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Applied and Environmental Microbiology, November 2001, p. 4955-4962, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.4955-4962.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Input of Protein to Lake Water Microcosms Affects Expression of
Proteolytic Enzymes and the Dynamics of
Pseudomonas spp.
Jakob
Worm* and
Ole
Nybroe
Section of Genetics and Microbiology,
Department of Ecology, The Royal Veterinary and Agricultural
University, DK-1871 Frederiksberg C, Denmark
Received 30 April 2001/Accepted 24 August 2001
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ABSTRACT |
The objective of this study was to determine how an input of
protein to lake water affects expression of a proteolytic potential and
influences the abundance and composition of a specific group of
bacteria. Pseudomonas spp. were chosen as a target group
that can be recovered on selective growth media and contain both
proteolytic and nonproteolytic strains. Amendment with 2 mg of casein
per liter increased total proteinase activity (hydrolysis of
[3H]casein) by 74%, leucine-aminopeptidase activity
(hydrolysis of leucine-methyl-coumarinylamide) by 133%, bacterial
abundance by 44%, and phytoplankton biomass (chlorophyll
a) by 39%. The casein amendment also increased the
abundance of culturable Pseudomonas spp. by fivefold
relative to control microcosms but did not select for proteolytic
isolates. Soluble proteins immunochemically related to the
Pseudomonas fluorescens alkaline proteinase, AprX, were detected in amended microcosms but not in the controls. The expression of this class of proteinase was confirmed exclusively for proteolytic Pseudomonas isolates from the microcosms. The population
structure of Pseudomonas isolates was determined from
genomic fingerprints generated by universally primed PCR, and the
analysis indicated that casein amendment led to only minor shifts in
population structure. The appearance of AprX-like proteinases in the
lake water might thus reflect a general induction of enzyme expression
rather than pronounced shifts in the Pseudomonas
population structure. The limited effect of casein amendment on
Pseudomonas population structure might be due to the
availability of casein hydrolysates to bacteria independent of their
proteinase expression. In the lake water, 44% of the total proteinase
activity was recovered in 0.22-µm-pore-size filtrates and thus
without a direct association with the bacteria providing the
extracellular enzyme activity. Since all Pseudomonas isolates expressed leucine-aminopeptidase in pure culture, proteolytic as well as nonproteolytic pseudomonads were likely members of the
bacterial consortium that metabolized protein in the lake water.
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INTRODUCTION |
Bacterial growth in pelagic
ecosystems is supported by a complex mixture of organic compounds
(43), among which proteins appear to be important
(7, 27, 40). Bacteria cannot assimilate proteins directly
(37) but depend on extracellular and/or cell-associated enzyme systems to liberate protein-bound amino acids for assimilation and metabolic processes (5). During a so-called
proteolytic cascade, proteins are broken into smaller fragments by
proteinase enzymes (endopeptidases), and these peptides serve as
substrates for exopeptidases (e.g., aminopeptidase) with affinities to
release terminal amino acids (28). Extracellular
proteinase activity is thus important for the initial cleavage of proteins.
Addition of protein to sea or lake water stimulates bacterial growth
and leucine-aminopeptidase (LAP) activity (13, 38, 50).
Following an input of protein, Pinhassi et al. (38) found that five populations of bacteria proliferated, while the abundance of
another 10 populations was more stable, as evident from a whole-genome hybridization between environmental DNA and DNA from pure cultures. The
authors proposed a link between the shift in the structure and function
of the community. However, they also recognized that proteolytic
activity should be traced directly to the enzyme-producing populations
to prove causal relationships. As yet, no studies have done that for
proteolytic enzymes in aquatic environments.
Pseudomonads are found in many aquatic ecosystems by both
culture-dependent (11, 12, 17) and culture-independent
techniques (9, 10, 16, 25, 39). Pseudomonads are known as
early colonizers of "new" habitats, such as developing root systems and food products, indicating an opportunistic growth strategy in
response to available nutrient resources. In general, they are also
easy to culture on nutrient-rich agar media. The genus Pseudomonas comprises both proteolytic and nonproteolytic
strains (53). Several proteolytic strains are well
characterized due, e.g., to their deterioration of milk
(8) and meat products (29). Several
proteinase enzymes have been characterized (14), and
antibodies have been raised to some of them (2, 33, 47). The above properties make Pseudomonas an attractive target
group for studies that address how protein amendment can affect the expression of a proteolytic potential and influence the dynamics and
composition of specific bacterial populations.
