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Applied and Environmental Microbiology, November 1998, p. 4467-4476, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Small, Dilute-Cytoplasm, High-Affinity, Novel Bacterium
Isolated by Extinction Culture and Having Kinetic Constants
Compatible with Growth at Ambient Concentrations of Dissolved
Nutrients in Seawater
D. K.
Button,1,2,*
Betsy R.
Robertson,1
Paul W.
Lepp,3 and
Thomas M.
Schmidt3
Institute of Marine
Science,1 and
Department of Chemistry
and Biochemistry,2 University of Alaska
Fairbanks, Fairbanks, Alaska 99775, and
Department of
Microbiology, Michigan State University, East Lansing, Michigan
488243
Received 29 June 1998/Accepted 2 September 1998
 |
ABSTRACT |
Dilutions of raw seawater produced a bacterial isolate capable of
extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg
dry mass were similar to values for many naturally occurring
aquatic organotrophs, but water and DNA comprised a large
portion of this small chemoheterotroph, as compared to
Escherichia coli. The isolate used only a few aromatic
hydrocarbons and acetate, and glucose and amino acid incorporation were
entirely absent, although many membrane and cytoplasmic proteins were
inducible; it was named Cycloclasticus oligotrophus. A
general rate equation that incorporates saturation phenomena into
specific affinity theory is derived. It is used to relate the kinetic
constants for substrate uptake by the isolate to its cellular proteins. The affinity constant KA for toluene was low at
1.3 µg/liter under optimal conditions, similar to those measured in
seawater, and the low value was ascribed to an unknown slow step such
as limitation by a cytoplasmic enzyme; KA
increased with increasing specific affinities. Specific affinities,
a°s, were protocol sensitive, but under
optimal conditions were 47.4 liters/mg of cells/h, the highest reported
in the literature and a value sufficient for growth in seawater at
concentrations sometimes found. Few rRNA operons, few cytoplasmic
proteins, a small genome size, and a small cell size, coupled with a
high a°s and a low solids content and the
ability to grow without intentionally added substrate, are consistent
with the isolation of a marine bacterium with properties typical
of the bulk of those present.
| |
INTRODUCTION |
Bacteria are the dominant
transthreptic (across-surface-feeding) group of chemoheterotrophic
organisms in aquatic systems. They regulate the concentrations of
dissolved biogenic organics at nanomolar concentrations in the oceans
(7) and help support food webs (48). These
diverse (3, 19, 39), mostly planktonic oligobacteria are
infrequently studied because few regarded as typical in the environment
have been successfully cultivated (54, 55). Their nutrient
collection ability remains in question because the specific affinities
reported for cultured bacteria are too small to support presumed rates
of growth in pelagic systems (6). Extinction culture, i.e.,
dilution of marine populations with unamended sterile seawater to a few
organisms, often produces pure cultures (13). The first
culture obtained by this technique was isolate RM 1, later referred to
as RB1 (57). It persisted through numerous subcultures in
Resurrection Bay seawater without substrate addition, allowing further
characterization, and a 5.7-kb chromosomal DNA fragment was sequenced
(57).
In this communication, we show that specific affinities are
sufficiently large to support growth at ambient hydrocarbon
concentrations in seawater, but values vary with experimental protocol.
To sharpen the kinetic analysis, specific affinity theory, where
nutrient uptake is specified by a rate constant that derives from the
amount of permease or initial enzyme, is extended to accommodate
saturation phenomena and constants related to organism composition. The
isolate is phylogenetically characterized and its nutritional and
physiological aspects are described by using the theory along with new
flow cytometric methods (42) to help understand the ability
of aquatic bacteria to persist in a dilute, predator-inhabited environment.
| |
MATERIALS AND METHODS |
Culture and analyses.
Extinction culture RB1 was obtained as
a 108 dilution of Resurrection Bay seawater (13)
and maintained in glycerol at 
50°C. Dilution was done with
unamended seawater from Resurrection Bay, Alaska, prepared by
filtration through fired Gelman A/E 47-mm-diameter filters,
autoclaving, refiltering through other fired Gelman filters, and
aseptically siphoning the sterile filtrate into incubation chambers.
