruption mutagenesis.
Construction of an
EPA-deficient strain of P. profundum SS9 was performed via
gene disruption mutagenesis with the mobilizable suicide plasmid
pMUT100 (encoding kanamycin resistance) (5). An 885-bp
internal fragment of an SS9 EPA biosynthetic open reading frame
(designated ORF3/4) was PCR amplified from strain DB110 genomic DNA
with the following primers, designed from the known EPA ORF3/4 gene
sequence of Shewanella sp. strain SCRC-2738 (59): 5'-CUACUACUACUAACAGCGAAATGCTTATCAAG-3' (primer 1) and
5'-CAUCAUCAUCAUGCCACCAAAACCAAATGAGCTAATAC-3' (primer 2). The
PCR product was first cloned into pAMP1 by using a Gibco BRL CloneAmp
system (Life Technologies, Gaithersburg, Md.) and subsequently
subcloned into pMUT100 as an EcoRI-BamHI fragment, yielding pEA30. Due to an internal EcoRI site
within the SS9 ORF3/4 PCR product, pEA30 contains a 710-bp fragment. Bacterial conjugations were used to transfer plasmid pEA30 from E. coli into P. profundum DB110 as described by
Chi and Bartlett (6). Kanr exconjugants arose
from integration of the pMUT100 plasmid into the genome of P. profundum SS9. These experiments yielded the EPA-deficient strain
P. profundum EA10, containing a disruption in EPA ORF3/4
which was confirmed by Southern blot analysis (45). Genomic
DNA from P. profundum EA10 and DB110 was digested with restriction enzyme PstI, HindIII,
BglII, HpaI, or KpnI and probed with
the internal fragment of ORF3/4 harbored on pEA30, labelled with
[
-32P]dCTP by random priming (Life Technologies).
Evidence for gene disruption was revealed by the replacement of
discrete fragments in the strain DB110 genome by fragments 6.2 kb
larger in the case of strain EA10. Killing experiments were conducted
at an extremely high pressure (100 MPa) and an extremely low
temperature (
20°C) to assess the susceptibility of strain EA10 to
such extremes compared to the parental strain, DB110. Stationary-phase
cultures of strains EA10 and DB110 were diluted to a density of
approximately 107 cells ml1 and incubated
either at 100 MPa (9°C) or 20°C (0.1 MPa) for up to 6 h.
Every 30 min for 100-MPa incubations and every 45 min for 20°C
incubations, CFU of both strains per milliliter were determined at
15°C (0.1 MPa) following culture dilution and plating onto 2216 marine agar.
Fatty acid analyses and lipid extracts.
Cells grown at
various hydrostatic pressures or temperatures were harvested in late
exponential phase via centrifugation at 5,000 × g,
washed in an equal volume of 50% artificial seawater (16 g of Sigma
sea salts per liter; Sigma Chemical Co.), frozen at 70°C, and
lyophilized prior to fatty acid methyl ester (FAME) derivatization.
Whole-cell methanolysates were used throughout the study for FAME
preparation and analysis. FAMEs were prepared by reacting 10 mg (dry
weight) of a lyophilized cell sample with 5%
H2SO4 in anhydrous methanol at 90°C for 90 min in 1.5-ml sample vials with Teflon-lined caps (Wheaton, Millville,
N.J.). Samples were allowed to cool, and FAMEs were extracted twice
with hexane and nonesterified fatty acids saponified with 10% NaCl.
Following FAME derivatization, the hexane layer was removed and
evaporated completely under a gentle stream of N2. The FAME
residue was then redissolved in 25 µl (final volume) of hexane
containing a known concentration of 19:0 methyl ester as an internal
standard and stored at 70°C until analysis.
1. Helium was used as the
carrier gas, and the injector was maintained at 250°C. Peak areas
were quantified, and mass spectra were acquired and processed with
Hewlett-Packard G1034C MS ChemStation software operated in the scan
acquisition mode. MS operating conditions were as follows: electron
multiplier, 1,800 V; transfer line, 250°C; electron impact energy, 70 eV; scan threshold, 50; 1.3 scans s1 with a mass range of
50 to 550 atomic mass units; and solvent delay, 2.35 min. Compounds
were identified by comparison of their retention times with those of
known standards, and sample mass spectra data were compared to the mass
spectra data of 75,000 compounds in the Chemstation NBS75K library. EPA
production was confirmed by comparison of mass spectra data of the EPA
methyl ester standard (Sigma Chemical Co.) with that of the
corresponding peak from P. profundum SS9. Fatty acids are
denoted as ratios of the number of carbon atoms to the number of double bonds.