In this study we aim to determine how protein amendment affects
expression of a proteolytic potential and influences the abundance and
population structure of Pseudomonas spp. in lake water.
Microbial dynamics (direct and culture-dependent estimates of microbial abundance) and enzyme activities (proteinase and LAP) were followed in
lake water microcosms. The abundance of Pseudomonas spp. was followed specifically (Gould S1 agar), and expression of the AprX-like Pseudomonas proteinase was detected immunochemically in the
microcosms. The population structure of Pseudomonas was
examined using genomic fingerprints generated by universally primed PCR
(UP-PCR).
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MATERIALS AND METHODS |
Sampling.
On 3 April 2000, water was collected from
mesotrophic Lake Esrum, Denmark (23). Within 2 h,
microbiological analyses (see below) were initiated in the laboratory,
and subsequently microcosms of 2.5 liters of lake water were
established. Duplicate microcosms were amended with 2 mg of casein
sodium salt (Sigma, St. Louis, Mo.) per liter to increase the pool of
biodegradable dissolved organic matter by approximately 2.5-fold
relative to the natural level (44), assuming a C:N ratio
of 5 for casein. The casein stock solution had been dialyzed against
water from the Milli-Q purification system (Millipore Corporation,
Bedford, Mass.) to remove eventual low-molecular-weight compounds.
Another two unamended microcosms served as controls. The microcosms
were incubated at 15°C in a 16-h light-8-h dark cycle on a shaker at
100 rpm and sampled daily during the following 4 days.
Abundance of phytoplankton and bacteria.
Phytoplankton were
collected on Whatman GF/C filters and frozen. Chlorophyll a
was extracted with 96% ethanol and measured spectrophotometrically to
indicate the phytoplankton biomass (20).
Samples for direct counts of bacteria were stained for 15 min with the
DNA-binding fluorophore SYBR green (Molecular Probes, Leiden, The
Netherlands), fixed in 2% buffered formaldehyde (final concentration),
and stored at 5°C for less than 2 weeks. Bacteria were counted with a
FacsCalibur flow cytometer (Becton Dickinson, Brøndby, Denmark) using
a fixed concentration of 2-µm-diameter fluorescent beads (Molecular
Probes) as internal standards for the volumes analyzed.
Proteinase and aminopeptidase activity.
Proteinase activity
(PRTase) was measured as the turnover of
3H-methylated casein (% h
1) according to Keil and Kirchman
(24). 3H-methylated casein (0.5 µCi µg1) was generated by reductive
methylation (48) of dialyzed (0.2 M borate buffer [pH
8.9]) casein sodium salt (Sigma) with 32 Ci mmol of
B3H41
(Amersham Pharmacia Biotech., Little Chalfont, England) and
formaldehyde (Sigma). Subsequently, protein-bound
3H was rinsed by several washes in 10-kDa-cutoff
Microcon microconcentrators (Amicon, Beverly, Calif.). Triplicate
samples of lake water were amended with
3H-methylated casein (ca. 10 µg of C
liter1) and incubated for 3 h at 20°C.
Incubations were stopped by additions of nonlabeled casein (2 g
liter1) and trichloroacetic acid (TCA) (5%
final concentration) to precipitate unhydrolyzed protein on ice for
>20 min. TCA-soluble radioactivity (protein fragments and other
degradation products) was separated from precipitated protein by
centrifugation (10,000 × g, 10 min, 5°C) and
quantified with a Beckman LS 1801 liquid scintillation counter (Beckman
Instruments, Inc., Fullerton, Calif.) by use of Lumasafe (Lumac LSC
B.V., Groningen, The Netherlands). Parallel blank samples were
incubated for either <1 min with lake water or 3 h with
autoclaved Milli-Q water. These two approaches gave similar background
levels of radioactivity in the assay.