Culture purity was ascertained from flow cytometry patterns and by
microscope observation of colonies on agar plates following incubation
for 10 days at 20°C. Population density, dry weight, DNA content, and
biomass were determined by flow cytometry (10). The standard
curve for biomass was taken from light scatter theory (28)
after corrections for system geometry, axial ratio, and formaldehyde
sorption, and calibration from the radioactivity of
14[C]acetate-grown cells together with CHN analyses
(42). Cell volume was determined by electrical impedance
(model ZBI Coulter counter), and buoyant density was
determined by centrifugation in Percoll (Sigma Chemical Co., St. Louis,
Mo.). DNA was determined from the DAPI (4',6-diamidino-2-phenylindole)
fluorescence of single cells (10) by using Escherichia
coli containing integral genome copies with a size of 4.7 Mbp
(43) or 5.17 fg and a GC content of 50 mol% (29)
as a standard. The AT bias of DAPI (59) was corrected in
accordance with the GC content of the isolates, 52.7 mol% for
Marinobacter sp. strain T2 (20) and 41.6 mol% for isolate RB1 (20). Light scatter and fluorescence axes of the cytograms were converted to dry mass and DNA per cell by use of
formulations of the standard curves. Phospholipid fatty acids were
determined by Microbial Insights, Inc., Knoxville, Tenn.
Substrate usage.
Autoclaved synthetic seawater
(13) was amended with individual substrates or mixtures of
substrate at 100 mg/liter. The mixture contained 30% carbohydrates
(glucose, fructose, fucose, galactose, rhamnose, and arabinose), 20%
organic acids (acetate, lactate, glycolate, and succinate), 10%
alcohols (mannitol, glycerol, and ethanol), 20% organic acids
(acetate, lactate, glycolate, and succinate), 20% casein hydrolysate,
and 20% peptone. Volatiles were added as vapors. Flasks were
inoculated with 104 to 106 of acetate-grown
cells/ml at 25°C and observed for change in population density, cell
size, and DNA per cell by flow cytometry. For amino acids and glucose
uptake, acetate (100 mg/liter)-grown cells were washed three times by
centrifugation, filtered (pore size, 1 µm) to remove aggregates, and
incubated with radiolabeled substrate at 25°C. Transformation rates
of toluene were measured with purified [U-14C]hydrocarbon
(NEN Life Science Products) by toluene-grown washed cells as previously
described (41). Cell production by continuous culture
(31) was limited by injection of toluene to 20 mg/liter into
the feed carboy headspace and operated at a dilution rate of 0.1/h.
Cells were collected from the reactor by syringe, aerated for 20 min to
remove residual toluene, and used directly for uptake measurements.
Kinetic constants were obtained by using programs written to give a
best fit to v versus S plots (see Table 1), logarithmic transformations, and affinity plots in combination.
DNA purification and analysis.
Genomic DNA was purified from
isolates after detergent lysis (22). The small-subunit
rRNA-encoding genes (rDNA) were amplified from positions 27 to 1492 (E. coli numbering) as described previously (25).
PCR-amplified products were either cloned into the pCRII vector
(Invitrogen, Inc., Carlsbad, Calif.) or purified on Wizard columns
(Promega, Madison, Wis.). Clones and PCR products were sequenced by
using the ABI Catalyst 800 for Taq cycle sequencing and the
ABI 373 A sequencer for analysis of products. A collection of 10 primers was used for rDNA sequencing, resulting in an average redundancy of 2.5 per nucleotide position. Sequences were aligned manually on the basis of conserved regions of primary sequence and
secondary structure. A phylogenetic tree was inferred from 1,272 positions by using fastDNAml (38).
The number of 16S rDNA genes was estimated by restricting purified
genomic DNA with three restriction enzymes (PvuII,
PstI, and SacI) and separating the fragments on a
1.0% agarose gel. Southern analyses of the restriction digests were
performed by using a digoxigenin-dUTP-labeled probe complementary to a
5' region (positions 8 to 519) of the E. coli B strain 16S rDNA.
Homologs to
tfdA genes were amplified by using conserved
primers and stringent reaction conditions that provided specificity
with divergent bacterial isolates (
25).
Protein separations.
Acetate- or toluene-grown cells were
disrupted with lysozyme in a French pressure cell (16)
and separated into soluble cytoplasmic and membrane fractions by
centrifugation at 155,000 × g. The membrane fraction
was taken up in phosphate-ethanol-glycerol buffer and separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as
reported previously (56) and modified as described in the
Millipore Investigator manual, and the proteins were quantified by
densitometry (Alpha Innotech IS-1000; Innotech, San Leandro, Calif.) of
silver-stained gels.
| |
RESULTS |
Kinetic theory.