IM and OM separation and analysis.
Isolations of inner (IM)
and outer (OM) membrane fractions were performed with P. profundum DB110 grown at 15°C in 2216 marine medium at
atmospheric pressure according to the methods of Schnaitman (47). Cells (500 ml) were harvested in late exponential
phase at a density of approximately 108 ml1
by centrifugation at 5,000 × g for 10 min. Cell
pellets were weighed and resuspended in a volume of ice-cold sucrose
buffer (200 mM Tris [pH 7.8], 5 mM EDTA, 0.25 M sucrose, and 0.5 mg
of lysozyme ml1) such that 30% of the total volume was
cells. The cells were then transferred to an Erlenmeyer flask and lysed
by being subjected to six cycles of alternating freezing (in a dry
ice-ethanol bath) and thawing (in a room-temperature water bath). The
cell lysate was then poured into 20 volumes of cold 200 mM Tris, pH
7.8, containing 0.5 mM MgCl2 and 0.1 mg of DNase I
(Calbiochem, La Jolla, Calif.) ml1. This material was
then passed through an 18-gauge needle for 10 min to shear the DNA and
disperse the cell debris. Unbroken cells were removed by centrifugation
twice at 7,000 × g, with the supernatant being
decanted between clearing spins. The envelope material, consisting of
IM and OM, was recovered by centrifugation at 27,000 × g for 30 min. The membrane pellet was dried under vacuum
without heat and resuspended in 1 ml of 25% (wt/wt) sucrose containing
5 mM EDTA. Sucrose gradients, each containing 5 mM EDTA, were prepared
as follows in SW41 ultracentrifuge tubes (Beckman Instruments,
Fullerton, Calif.): 0.5 ml of 55% (wt/wt) sucrose, 2.1 ml of 50%
sucrose, 2.1 ml of 45% sucrose, 2.1 ml of 40% sucrose, 2.1 ml of 35%
sucrose, and 2.1 ml of 30% sucrose. The 1-ml membrane suspension in
25% sucrose was layered onto the gradient, and the gradient was spun
for 14 h, in an SW41 ultracentrifuge rotor at 92,000 × g and 4°C. The OM appeared as a pair of similar opalescent bands at about 50% (wt/wt) sucrose. The IM was a translucent, yellowish band at about 36% (wt/wt) sucrose. Fractions were removed from the gradients and analyzed for purity of composition by (i) sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and (ii)
enzyme assays for both D-lactate dehydrogenase activity and
succinate dehydrogenase activity. SDS-PAGE analysis was used for
comparison of the OM fractions to OM proteins isolated by a Triton
X-114 detergent extraction method (4) modified as described
by Chi and Bartlett (6). OM fractions appeared nearly identical to the Triton X-114 extraction OM preparations. Bicinchoninic acid protein assays (Pierce, Rockford, Ill.) were performed to quantitate the total protein present in the membrane fractions, and
D-lactate and succinate dehydrogenase activity assays were used as markers for the cytoplasmic membrane fractions as described by
Osborn et al. (42). Fractions identified as IM had
D-lactate and succinate dehydrogenase specific activities
(in micromoles per minute per milligram of protein) of 1.2 and 0.7, respectively. Fractions identified as OM had D-lactate and
succinate dehydrogenase activities of 0.09 and 0.01, respectively,
indicating that the level of contamination of the OM with IM was very
low. Once membrane fractions were confirmed and their purity was
assessed, fatty acid analysis was performed as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Fatty acid composition of P. profundum SS9.
The
major fatty acids produced by P. profundum SS9 include
(shown as the systematic name followed by the common name) 12:0 (dodecanoic acid; lauric acid), 14:0 (tetradecanoic acid; myristic acid), 14:1 (cis-7-tetradecanoic acid; myrisoleic acid),
iso-16:0 (14-methyl-pentadecanoic acid), 16:0 (hexadecanoic acid;
palmitic acid), 16:1 (cis-9-hexadecanoic acid; palmitoleic
acid), 12-OH (3-hydroxydodecanoic acid; 
-hydroxylauric acid), 18:0
(octadecanoic acid; stearic acid), 18:1 (cis-11-octadecanoic
acid; cis-vaccenic acid), and 20:5 (all-cis-5, 8, 11, 14, 17-eicosapentaenoic acid; EPA). The fatty acid profile of
P. profundum SS9 cultivated aerobically at 15°C (0.1 MPa)
was similar to that recently reported by Nogi et al. (39)
under similar conditions. Included in Tables
2 and 3 and
Fig. 1 are the unsaturation index values
(sums of the percentages [by weight] of UFA species multiplied by the
numbers of double bonds) (12) and the UFA/SFA ratios for the
various strains and conditions used in this study in order to display the relative degree of membrane unsaturation.