Coefficients of variation averaged 3.4% (standard deviation [SD] = 2.2%) for triplicate samples or blanks. Corrected for blank
values,
radioactivity in supernatants was divided by total radioactivity
added
and normalized to incubation time. No corrections were done
for
bacterial assimilation of
3H in accordance with
Keil and Kirchman (
24). The turnover rate
(% h
1) measured under these conditions was
constant during an incubation
of at least 4 h (data not
shown).
LAP activity was measured with
L-leucine-4-methyl-coumarinylamide hydrochloride (ICN,
Costa Mesa, Calif.) (Leu-MCA). Cleavage
of the peptide bond between Leu
and MCA releases fluorescent 7-amino-4-methylcoumarin
(AMC) with
proportional increases in fluorescence, as described
by Hoppe
(
19). Samples were amended with 0.25 mM Leu-MCA and
incubated in the dark at 20°C. Fluorescence was read with a Kontron
SFM 25 fluorometer (Kontron AG, Zürich, Switzerland) and
calibrated
against standard solutions of free AMC (ICN). The formation
of
hydrolysates was normalized to the time of incubation to express
hydrolysis rate as micromolar per hour. No increase in fluorescence
was
observed in parallel blank samples of autoclaved Milli-Q
water.
Immunochemical detection of AprX-like Pseudomonas
proteinase.
To test whether Pseudomonas contributed to
the proteolytic activity in the microcosms, proteins in the water were
concentrated, and the presence of the alkaline Pseudomonas
proteinase (AprX) was estimated immunochemically. Polyclonal rabbit
immunoglobulin G (IgG) (antibody) raised toward a purified 46-kDa
proteinase of P. fluorescens UP206 was provided by A. C. Magee and D. A. McDowell, University of Ulster, United Kingdom
(33).
The specificity of the antibody was tested by Western blot analysis of
proteolytic culture supernatants of the following strains:
P. fluorescens strain ON2 (
54),
P. aeruginosa
DSM 50071,
Erwinia chrysanthemi DSM 4610,
Serratia
marcescens ATCC 990, and
Proteus mirabilis S31F2
(Department of Veterinary Microbiology, The Royal
Veterinary and
Agricultural University, Frederiksberg, Denmark).
These bacteria
produce proteinases homologous to the AprX proteinase
of
P. fluorescens CY091 (
31,
54). Western blot analysis was
also performed with supernatants from selected proteolytic and
nonproteolytic supernatants from
Pseudomonas isolates from
Lake
Esrum. Nonproteolytic control strains included
P. fluorescens DF57 (
45),
P. fluorescens
ON2-pd5 (
54), and
Escherichia coli DSM 498. The
strains were grown overnight in ZoBell medium, Luria
broth, or minimal
medium with glucose, glutamine, and casein to
induce expression of
proteolytic activity (
54). Culture supernatants
were
harvested following centrifugation (6,000 ×
g, 5 min),
and
protein concentration (bicinchoninic acid protein assay; Pierce,
Rockford, Ill.) and proteinase activity (
41) were measured
before
Western blot
analysis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed in 12% acrylamide gels (Novex, San Diego,
Calif.).
Details for SDS-PAGE and Western blotting are described
by Kragelund et
al. (
26). Indigenous phosphatase activity was
inactivated
by a 30-min incubation in phosphate-buffered saline
(pH 2.6). Following
washing and blocking steps, nitrocellulose
membranes with transferred
proteins were incubated overnight with
the rabbit antibody at dilutions
of 1:5,000 or 1:10,000. Rabbit
antibody was detected following 3-h
incubations with a 1:1,000
dilution of anti-rabbit immunoglobulins
conjugated with alkaline
phosphatase (Dako, Glostrup, Denmark) and a
color reaction with
5-bromo-4-chloro-3-indolylphosphate (Sigma) and
nitroblue tetrazolium
(Sigma).
For the detection of the AprX-like
Pseudomonas proteinase in
the microcosms, water samples were passed through 0.22-µm-pore-size
Stericup-GS filter system (Millipore) and concentrated approximately
400-fold by use of Ultrafree-CL and Centriplus centrifugal filter
units
(Millipore) with nominal molecular size limits of 30 and
10 kDa,
respectively. Subsequently, SDS-PAGE, blotting, and immunodetection
were carried out as described
above.
Cultivation of bacteria.