The collision frequency of substrate molecules
with a cell can be described by a per-particle rate constant,
k (51). For a substrate of molecular mass
M, concentration S, and diffusion constant
D, and a cell of radius rx (Table
1), k = 4
rxD/1,000 (1). For an organism of
density 1.04 g cm3, the equation k = 10 DM/rx2 describes substrate collection by a
perfectly collecting sphere and is the maximal value for specific
affinity (4) a°max. At subsaturating concentrations, the observable rate of uptake,
v, for a cell population of biomass X is smaller
than that described by a°max in proportion to
the surface area unavailable to colliding molecules for transport, and
entrance is resisted by the cell envelope more than it would be if the
cell were comprised of water alone. This resistance can be specified in
terms of an absorbability constant, 
, so that the uptake rate is:
|
(1)
|
Converting absorbability back to resistivity, R, where
= 1
R, and with R = (
a°
max a°
S)/
a°
max, the rate equation
may
be written in terms of the base or saturation-independent value
of
the specific affinity
a°
S:
|
(2)
|
Assuming that uptake requires interaction with a permease or
initial enzyme, the specific affinity depends on the number
or
aggregate effective area of the active sites of these proteins
and is
reduced by saturation phenomena to some value,
aS. At small
substrate concentrations where
v is unaffected by saturation,
aS
approaches
a°
S. The population of molecules in
the organism
devoted to substrate uptake may be approximated by
comparing this
rate constant for the total active-site area of the
protein in
question with the corresponding rate constant for the whole
cell.
In the case of N substrate-collecting molecules with effective
site radius
rs:
|
(3)
|
As substrate concentrations increase, resistivity is increased by
saturation and, where the resistance is hyperbolic, the
rate equation
is completely specified by
a°
S and
Vmax (
9).
Rearranging the
Michaelis-Menten equation by substituting
a°
S,
the initial slope of the
v versus
S curve, for
Vmax/
Km:
|
(4)
|
and a
v/
S versus
v or affinity plot gives
the two kinetic constants
a°
S and
Vmax as intercepts. Where
Vmax is experimentally
indeterminant
(
40) or the kinetics are nonhyperbolic (
34),
the
v/
S intercept remains the base value of the specific
affinity,
and the saturation-dependent affinity,
aS is
v/
S, i.e., the value
whose
product is rate at any substrate concentration,
S. Specific
affinity is obtained as units of liters per gram of cells per
hour by
taking
N as the number of permease molecules,
k
cat as
their catalytic constant expressed as a residence
time
= 1/k
cat,
and
aS as
v/
S from equation 2. Surface area is converted to wet
mass
by use of cell density, and choosing
c = 5
DMrs2/2
rX4 liters
cell (g of cells site h)
1 gives the following:
|
(5)
|
The numerator describes the unsaturated or maximal observable
value of the specific affinity as
a°
S =
Nc.
Inspection shows
that the saturation-dependent specific affinity,
aS, approaches
a°
S at
low concentrations and zero at high concentrations, the
affinity
constant
KA is
S at
a°
S/2,
Km remains
S at
Vmax/2, and
the general rate equation
becomes:
|
(6)
|
Characteristics.
RB1 populations diluted to a single cell in
unamended sterile seawater attained 105 organisms/ml. These
small populations were sufficient for phylogenetic analysis that
suggested a previously undescribed genus and for characterization by
flow cytometry. Addition of sugars, amino acids, organic and fatty
(lipoic) acids, alcohols, polyols, glucan, and lignin derivatives such
as anisoin (4,4'-dimethoxybenzoin) facilitated little further increase
to the base population of 105/ml. A complex mixture of
common substrates transformed the inoculum into cells with low DAPI-DNA
fluorescence (Fig. 1). Natural media such
as yeast extract gave slightly larger cell populations, but cytograms
plotting DNA versus biomass remained poorly resolved, suggesting that
the additions to media were also unhelpful in providing larger
populations. The low-fluorescence or "dim" cells produced often
appear in old cultures (42) and in freshwater and seawater
populations as well (11). Since the dry biomass of these dim
cells is often the same as that for growing cells (Fig. 1A is an
exception), much of the DNA may be degraded (26) but
retained. Flow cytometry has been used previously to detect apoptosis
in eukaryotic cells (36), changes which include DNA fragmentation (17), and data show that it can be used to
indicate the DNA status of bacterial cells and for development of
suitable growth conditions as well.