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Dependence of fatty acid composition on growth phase. The percentages of the major fatty acid species were found to vary depending on the phase of growth. This was characterized for P. profundum DB110 grown aerobically at 15°C and 0.1 MPa (Fig. 1). Across the spectrum of time periods examined, the most dramatic regulation involved the progressive increase in the branched-chain fatty acid iso-16:0 as a function of culture age. iso-16:0 levels were consistently undetected until late exponential phase and then increased throughout stationary phase, reaching nearly 11% of the total fatty acid (by weight) in advanced stationary-phase cultures. 18:1 and EPA levels increased from early to late log phase, whereas 14:0 decreased from early log phase to later growth stages. The unsaturation index values and UFA/SFA ratios were lowest during early exponential phase and highest during late log phase. In light of these results, care was taken to analyze the fatty acid contents of all P. profundum strains within a particular growth phase, specifically late exponential phase (corresponding to an optical density at 600 nm of approximately 0.7), so that the data would not be skewed as a result of growth phase differences.
Analysis of IM and OM composition.
Fatty acid composition
differences as a function of cellular location were also investigated
(Table 4). IM and OM were isolated by
density gradient centrifugation from strain DB110 grown at 15°C.
Compared to the IM fraction, the OM fraction was enriched with the
hydroxylated fatty acid 12-OH (3.2% versus 12.3%, respectively). The
specific enrichment of the OM with 12-OH is likely to derive from the
lipid A component of the lipopolysaccharide layer (44). In
addition, the OM contained a higher percentage of the shorter-chain SFAs, specifically 12:0 (7.6% in OM versus 2.9% in IM) and, to a
lesser extent, 14:0 (7.2% in OM versus 5.1% in IM). This higher percentage of SFAs in the OM is consistent with findings in E. coli (24). UFA types exhibited no dramatic differential
localization between the membrane fractions.
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Temperature- and pressure-dependent regulation of fatty acid composition. Comparison of the fatty acid profiles of P. profundum DB110 grown under different conditions indicated that low temperature and high pressure both enhanced UFA levels, but to different extents and with different selective effects (Fig. 2). It should be noted that the 15, 9, and 4°C cultures were grown aerobically, whereas all experiments in pressurizable bulbs necessitated growth under fermentative conditions with glucose and buffer added to the cultures. With decreased temperature, moderate to slight increases in the proportions of 16:1 and 18:1 occurred along with a significant increase in EPA and a dramatic reduction in the proportion of iso-16:0. Changes in pressure resulted in an even more dramatic effect on the fatty acid composition. The proportion of 18:1 increased from 3.6 to 16.2% of total fatty acids as cells were pressurized from 0.1 MPa (9°C) to 28 MPa (9°C), with an additional increase to 25.1% upon progression from 28 MPa (9°C) to 50 MPa (9°C). Over the pressure range tested, the proportion of EPA also increased, from 2.7% at 0.1 MPa (9°C) to 11.4% at 50 MPa (9°C). No iso-16:0 was produced in any of the cultures grown in the pressurizable bulbs regardless of the growth phase. Akin to observations made for other piezophilic species (12, 21), the relative degree of fatty acid unsaturation in P. profundum SS9 increased in response to decreased temperature or increased hydrostatic pressure, as indicated by the general increase in unsaturation index values and UFA/SFA ratios as a function of decreased temperature or increased pressure (Table 2).
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9.87 atm). Data represents mean percentages
(by weight) of fatty acid species, ± standard deviations, derived from
triplicate samples harvested in the late exponential phase of growth.
See Materials and Methods for cultivation conditions.
Effect of cerulenin treatment on growth at high pressures and low
temperatures.