Bacteria were cultured on two solid
media, Gould S1 (15) and 1/10 strength ZoBell 2216E agar
(0.5 g of peptone, 0.1 g of yeast extract, 15 g of agar per
liter of lake water) following 10-fold dilutions in 0.9% NaCl. Gould
S1 is highly selective for Pseudomonas spp. due to the
antibiotic trimethoprim and the detergent sodium lauryl sarcosine
(15, 21, 22, 26), while the ZoBell medium is a general
medium supporting the growth of a wide variety of bacteria.
Phenotypic and genotypic characterization of
Pseudomonas.
A collection of Pseudomonas
isolates was established for subsequent phenotypic and genotypic
characterization. At the start of the experiment, 96 colonies from the
Pseudomonas-specific Gould S1 agar were streaked to purity.
On day 5, this procedure was repeated for the four microcosms to
provide a collection of 480 Pseudomonas isolates. The strain
collection was stored in equal volumes of ZoBell medium and glycerol at
80°C.
Pseudomonas isolates were screened for extracellular
proteinase activity on casein in skim milk agar (
41). To
screen for
LAP activity, cultures grown overnight in liquid ZoBell
medium
were incubated with 0.25 mM Leu-MCA for 3 h in the dark at
20°C.
Fluorescence was quantified before and after the incubation by
use of an LS 50B luminescence spectrometer (Perkin Elmer Ltd.);
see
details for LAP activity above. Additionally, some isolates
were
characterized by their ability to utilize 95 distinct carbon
sources in
Biolog GN2 assays (Biolog Inc., Hayward, Calif.). Strains
were streaked
on tryptic soy broth agar (Difco Laboratories, Detroit,
Mich.) prior to
the inoculation in duplicate Biolog GN2 plates
at a final cell density
of optical density at 600 nm (OD
600) =
0.1. Color reactions following the utilization of specific carbon
sources
were quantified after 24 h of incubation
(OD
590 read by
EL312 automated microplate reader;
Bio-Tek Instruments Inc., Winooski,
Vt.). Wells with
OD
590 values 40% above the blank wells were
scored
as positive if a purple color response was also visible. Strains
were identified by matching the patterns of sole-carbon-source
utilization to the GN database release 4.01C running under the
Biolog
MicroLog 1 system release 4.01B (Biolog Inc.).
Genomic fingerprints of the isolates were obtained by UP-PCR
(
4). A universal primer consists of a universal sequence
(5'
end) and a variable sequence (3' end) without homology to any
known
gene position. This design stabilizes the primer annealing
at
relatively high temperatures and the generation of reproducible
PCR
products without prior knowledge of the target organism
(
4).
DNA was extracted from cultures grown overnight in
liquid ZoBell
medium by 10 min of incubation at 94°C and freezing at
20°C.
Then 1 µl of cell extract was mixed with 40 ng of L15/AS19
primer
(
32), 0.6 U of F-501 DyNAzyme II DNA polymerase
(Finnzymes,
Espoo, Finland), and 18 µl of reaction buffer. The
reaction buffer
contained 1× F-511 Dynazyme buffer (Finnzymes), 2 mM
MgCl
2, and
0.4 mM deoxynucleoside triphosphate
mix (New England Biolabs Inc.,).
The PCR was carried out in a GeneAmp
PCR System 9700 thermal cycler
(Perkin Elmer, Norwalk, Conn.) with the
following settings: 94°C
for 3 min, followed by 33 cycles of 92°C
for 50 s, 53°C for 70
s, and 70°C for 60 s, and a
final extension prolonged by 2 min.
PCR products were separated by
electrophoresis in 2% (wt/vol)
agarose gels. Gels were loaded with
100-bp ladders (Amersham Pharmacia
Biotech Europe GmbH, Denmark) as
molecular size standards. PCR
products were grouped manually by use of
digitized photographs
overlaid by a size
marker.
Data analysis.
Abundances and biochemical measures in
duplicate microcosms were compared by t test (two-tailed at
P < 0.05) following logarithmic transformations to
equalize the variances (42). Distributions of
Pseudomonas isolates among UP-PCR groups were analyzed by
the following indices (3): diversity H = 
pi ln
pi, where pi is the frequency of isolate in the ith UP-PCR group.