With acetate present in high (100-mg/liter) concentrations, smooth
translucent, colorless microscopic colonies appeared on
agar plates in
10 days and turbid cultures were produced in liquid
culture. Liquid
cultures resulted in cytograms (Fig.
2)
showing
dry weights of from 15 to 45 fg (dry weight)/cell comprised of
mostly B-phase cells with some D-phase (two-chromosome) cells
(
15) and a few replicating C-phase cells. Cell shape was
that
of a short rod defined by a thin cell envelope (Fig.
3),
and flagella
appeared to be present. Mean
values for the cells of cultures
in various stages of growth, as
indicated by the range in single-chromosome
content, are compared for
three species (Table
2). Both the dry
weight and the genome size of RB1 were comparatively small, but
the DNA
content was large, as shown in Table
2.
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FIG. 2.
Cytogram showing cells before (B), during (C), and
following (D) replication. s, standard spheres.
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Amphiphile kinetics.
The maximal growth rate,
µmax, on acetate was 0.19/h in batch culture with a cell
yield of 1.24 mg (wet weight) of cells produced per mg of acetate
consumed. Washed cells incubated with [14C]acetate gave
linear rates of incorporation for an hour, but the specific affinity
(Fig. 4) was only 9 × 102 liters/g of cells/h. Uptake rates were also linear
with substrate concentration to give a concave-side up affinity plot
(Fig. 4A, inset).
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FIG. 4.
Acetate (substrate A) uptake kinetics. (A)
Rate vA of [14C]acetate uptake by
washed cells at 1.2 mg of cells/ml over 2 min. The slope gives a
specific affinity of a°A = 0.09 liters/mg of
cells/h; other constants from the affinity plot (inset) are
indeterminant. (B) Uptake rates of acetate vA (=
µY) from the growth rates µ at 12 to 195 mg of acetate/liter
over 30 h and cell yield Y (=1.24 g of cells [wet
weight]/g of acetate used). The kinetic constants are
a°A = 1.5 liters/g of cells/h and
Vmax = 125 mg of acetate/g of cells/h from the
affinity plot intercepts (inset).
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|
In batch culture, the acetate concentration remained essentially
unchanged during 50 h of substrate-limited growth because
of the
low affinities, and the kinetic constants could be evaluated.
The
resulting rates of growth, together with cell yield, transformed
into a
linear dependency of uptake rate on concentration (Fig.
4B). Two
populations appeared at moderate rates of growth (Fig.
2). The ratio of
cells with two, as compared to one, chromosomes
increased with growth
rate µ according to µ =
kD/
B, where
k is
1.74/day, as the acetate concentration increased from 12 to 107
mg/liter. This demonstrated the range of acetate concentrations
that
was effective in growth rate control. The associated specific
affinity
of 1.5 liters (g of cells h)
1 was small but 16 times the
value directly observed from [
14C]acetate uptake. At
higher concentrations, the increase in uptake
rate was truncated by
µ
max.
Growth substrates.
When washed acetate-grown cells were
incubated with glucose or an amino acid mixture, incorporation was
negligible (Table 3). The highest
specific affinity attained was 0.003 liters/mg of cells/h, indicating a
resistivity, R, to incorporation of only 1 × 109 to
5 × 109, or very nearly unity as compared with a
perfect resistance of exactly unity. Growth strategies of oligobacteria
often include the concomitant use of multiple substrates to
increase the usable concentration of substrate (5).
However, data show an absence of incorporation of trace amounts of
radioactivity from hydrolysis products of carbohydrates and amino acids
by active cultures.
Dilution to about 1 cell of a culture that was maintained on acetate
for 3 years into 20 ml of unsupplemented seawater resulted
in cultures
with usual characteristics (Table
4),
showing that
the ability to grow in very dilute media is a constitutive
property
of RB1. The DNA content of the 21-day unamended seawater
culture
is consistent with a growth rate of 0.9/day, neglecting a small
C-phase population. A number of hydrocarbons were found to support
populations in the milligram-per-liter range from an
acetate-grown
inoculum including naphthalene, phenanthrene,
biphenyl, and toluene.
Various monoterpenes, dodecane, and methane did
not support growth.
Toluene uptake.
14[C]toluene supplied to washed
cells appeared as CO2 (Fig.