As a first step in assessing the role of UFAs in the
growth of P. profundum SS9, the effect of the antibiotic
cerulenin was tested. Cerulenin irreversibly inhibits fatty acid
biosynthesis enzymes -ketoacyl-acyl carrier protein synthases I and
II in a variety of bacteria and fungi, perturbing the formation of UFAs (41). Cerulenin treatment allowed us to specifically
determine the effect of diminished monounsaturated fatty acid (MUFA)
levels (specifically 16:1 and 18:1) on growth of P. profundum SS9. As shown in Table 2, analysis of the fatty acid
composition of cerulenin-treated strain DB110 under the various
temperature and pressure growth conditions revealed a nearly complete
inhibition of 18:1 production and moderate reductions in 16:1. In
addition, cerulenin elicited dramatic increases in the proportions of
12:0, 14:0, and 14:1 as well as moderate increases in EPA. The fact
that EPA levels rose while 18:1 values declined is consistent with
separate pathways directing the biosynthesis of the two UFAs, with the
pathway responsible for 18:1 production being far more sensitive to the
antibiotic than that of the EPA pathway.
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, 15°C, without cerulenin; 
, 15°C, with
cerulenin; 
, 4°C, without cerulenin; 
, 4°C, with cerulenin.
(B) Growth characteristics and fatty acid analyses of oleic acid-auxotrophic mutants. A second line of experimentation which was performed to address the importance of particular UFAs for growth at low temperature or high pressure involved the generation of a collection of chemical mutants exhibiting an auxotrophic requirement for oleic acid (18:1). We predicted that many of these mutants would be altered in the production of certain UFAs. Of the 6,345 NG-mutagenized colonies screened, 5 displayed a requirement for oleic acid, exhibiting negligible or complete lack of growth on unsupplemented 2216 agar. These mutant strains were designated P. profundum EA1 to EA5. All of these mutants were verified to be derivatives of P. profundum SS9 based on Coomassie blue R staining of whole-cell proteins and SDS-PAGE analysis with comparison to strain DB110 proteins. All of these mutants exhibited a specific enhancement of growth in the presence of UFAs, since SFAs such as palmitic acid (16:0) and lauric acid (12:0) failed to compensate for their growth defects. Only mutant EA5 exhibited an absolute requirement for oleic acid for growth.
The fatty acid profiles of mutants EA1 to EA4 were obtained. However, mutant EA5 could not be accurately analyzed due to its obligate requirement for exogenous 18:1. Of the five mutants isolated, mutants EA1 and EA4 exhibited the least-stringent requirements for exogenous 18:1 and displayed wild-type levels of all fatty acids. However, mutants EA2 and EA3 exhibited markedly altered fatty acid profiles relative to strain DB110 and were therefore selected for further analysis. The fatty acid compositions of strains EA2 and EA3 grown at various temperatures and pressures are listed in Table 2. Strain EA3 exhibited severely diminished 16:1, 18:1, and EPA levels as well as elevated proportions of 12:0, 14:0, and 14:1 under routine culture conditions of 15°C (0.1 MPa). At low temperature, strain EA3 produced fivefold-higher EPA levels, but little difference in fatty acid composition was evident in cells grown at low and high pressures. Figure 4 shows the growth characteristics of strain EA3 at various pressures and temperatures in the presence or absence of exogenous 18:1 in the form of Tween 80 at a concentration of 0.025%. Without 18:1 supplementation, strain EA3 exhibited dramatic low-temperature and high-pressure sensitivities, exhibiting virtually no growth at 4°C (0.1 MPa) or 28 MPa (9°C). However, strain EA3 displayed dramatically enhanced growth at a low temperature (4°C) or an elevated pressure (28 MPa) in the presence of Tween 80. These results are qualitatively consistent with the cerulenin studies, although the influence of exogenous 18:1 on growth at low temperature was substantially more dramatic for strain EA3 than for cerulenin-treated cells. The growth characteristics of strain EA3 were also similar to those previously reported for strain EA5 (2).
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, 50 MPa; Growth characteristics and fatty acid analysis of strain EA10, a P. profundum SS9 mutant defective in EPA production. The results presented above indicate a role for MUFAs in the growth of P. profundum SS9 at low temperatures and at elevated pressures (cerulenin and strain EA3 data), as well as a possible role for EPA under similar conditions, at least when the proportions of certain other fatty acids have been altered by mutation (strain EA2 data). To assess the role of EPA more directly, a reverse-genetics methodology was employed to construct a mutant unable to produce EPA. This first entailed the cloning of an internal fragment of a P. profundum SS9 EPA biosynthesis gene by making use of the previously published sequence of a cluster of genes required for EPA biosynthesis from Shewanella sp. strain SCRC-2738 (59) and sequence information obtained from Vibrio marinus of genes involved in the production of DHA (22:6n-3) (22). From these reports seven open reading frames (ORFs) were determined to be necessary for imparting the ability to produce PUFAs to recombinant E. coli strains.