H was normalized by the richness in groups (S) to
obtain an index of evenness E = H/ln
S. Diversity indices were compared by t tests
(42) where continuity of the variables was assumed from
the relatively high number of isolates being distributed among a
relatively high number of UP-PCR groups. The distribution of UP-PCR
groups was also compared by 
2 tests. We
focused specifically on the 10 most abundant UP-PCR groups, but for
2 statistics, relevant joint groups had to be
defined to meet the required minimum of five observations per class in
each sample (42).
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RESULTS |
Microbial dynamics in microcosms.
Initially, the lake water
contained 14 µg of chlorophyll per liter. In the control microcosms
(dashed lines), chlorophyll increased and peaked at 39 µg
liter1 on days 3 to 4 and decreased to 31 µg
liter1 at the end of the experiment (Fig.
1A). Direct counts of bacteria increased
from 1.8 × 106 to 5.3 × 106 ml1 from days 1 to 3 and declined subsequently to 3.3 × 106
ml1 (Fig. 1B). In parallel, colony counts on
ZoBell agar increased from 5.2 × 103 to
20 × 103 CFU ml1,
whereas colony counts of Pseudomonas (Gould S1) decreased
from 13 to 5.4 CFU ml1 (Fig. 1C). Finally,
enzyme activities were stimulated: PRTase increased gradually from 5.8 to 11.5% h1, and LAP activity increased from
0.5 to 2.2 µM h1 (Fig. 1D).
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FIG. 1.
Microbial dynamics in control ( ) and casein-amended
( ) microcosms. Shown are chlorophyll (A), direct counts of bacteria
(B), CFU on ZoBell agar and the Pseudomonas-specific
Gould S1 agar (C), enzyme activity of PRTase and LAP (D). Values are
averages of duplicate microcosms, with standard deviations shown by
vertical lines (except day 1). Significant differences between
casein-amended and control microcosms are indicated by asterisks
(two-tailed t test, P < 0.05).
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Amendment with 2 mg of casein per liter increased PRTase activity from
6 to 12% h
1 by day 2, and a significantly
higher level than in the control
microcosms persisted until day 5. LAP
also increased significantly
but peaked 1 day later at 3.4 µM
h
1. Subsequently, LAP activity was similar to
the control (ca. 2
µM h
1) (Fig.
1D). By day
3, direct and culturable counts of bacteria
responded to the casein
enrichment with peaks at 7.6 × 10
6 cells
ml
1 and 5.3 × 10
4
CFU ml
1, respectively, but this significant
difference relative to the
control microcosms disappeared during the
subsequent days (Fig.
1B and C). In contrast, counts of
Pseudomonas remained fairly
constant from days 3 to 5 at 30 CFU ml
1, which is a fivefold higher abundance
than in the control microcosms
(Fig.
1C). Finally, chlorophyll peaked
at a significantly higher
level of 52 µg
liter
1 on day 4 (Fig.
1A). The casein effect
relative to control microcosms
was replicated in an independent
experiment (data not
shown).
PRTase and LAP activity was measured in unfiltered and
0.22-µm-pore-size-filtered samples to address the significance of
cell-associated
enzyme activity. Both at day 1 and at day 5, PRTase and
LAP in
the filtrates equaled 44% ± 17% and 3.8% ± 4.7% of the
total activities,
respectively. The different distributions of enzyme
activities
were confirmed on more sampling occasions (data not
shown).
Immunochemical detection of AprX-like Pseudomonas
proteinase.
PRTase activity in the microcosms was derived from a
diverse microbial community. To address whether pseudomonads express a
proteolytic potential in lake water, we set out to detect the AprX-like
Pseudomonas proteinase immunochemically. An antibody to the
AprX proteinase of P. fluorescens CY091 (33)
reacted with 50- to 60-kDa proteins in proteolytic culture supernatants from Pseudomonas isolates obtained from Lake Esrum (Fig.
2). These isolates represented the
proteolytic UP-PCR groups A+, D+, E+, F+, G+, H+, and I+ (see below). A
band of approximately 51 kDa was found for the proteolytic control
strain P. fluorescens ON2 (54), and a
relatively broad band of approximately 55 kDa was found for a mixed
culture supernatant derived from the 96 Pseudomonas isolates
obtained from Lake Esrum at day 1 (Fig. 2).