5), cell material, and nonvolatile
metabolic products (41) distributed among several compounds
that remain unidentified in dilute-substrate experiments
(
max for the mixture was 401.5 nm). Use of small populations was required to avoid premature depletion during the several minutes needed to establish a time course for substrate uptake
by these very active cells. Time course curves for
14CO2 evolution were linear and without
significant intercept values at time zero. The accumulation rate of
14C-labeled nonvolatile metabolic products was more erratic
and may have been affected by resorption; 70% of the product
radioactivity accumulated over the first few minutes was lost in
high-biomass experiments. Radioactivities collected as cells on
membrane filters (data not shown) were inconsistent, giving an error of
approximately 30% due to the large membrane filter/biomass ratio
used, but values were near those for 14CO2.
From these and other data, we estimate a yield of cells-carbon dioxide-products of 1:1.00:1.95, similar to that for Marinobacter arcticus (41). Specific affinities calculated from
uptake rates and cell yield increased substantially when uptake was
observed over longer incubation times, as facilitated by reducing
biomass to nanogram-per-milliliter quantities (Table
5). A large value was obtained from
continuously grown cells as well. Competitive displacement of label
from residual toluene was estimated at 90% after sparging for 2 min
from the increase in specific affinity and decrease in radioactivity;
20 min was allowed for complete removal. Passage through
1.2-µm-pore-size filters had little effect on observed specific
affinities and precluded overestimation of values due to the presence
of small clumps undetected by flow cytometry, although these were
presumed to be absent from the low value of the above-scale particle
count.
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FIG. 5.
Toluene (substrate B) uptake kinetics. (A)
Uptake over 4 min with 2,590 µg of cells/liter; (B) uptake over 90 min with 2.4 µg of cells/liter. [14C]toluene uptake was
from the rate of liberation of oxidation products
(VPB) ( ) and CO2
(VQB) ( ) and was calculated as toluene mass;
toluene uptake rates from CO2 recovered from continuously
grown cells ( ) are also shown (31). Rates were calculated
from the appearance of cell material, CO2, and metabolic
products.
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Taxonomy.
The 16S rDNA of the isolate is phylogenetically
related to the recently described Cycloclasticus pugetii
(18) and used to designate the organism as
Cycloclasticus oligotrophus (57). Differentiation
is based on small size and essentially absolute inability to utilize
polar substrates such as amino acids (Fig. 1 and Table 3). The
enrichment culture isolate used for physiological comparison (Table 2)
was obtained from ballast water of the tanker Mobile Arctic
in Port Valdez, Alaska, and also grows on toluene (a°B = 0.63 liters/mg of cells/h and
KA = 66 µg/liter [41]) but
uses amino acids (a°S = 4.1 liters/mg of
cells/h, KA = 136 µg/liter for a mixture
[unpublished data]). It is phylogenetically similar to
Marinobacter hydrocarbonoclasticus (20) but grows on glucose and is referred to as Marinobacter
arcticus. Phylogenetic locations relative to some other
aquatic bacteria are shown in Fig. 6.
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FIG. 6.
Phylogenetic tree of Proteobacteria inferred
by maximum-likelihood analysis of 16S rRNA gene sequences. The
oligobacteria discussed are indicated by boldface type. The marker bar
represents evolutionary distance.
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|
Structural genes.
Five open reading frames sequenced from
chromosomal DNA (57) included homologs with the large and
small subunits of biphenyl dioxygenase as well as an unidentified
membrane protein. Only a single copy of the rRNA operon
(44) was present in C. oligotrophus. Both
C. oligotrophus and M. arcticus contained genes
that were amplified when tfdA-specific primers were used
in the PCR. The tfdA gene product has been associated with
the metabolism of chlorinated hydrocarbons (25). The PCR
products from C. oligotrophus were approximately 400 and 450 bp, while the product from M. arcticus was about 600 bp, compared to only 360 bp for the fragment from Alicaligenes
atrophies (25).
Cytoarchitecture.
Indigenous marine bacteria are comparatively
small, a morphological characteristic that has impeded observation.
They also are thought to be quite dense, with carbon content
increasing to 0.38 g/cm with decreasing size (33); however,
the required cell volume data are difficult to measure by microscope
due to their small size. Flow cytometry and radioactivity of
14[C]acetate-grown C. oligotrophus
(42) gave the expected low dry weight for a typical marine
bacterium (Table 2), but the total solids content was only 16% as
calculated from cell volume according to Coulter Counter and buoyant
density. While the CHN ratio was normal at 47:5.6:9.2% by CHN
analysis, the carbon content was also low at 7% of wet cell mass
according to the radioactivity measurements. Dry weight was
corroborated by comparatively low equilibrium density measurements.