An internal fragment of a homologue of Shewanella ORF3/4 from P. profundum SS9 was amplified and cloned by PCR with primers derived from SCRC-2738 ORF3/4 positions 542 to 561 and 1403 to 1428. This amplification yielded a product of the expected size whose sequence possessed a high degree of relatedness, 83 and 87% identity at the DNA and deduced protein levels, respectively, to ORF3/4 from Shewanella sp. strain SCRC-2738. Construction of a P. profundum SS9 EPA mutant, designated strain EA10, followed the introduction of the SS9 sequence into a suicide plasmid and its delivery into P. profundum DB110 by conjugal transfer (described in Materials and Methods). Kanamycin-resistant exconjugants were screened initially by examining their fatty acid profiles at 15°C (0.1 MPa) for the lack of EPA production. One of these mutants was subsequently confirmed by Southern blot analysis to be a gene disruption mutant and designated P. profundum EA10 (data not shown). The fatty acid profile of strain EA10 grown under various conditions is listed in Table 2. By comparison with strain DB110, under identical conditions strain EA10 considerably upregulated its proportion of MUFAs (18:1, 16:1, and 14:1) while 16:0 levels tended to remain lower than those of strain DB110. At a low temperature (4°C), strain EA10 exhibited markedly reduced SFA content, with significant increases in 16:1 and 18:1, relative to the level obtained by a 15°C cultivation. The most dramatic effect of high pressure (28 MPa, 9°C) on strain EA10 was to increase the proportion of 18:1 fatty acid from 12.7 to 25.4%. Surprisingly, when the growth of strain EA10 was examined at a low temperature and high pressure, no significant deviations from wild-type growth were evident, except for a modest reduction in growth yield under all conditions tested. The growth characteristics of strain EA10 versus strain DB110 at various pressures are shown in Fig. 6. Strains EA10 and DB110 exhibited nearly identical growth rates at 4 and 15°C (0.1 MPa) or at 0.1, 28, and 50 MPa (9°C). Moreover, the combined effects of increased pressure and decreased temperature (28 MPa, 4°C) resulted in nearly identical growth abilities for the two strains. Under these conditions (28 MPa, 4°C), strain EA10 displayed a UFA/SFA ratio of 8.38, with 18:1 and 16:1 comprising 27.2 and 58% of the cellular fatty acids, respectively. This is in comparison to strain DB110, which, under identical conditions, exhibited a UFA/SFA ratio of 3.1, with 18:1, 16:1, and EPA comprising 20, 37, and 14.8% of the total cellular fatty acids, respectively. These results indicate that under the laboratory conditions used in this study, EPA is not vital to the growth of P. profundum SS9 over the course of many generations, even under low-temperature or high-pressure conditions, situations in which its levels are typically upregulated. Likewise, no differences between the two strains with regard to survival at extremes of temperature (20°C, 0.1 MPa) or pressure (100 MPa, 9°C) were identified (data
not shown).
|
, EA10, 50 MPa; | |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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The present study has evaluated the types of fatty acids produced by the psychrotolerant, piezophile P. profundum SS9, their distribution between the IM and OM, and their regulation as a function of growth phase, temperature, and hydrostatic pressure. In addition, the significance of UFAs in growth at low temperatures and elevated pressures was explored through the use of a fatty acid synthesis inhibitor and by mutant analysis.
In general, the cellular distribution of fatty acids in P. profundum SS9 is similar to that reported for other gram-negative bacteria. For example, the high proportion of the hydroxylated fatty acid 12-OH, derived from the lipid A component of the lipopolysaccharide layer, and the elevated levels of shorter-chain SFAs present in the OM are consistent with findings for other gram-negative bacteria (24, 44). In addition, MUFAs and EPA are present in large amounts in both membranes, revealing no dramatic differential localization.