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FIG. 2.
Western blot to detect AprX-like
Pseudomonas proteinase in supernatants from
overnight-grown Pseudomonas cultures. Proteolytic
activity was detected in supernatants from all proteolytic strains.
Lanes 1 to 10, representative isolates of the 10 most abundant UP-PCR
groups, named A through J, with suffixes to indicate proteolytic
activity on skim milk agar (+ or ). Lane 11, mixture of supernatants
from 96 proteolytic and nonproteolytic strains isolated at day 1. Lane
12, proteolytic supernatant from P. fluorescens ON2. At
the left is shown the migration of protein size standards. Bitmap
pictures from separate digital gel scans were combined graphically.
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Nonproteolytic supernatants from
Pseudomonas isolates
belonging to UP-PCR groups B
, C
, J
, and W
did not contain
material
that reacted with the antibody. Furthermore, no antibody
reactivity
was observed for the nonproteolytic control strains
P. fluorescens ON2-pd5,
P. fluorescens DF57, and
E. coli DSM 498 or for the proteolytic
supernatants of
P. aeruginosa,
E. chrysanthemi DSM 4610,
S. marcescens ATCC 990, and
P. mirabilis, all of which
produce proteinases with
some homology to the
P. fluorescens
AprX proteinase (see Materials
and
Methods).
In the lake water sampled at day 1, the proteinase antibody reacted
with material migrating at 73 kDa in concentrated samples
of
0.22-µm-pore-size-filtered lake water. At day 5, this band
was less
intense, but a double band of approximately 50 to 55
kDa appeared in
water from the casein-amended microcosms (Fig.
3). This doublet did not appear in the
control microcosms (data
not shown). The 50- to 55-kDa doublet was
similar to that observed
by Western blot analysis of proteinases from
Pseudomonas isolates
from Lake Esrum (Fig.
2). Hence, these
data indicate that the
AprX-like
Pseudomonas proteinase was
expressed in the casein-amended
microcosms.
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FIG. 3.
Western blot to detect AprX-like
Pseudomonas proteinase in approximately
400-fold-concentrated 0.22-µm-pore-size-filtered water from the
microcosms. Lane 1, Lake Esrum at day 1. Lanes 2 and 3, duplicate
microcosms amended with casein at day 5.
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Pseudomonas population structure.
To examine
the population structure of Pseudomonas in the microcosm
experiment, 469 isolates from Gould S1 agar were screened for
proteinase (skim milk assay) and LAP activity and grouped by UP-PCR
genomic fingerprints. The population structure for
Pseudomonas was highly diverse, as 138 distinct UP-PCR
groups were defined for these isolates. The 10 most abundant UP-PCR
groups consisted of between 80 and 7 isolates and were named A+, B,
and C through J (Fig. 4). The suffix
specifies the presence or absence of proteolytic activity on skim milk
agar (+ or ). UP-PCR groups were specifically proteolytic or
nonproteolytic, as 97% of the isolates matched the prevailing
phenotype of a given group, whereas LAP was expressed by all isolates
grown on liquid ZoBell medium and could not be used to differentiate
the isolates (Table 1). The remaining 212 isolates were distributed in 128 groups. For statistical analyses, these 128 groups were joined as proteolytic and nonproteolytic residual
groups, i.e., Res+ and Res, respectively. A robust identification by
the Biolog GN2 assay (similarity index > 0.5) was obtained for
isolates representing some of the major UP-PCR groups: P. fluorescens biotype F (A+), P. fluorescens biotype G
(G+), P. currogata (C), and P. synxantha (D+,
E+, and F+).
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FIG. 4.
UP-PCR genomic fingerprint of representative isolates of
the most abundant UP-PCR groups. The groups are named A through J with
a suffix for their ability to produce proteinase activity on skim milk
agar (+ or ). The migration of size standards is indicated by
horizontal dotted lines. Bitmap pictures from separate digital
gel scans were combined graphically.
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At day 1, 84% of the
Pseudomonas isolates were proteolytic.