Also, the refractive index by minimal optical density in serum albumin
was 1.025, to give a low dry weight (27) of 19%. This
compares with a refractive index of 1.039 for E. coli and a
dry weight content of 27 to 30%, and the organism was more dilute than
expected for marine bacteria (35). One result of the low dry
weight was a DNA content six times that of E. coli on a dry
mass basis despite a genome that is little more than half the size.
Two-dimensional electrophoretic patterns suggested fewer proteins in
C. oligotrophus than in
M. arcticus and
E. coli (Table
2), consistent with organism simplicity. Among the 210 cytoplasmic
and 170 membrane peptides resolved, about 50 in each
appeared
as unique toluene-inducible spots. Among the major membrane
peptides,
the concentration of at least four more doubled in the
presence
of acetate, and induction by toluene was equally strong for 11
others (Fig.
7). Two cytoplasmic
toluene-inducible proteins, according
to their N-terminal amino acid
sequences, were related to catechol
2,3-dioxygenase and
dihydroxynaphthalene dioxygenase (unpublished
data). Data show that
although few growth substrates have been
identified, this small low-DNA
organism retains the ability to
adjust its protein composition to
environmental conditions.
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FIG. 7.
Densitometry scans of one-dimensional SDS-PAGE gels from
the membrane protein fractions of acetate- and toluene-grown cells.
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|
The unsaturated fatty acid content of acetate-grown
C. oligotrophus increased with growth on toluene at the
expense of stearic
and oleic acids (Table
6) and the major
trans fatty
acid, 16:w7t,
present at 2.4%, was nearly eliminated with growth on
toluene.
| |
DISCUSSION |
Composition.
The dry cell mass of C. oligotrophus
was 15 to 20 fg/cell when growth rates were restricted, maximal at 80 fg/cell, and similar to values for populations from the Gulf of Alaska,
where means ranged from 30 to 37 fg/cell between those collected at the
sea surface and those collected at a depth of 1400 m (unpublished data). Mass was at the 15th percentile of bacteria from the Gulf profile; i.e., 85% of the particles having >1.5 fg of DNA and a 7-fg
dry cell mass were smaller (unpublished data). Yet the organism was far
smaller than most aquatic bacteria that have been isolated and
perpetuated in pure culture. The DNA content of 3.2 fg (2.9 Mb)/genome
was the same as the mean value of 3.1 to 3.2 fg for total DNA in the
depth series. The large DNA content retained following cultivation in
unamended seawater (Table 4, 21-day culture) is, in the absence of a
long lag phase, indicative of a growth rate that is higher than
justified by initial and final cell populations. Chromosome
runout, or production of some two-chromosome-containing cells
following exhaustion of the natural substrate (2), occurs in
the isolate (47) and provides an explanation for this
apparent discrepancy between growth rate and cell cycle theory
(23). DNA comprised much of the cytoplasm of smaller
bacteria in seawater, so these organisms may approach a minimum dry
weight for their genome size. Induction (41) and regulation
(46) of proteins for hydrocarbon metabolism are known and,
although the genome size of the isolate is comparatively small (Table
2), the ability to regulate was encoded within this small genome. Only
about 5% of the in situ forms from the depth series contained less
than 1.5 fg of DNA, which suggests a lower limit for pelagic marine
bacteria. Small size and low dry weight maximize the surface-to-volume
ratio for effective nutrient accumulation, minimize endogenous costs,
and reduce value to predators. However, the small genome size could
restrict metabolic flexibility. Organisms with very low amounts of DNA
have not yet appeared in our extinction cultures. The only other known
extinction culture isolate, Sphingomonas sp. strain RB2256,
also has a high specific affinity for substrate and but one rRNA operon
copy (45). Because DNA is a major fraction of the dry weight
of C. oligotrophus, minimizing it represents a way of
allowing small size. Similarity in size and DNA content to in situ
organisms and the retained ability to grow on unamended seawater
suggest that the isolate may be rather typical of marine bacteria.
Nutrition.
The low specific affinity for acetate indicated
that uptake of this substrate was incidental and of little use to the
organism in the environment, and the large affinity constant with
specific affinity abruptly truncated by µmax suggested
that acetate was not actively transported. The increase in
trans fatty acids during growth on this low-affinity
substrate is consistent with increased stress (58) and that
less fluid membranes are not used for defense against 30-mg/liter
concentrations of toluene used in culture media. Acetate is, however,
advantageous for use in laboratory media since large concentrations of
the substrate can be added without substrate inhibition. Potential
difficulties from substrate-accelerated death due to unintentionally
added polar substrates in inhibitory concentrations during isolation
were avoided by an essentially perfect resistance to their
incorporation, as determined by glucose and amino acid uptake data,
possibly contributing to our success in isolation.