P. profundum SS9 was found to exhibit markedly increased levels of the branched-chain fatty acid iso-16:0 concomitant with entry into stationary phase. The nature of growth phase regulation of fatty acid production depends on the microorganism being studied (14, 25, 29, 36, 40). Our results are similar to those obtained in studies of another gram-negative marine isolate, which exhibited progressive increases in branched-chain fatty acids with culture age (40), and are also similar to the observed induction of cyclopropane fatty acids with the onset of stationary phase in E. coli (25, 29).
Modulation of membrane lipids by temperature or pressure is well established among many poikilothermic organisms. Such adjustments most notably include changes at the level of fatty acyl chain composition. The responses of bacteria to a reduced temperature or elevated pressure frequently entail the increased incorporation into membrane phospholipids of UFAs, which include PUFAs in those organisms capable of their production (11, 12, 15, 25, 35, 36, 54). Fatty acid profiling of P. profundum SS9 revealed a pronounced regulation of cellular fatty acid composition in response to changes in temperature or pressure, most notably a greater proportion of 16:1 and EPA at low temperatures and an increased proportion of 18:1 and EPA at elevated pressures.
Many deep-sea isolates exhibit substantial increases in MUFAs in response to increased cultivation pressure. The piezophilic bacterium CNPT3, which grows optimally at hydrostatic pressures of 30 to 50 MPa, does not produce PUFAs yet is capable of growth at pressures up to nearly 70 MPa (11). As a function of increasing pressure, CNPT3 exhibits higher proportions of 16:1 and 18:1 while the relative amounts of 14:1, 16:0, and 14:0 decrease. Likewise, it was suggested that the pressure-induced increases in the MUFA iso-17:1 contributed to the piezotolerance of the deep-sea bacterium RS103 (21).
Thus, a correlation between the degree of fatty acid unsaturation and
the cultivation temperature or pressure has frequently been drawn.
However, to address the possible adaptive role of UFAs in membrane
function in vivo at low temperatures or high pressures, physiological
and genetic experiments are required. Here the functional significance
of UFAs was examined by using the -ketoacyl-acyl carrier protein
synthase I and II inhibitor cerulenin to preferentially inhibit MUFA
synthesis and by obtaining mutants altered in UFA synthesis. Figure
7 summarizes the 16:1, 18:1, and EPA
levels of the various P. profundum strains (DB110, EA3, EA2,
and EA10) and cerulenin-treated strain DB110 at 15°C (0.1 MPa) along
with their low-temperature (4°C) and high-pressure (28 MPa) growth
phenotypes. Those strain-treatment combinations which resulted in
reduced MUFA (18:1 and 16:1) levels without dramatic compensatory
increases in the proportion of EPA (i.e., strain EA2) exhibited both
low-temperature and elevated-pressure sensitivity (cerulenin treatment
and strain EA3); conversely, those strain-treatment combinations which
exhibited wild-type (or higher) 16:1 and 18:1 levels (strains DB110 and
EA10) displayed low-temperature- and elevated-pressure-adapted growth.
These results, in conjunction with the fact that 18:1 supplementation
was able to complement the ability of strain EA3 to grow at low
temperature and high pressure and when cells were treated with
cerulenin, suggests that MUFAs are particularly important for growth of
P. profundum SS9 under low-temperature or high-pressure
conditions. Furthermore, the fact that a P. profundum SS9
mutant defective in EPA production retained both elevated-pressure- and
reduced-temperature-adapted growth via modulations solely in MUFAs is
consistent with the primary importance of MUFAs under these conditions.
|
A decrease in MUFA levels may be partially compensated for by EPA overproduction. Strain EA2 overproduces EPA while underproducing MUFAs. This strain is fascinating because of the shift in its growth ability at higher pressures. However, because the growth rate and yield of this strain are so reduced compared with those of wild-type P. profundum SS9, it would appear that EPA is a poor substitute for MUFAs.
What is the explanation for the need for MUFAs during growth at low temperatures and high pressures? Two potential hypotheses can be proposed. Foremost, it is possible that these fatty acids are required to maintain membrane fluidity or phase within an acceptable range for optimal growth. Studies of E. coli and Acholeplasma laidlawii (27, 43) have suggested that the ability of microorganisms which are unable to effectively regulate their membrane lipid fatty acid composition to grow at various temperatures may be determined by the phase state of their membrane lipids, as evidenced by the severe impairment of growth when cells exhibit more than about half of their lipids in the gel state (29). Indeed, it has been shown that membrane proteins of highly ordered gel-phase membranes are inactive or are excluded (50). To address this hypothesis, it will be necessary to directly measure physical properties of membranes of different P. profundum strains at various temperatures and pressures.