The most abundant UP-PCR groups (D+, F+, and H+) comprised between
6 and 9% of the isolates. Groups A+, B
, C
, E+, G+, I+, and J
each
accounted for 0 to 3% of the isolates. The remaining isolates
were
assigned to the groups Res+ (56%) and Res
(11%) (Fig.
5).
At day 5, proportions of proteolytic
pseudomonads were reduced
to 57% ± 1% and 60% ± 2% of the
isolates in the casein-amended
and control microcosms, respectively.
The decrease from the initial
84% was significant (
P < 0.001) but independent of the casein
amendment (
P = 0.47). Diversity, evenness, and richness decreased
between days 1 and
5, almost consistently at
P < 0.05. The lowest
diversity indices were found in casein-amended microcosms, but
they
were not significantly different from values from control
microcosms
(Table
1).
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FIG. 5.
Relative distribution of Pseudomonas
isolates grouped by similar UP-PCR genomic fingerprints at days 1 and 5 (control and casein amendment). Group names are indicated with a suffix
for their ability to express proteinase activity on skim milk agar (+ or ). Groups represented by small numbers of isolates were joined to
clarify the significant changes in the population structure; see text
for explanation and statistics. Day 1 and 5 samples comprise 90 and 189 of 190 isolates, respectively.
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The distribution of UP-PCR groups changed between days 1 and 5 (Fig.
5). Groups A+, B
, and C
became more abundant, whereas
Res+ became
less dominant (
P < 0.001). Consistent shifts were
not
evident for Res
or groups D+ through J
. Casein amendment
only
affected the abundance of group B
, as the higher frequency
of B
following the casein amendment was significant (
P < 0.02)
relative to the control microcosms. A statistical analysis of
the
distribution of UP-PCR groups in casein-amended and control
microcosms
is weakened by the high incidence of groups containing
less than five
observations. Consequently, joint groups were defined
to test for
broader similarities between replicates and treatments.
Two relevant
contingency tables, [Res+; Res
; (ADEFGHI)+; (BCJ)
]
and
[Res±; (ABC); (DEFGHIJ)], both showed similarity between replicate
microcosms (
P > 0.76), but significant differences
between casein-amended
and control microcosms (
P < 0.03). Hence, the population structure
of
Pseudomonas
shifted during the microcosm experiment, but only
minor compositional
differences were related significantly to
the casein
amendment.
| |
DISCUSSION |
Dynamics in microcosms.
The addition of protein (2 mg of
casein per liter) appeared to induce a cascade of events in the
microbial community. PRTase activity was rapidly mobilized, as
increased turnover rates of [3H]casein were
recorded in spite of the amendment with unlabeled casein (Fig. 1D). The
action of PRTase provided protein hydrolysates for exopeptidases, e.g.,
LAP, and the subsequent metabolism of protein-bound amino acids was
indicated by a transient increase in LAP and bacterial abundance.
Finally, the biomass of phytoplankton increased, which indicated that
casein-bound nitrogen was made available for phytoplankton growth (Fig.
1).
The culturable
Pseudomonas population never exceeded
10
2 CFU ml
1 on Gould S1
agar. For comparison, direct probing techniques have
revealed cell
abundances between 10
1 and
10
4 ml
1 for
species/serotypes of
Pseudomonas (
10,
39,
51)
or the
RNA group I genus (
25). Gould S1 agar is specific
for
Pseudomonas,
but plate counts may be lower than on media
without selective
agents (
15,
26,
35). Therefore, a
culturing bias is likely
to have affected the
Pseudomonas
recovery (
1).
The stimulation of bacterial abundance and LAP activity following an
amendment with protein was also found by Pinhassi et
al. in coastal
mesocosms (
38). To our knowledge, however, no
studies have
previously demonstrated the sequential mobilization
of PRTase and LAP
activity. Enzymes with affinity to cleave either
interior (PRTase) or
terminal peptide bonds (LAP and other exopeptidases)
are clearly
distinguished in the nomenclature of enzymes (
52).
Hence,
the cleavage of [
3H]casein and the peptide-like
Leu-MCA model substrates monitor
enzymes responsible for distinct steps
in the mobilization of
protein for bacterial
growth.