Organism sensitivity to environmental change is suggested by the large
increase in specific affinity and
Vmax for both
acetate
and toluene that is facilitated by continuous undisturbed
growth
in batch culture, long incubation times at extremely small
populations,
or by continuous culture. Such sensitivity is consistent
with
the even larger increase in the specific affinity observed for
phosphate when care was taken to use populations directly from
continuous culture with minimal manipulation (
40). These
phosphate
data also showed an extreme variability in
Vmax due to manipulation,
as reported here for
acetate and toluene. Affinities of transthreptic
organisms may often be
underestimated because the flux computed
from growth rates is difficult
to attain in uptake experiments
and presumed growth rates in the oceans
are higher than justified
by most measurements of specific affinity
(
6). Comparisons
of specific affinities with those from
other systems and with
expected flux requirements for growth may
help alleviate or explain
these
discrepancies.
Kinetics.
The maximal specific affinity for toluene use by
C. oligotrophus is uncertain, but the observed
value of 47.4 liters/mg of cells/h is the largest known
for various organism-substrate combinations (6). It compares
with a molecular collision frequency sufficient to attain a specific
affinity of about 6,000 liters/mg of cells/h, meaning that the
resistance, R, to unimpeded flow into the organism is 0.992, giving
an absorbability, , of 0.8% of the theoretical maximum, where all
molecular collisions with the cell surface are successful. The specific
affinity for continuously grown cells was only slightly lower, and the
standard error of the measurement was small (Fig. 6). The
reduction could have been due to the sensitivity of the organism
to unavoidable population disturbance during the aeration step
between cell harvest and uptake measurement or to unexpected
oxygen limitation since the process is electron acceptor sensitive
(32).
Rates of growth with in situ toluene concentrations measured at 1 µg/liter (
12) can be calculated from the data of
Table
5 and equation 4. To do this, we substitute
a°
B for
Nc in equation
5 to obtain
nutrient collection on an organism, rather than enzyme
molecule, basis;
set
KAc at unity so that the denominator
is
2 since the affinity constant
KA is the
substrate concentration
at which the specific affinity is half its
maximal value,
a°
B;
and with
KA =
B = 1.3 µg/liter, solve for
c to obtain (2
1)/1.3
× 10
6 = 0.769 × 10
6 liters
2/g
of cells/g of substrate/h. At
B = 1 × 10
6 g/liter, the specific affinity at 1 µg/liter is 47.4 × 10
3/[1 + (1 × 10
6)(0.769 × 10
6)] = 26.8 × 10
3 liters/g of cells/h. With
aB
determined, equation 6 may be written
in terms of the specific
rate of growth, µ
B, from the toluene
(substrate
B) component of the medium:
|
(7)
|
With a cell yield
Y of 0.46 mg (wet weight) of cells/mg
of toluene used from radioactivity incorporation, the specific
growth
rate is (26.4 × 10
3)(1 × 10
6)(0.46) = 0.012/h or 0.29/day. Specific
affinities for substrates
should be additive when rates involve
different rate-limiting
steps so that concomitant utilization of only
two other substrates
at equivalent affinities and concentrations should
allow the organisms
to exceed a doubling time of 1 day. The value
of reporting this
kinetic constant is suggested by literature
data from which specific
affinities may be calculated that give
insufficient values for
observed rates of culture growth at all but
very large concentrations
of substrates (
6). Inclusion would
help clarify the abilities
of populations to grow at small substrate
concentrations because
the scale is absolute rather than normalized to
Vmax, because
most aquatic microbial populations
are nutrient limited, and because
a°
s is
sensitive to some difficult-to-avoid systematic
errors.
Environmental applications.
Use of exogenous toluene in
seawater samples is too rapid (8) for likely support of
the required enzyme systems by anthropogenic sources, but
hydrocarbons might be formed as electron sinks during anaerobic
metabolism of complex organics from plant material deposited in
underlying sediments. The presence of a PCR product amplified by
tfdA-specific primers, the first enzyme in the pathway for the degradation of 2,4-dichlorophenoxyacetic acid, further suggests a
role for biogenic hydrocarbons in the nutrition of C. oligotrophus. Large quantities of bromoform occur in coastal
seawater (7), and chlorinated hydrocarbons can provide
a vehicle for chlorine removal in plants (21). The isolate
also contains glutathione-S-transferase (57), which may assist in dechlorination (24)
prior to exergonic metabolism of halogenated hydrocarbons, so there
appears to be potential supplies of both hydrocarbons and chlorinated
hydrocarbons to support hydrocarbon-oxidizing bacteria in the ocean.