Alternatively, the critical nature of MUFAs could be based on a role in
specific membrane protein interactions which, if disrupted, result in
altered growth ability at reduced temperatures or elevated pressures.
This suggests a need for MUFAs in local
as opposed to globalmembrane
processes. This latter hypothesis is consistent with the notion of a
lipid annulus surrounding individual membrane proteins (34,
53). However, despite evidence that many membrane-associated proteins have an absolute lipid requirement for activity
(46), we are unaware of any such proteins exhibiting a
strict functional requirement for phospholipids with particular fatty
acyl chains.
A major result of this work was the discovery that neither low-temperature- nor high-pressure-adapted growth mandates EPA production in P. profundum SS9, at least under the culture conditions employed. The ability of EPA-deficient strain EA10 to grow at a reduced temperature or an elevated pressure (Fig. 6) was essentially the same as that of the parental strain. This result was surprising. The few previous studies which have examined phenotypic effects associated with altered PUFA production, the majority of which having been conducted in cyanobacteria, plants, and fungi, have implicated PUFA production as being a necessary component for growth at low temperatures (19, 32, 48, 51, 52).
The distribution of EPA-producing bacteria in the environment has also been taken as evidence of a need for EPA for growth at low temperatures or high pressures. The discovery by DeLong and Yayanos (12) that numerous deep-sea bacterial isolates contain substantial quantities of omega-3 PUFAs, namely, EPA and DHA (22:6), led to the speculation that such polyenoic fatty acids are specifically involved in the adaptation of piezophilic bacteria to the high-pressure, low-temperature conditions prevalent in the deep-sea environment. Since then, it has been confirmed that PUFA production occurs in numerous bacterial species isolated from Antarctic regions as well as temperate marine environments. Nichols et al. (37) have compiled data regarding the percentage of EPA producers isolated from various environments. Analyses revealed that only 1.5% of temperate marine isolates (60, 61), approximately 14% of Antarctic isolates (37), and 27% of deep-sea isolates (12) produce EPA. More recently, Yano et al. (55) investigated the distributions of bacteria containing PUFAs (both EPA and DHA) in the intestines of deep-sea fish and shallow-sea poikilothermic animals. Not only did the intestinal microflora of deep-sea fish contain a higher proportion of PUFA producers, but the percentage of PUFAs within these isolates was also greater than that of shallow-water animals. These results all suggest that there is a preponderance of PUFA producers associated with high-pressure and low-temperature environments.
What then is the explanation for EPA-deficient strain EA10's apparent lack of low-temperature or elevated-pressure sensitivity? Foremost, its increased MUFA content may be capable of compensating for the absence of EPA. Increased unsaturation of a membrane phospholipid-bound fatty acid does not result in a linear decrease in the membrane phase transition temperature. Biophysical studies employing synthetic mixed acid phosphotidylcholines (PC) have shown that the introduction of a double bond into 18:0/18:0-PC, yielding 18:0/18:1-PC, lowers the gel-to-liquid-crystalline phase transition temperature by nearly 50°C, whereas incorporation of the PUFA 20:4n-3 to yield 18:0/20:4-PC lowers the phase transition temperature by only an additional 19°C (8). Thus, in the case of strain EA10, the increased levels of 16:1 and 18:1 may provide adequate compensation for the loss of EPA. Alternatively, EPA may be required only under certain physiological conditions not evaluated in our work.
A final possibility is that EPA (and possibly DHA) is not required for psychrotolerant or piezotolerant bacterial growth but is needed as a nutritional source by higher organisms with which the EPA-producing microorganisms have established symbiotic associations. DeLong and Yayanos have previously suggested a specific role for in situ secondary production of PUFAs by piezophilic bacteria (12). Indeed, many of the PUFA-producing microorganisms that have been discovered have been isolated from vertebrate or invertebrate sources (10, 12, 13, 55, 58, 59).
| |
ACKNOWLEDGMENTS |
|---|
We thank Stu Brody of the University of California, San Diego, Biology Department for advice on fatty acid analysis.
This work was supported by grant MCB96-30546 from the National Science Foundation to D.H.B.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, 4405 Hubbs Hall, University of California, San Diego, 8604 La Jolla Shores Dr., La Jolla, CA 92093-0202. Phone: (619) 534-5233. Fax: (619) 534-7313. E-mail: dbartlett{at}ucsd.edu.
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