The observed specific affinity for toluene is 100 to 1,000 times the
value for the bacterioplankton community toward amino
acids, both in
freshwater and marine systems (unpublished), and
values range over a
factor of 300 for various cultures (Table
7). Assuming minimal systematic effects
on the determination
of kinetic constants, some organisms are 300 times
better at collecting
toluene from low concentrations than others.
Ambient toluene may
be partitioned from low external
concentrations to higher concentrations
within the membrane
lipid (
50) and, because of the direct relationship
between
permeability and partition coefficient (
30), diffuse
unimpeded into the cytoplasm. The specificity of dioxygenases
is broad
(
53), and the quantity available for toluene oxidation
can
be estimated at 2,500 molecules per cell (
6), so permeases
may not be required. At
Vmax, the residence time
of toluene on
dioxygenase molecules could be as long as 250 ms, and if
the collection
area of the active site has a radius of 10 Å to give
c = 9.2 liters
cell (g of cells site
h)
1, the specific affinity is 84 liters/mg
of cells/h, only about
twice that observed. Solving equation 4 for
S gives an affinity
constant,
KA, of
4.6 µg/liter that is near the measured value
of 1.3 µg/liter. The
small affinity constant for this oligotroph,
compared to most measured
values for bacteria (
5), might be
attributed to small
amounts of enzyme in metabolic pathways, amounts
that are sufficient
for growth when nutrient concentrations are
low and rates are low but
small enough to increase residence time
in metabolic pathways. This may
be seen from equation 5, where
the saturation-dependent specific
affinity decreases with increasing
. At environmentally excessive
concentrations, overloaded pathways
from the large amounts of initial
enzyme produced to collect substrate
cause an increase in the effective
, i.e., residence time, based
on the rate of substrate passage
through the whole pathway including
steps catalyzed by small numbers of
enzyme molecules. Small specific
affinities may be associated with
large saturation constants and
maximal velocities for rapid metabolism
at high concentrations
as indicated by large
Vmax values (Table
7). The large specific
affinity is thought to be related to the amount of initial enzyme,
and
the small affinity constant is taken as flux limitation by
the amount
of downstream enzyme consistent with the concept of
flux-limiting
enzymes (
37). That
Km exceeds
KA is expressed
by the concave-side-up affinity
plots. Simulations show that these
kinetics are consistent with both
stimulation of slow steps such
as macromolecule synthesis through
positive feedback to give an
effective increase in
and the
concentration-dependent shift
in the control point of a metabolic
pathway mentioned above. The
low affinity constants for toluene
uptake are consistent with
in situ
Km values
(Table
7), and the decreased affinity with
increased saturation
constants is consistent with specific affinity
theory but not with the
application of Michaelis-Menten concepts
for enzymes to whole cells.
The growth substrates that sustain this organism in
unamended seawater media remain unknown. This is also true for
pelagic
bacteria, given observed specific affinities for common
substrates,
if one assumes a homogeneous distribution of
dissolved organics
and that most of the approximately
10
6 cells/ml are both metabolically active and reproducing.
Although
care was taken to exclude hydrocarbon vapors from the
incubation
chambers, complete elimination may not have taken
place. Alternatively,
untested substrate combinations may have been
used by this induction-capable
isolate. The kinetic constants
shown here are the first that are
consistent with growth in the pelagic
marine environment at measured
concentrations of organic
substrate.
| |
ACKNOWLEDGMENTS |
We thank Luis Pinto for electrophoretic analyses and Rosemary
Ruff, formerly of this institution, and Ya Chen, of the University of
Wisconsin Integrated Microscopy Resource, for electron microscopy.
Support was provided by the Ocean Sciences and Metabolic and Cellular
Biochemistry sections of the National Science Foundation and the U.S.
Environmental Protection Agency.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Marine Science, University of Alaska, Fairbanks, AK 99775. Phone: (907) 474-7708. Fax: (907) 474-7204. E-mail:
dkbutton{at}ims.alaska.edu.
| |
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Abstract
Introduction