Numerous studies have shown negligible levels of LAP activity in
0.2-µm-pore-size filtrates (
6). PRTase activity
associated
with cells (i.e., ectoenzymes) and larger particles was also
prevalent
in a study of oligotrophic seawater (
18). This
indicates a strategy
where bacteria tend to concentrate the formation
of hydrolysates
near the cell surface (
5,
18,
49). We also
found negligible
activity of LAP (4%) in 0.22-µm-pore-size
filtrates, but nearly
half of the total activity of PRTase (44%) was
detected in the
same size fraction. Hence, in our more eutrophic
system, PRTase
was not strongly cell associated, so that hydrolysates
of dissolved
proteins were available to virtually all bacteria
independent
of their expression of
proteinase.
Immunochemical detection of AprX-like Pseudomonas
proteinase.
Representative isolates of the proteolytic UP-PCR
groups A+ and D+ through I+ all produced a single extracellular protein in pure cultures with size and immunoreactivity (Fig. 2) comparable to
the AprX-like proteinase of P. fluorescens UP206
(33). The 50- to 60-kDa size range of these putative AprX
proteinases is consistent with those reported for purified proteinases
from P. fluorescens (14). Proteolytic
supernatants from nonpseudomonads with related proteinase systems
(31) did not react with the antibody, and lack of
reactivity was also observed for proteolytic supernatants from P. aeruginosa DSM 50071. Hence, the antibody was specific for a
subgroup of the alkaline proteinases in Pseudomonas, as
shown for comparable proteinase antibodies (2, 47).
Nevertheless, the grouping by UP-PCR profiles and subsequent Western
blot analysis of representative isolates strongly indicates that
AprX-like proteinases were widespread among proteolytic pseudomonads in
Lake Esrum, because the 10 most abundant UP-PCR groups accounted for
55% of the isolated strains.
Proteins with size and immunoreactivity comparable to the AprX-like
proteinase of
P. fluorescens UP206 (
33) were
found in
concentrated 0.22-µm-pore-size filtrates from the
casein-amended
(Fig.
3) but not from the control microcosms (data not
shown).
The antibody also reacted with 73-kDa proteins in the samples.
This larger size class of proteins was not recovered from pure
cultures, even in a mixture of supernatants from 96 strains isolated
at
day 1 (Fig.
2). Therefore, we consider the 50- to 55-kDa bands
in the
Western blot prime evidence that the AprX-like proteinase
of
Pseudomonas was present in the casein-amended microcosms.
Accordingly,
it appears that
Pseudomonas contributed to the
increase in bulk
proteinase activity following an input of protein
(Fig.
1). To
the best of our knowledge, no studies have previously
provided
comparable direct evidence for expression of proteinase by a
specific
group of bacteria in a complex
community.
The appearance of the AprX-like
Pseudomonas proteinase might
reflect that the expression of proteolytic activity from resident
pseudomonads was stimulated by the casein amendment (
5).
It
has also been suggested that changes in the expression of enzyme
activity might reflect changes in the composition of the population
(
34), i.e., that the AprX-like
Pseudomonas
proteinase was detected
because strains producing this class of enzymes
proliferated in
response to the casein amendment. These two fundamental
mechanisms
controlling the expression of enzyme activity were addressed
by
an analysis of the abundance and population structure of
Pseudomonas isolates.
Abundance and composition of Pseudomonas spp.
UP-PCR genomic fingerprints provided a specific and reproducible system
to discriminate isolates at the subspecies level without prior
knowledge of their identity (4). The grouping of isolates by UP-PCR fingerprints demonstrated a highly diverse population structure of Pseudomonas in Lake Esrum (Table 1; Fig. 5).
Complexity of pseudomonad populations has so far not been analyzed in
aquatic systems, whereas studies in soil, using fingerprints generated by repetitive sequence primed PCR, have revealed complex population structures (21, 30). A high diversity of
Pseudomonas has been explained by the ability of this group
of bacteria to differentiate in niches and by a flexibility regarding
exchange of mobile genetic elements and genomic recombination
(46).
Casein amendment led to a fivefold-higher abundance of
Pseudomonas than found in control microcosms (Fig.
1), but
the impact
on the
Pseudomonas population structure was
limited (Table
1 and Fig.
5). Between days 1 and 5, proportions of
proteolytic
phenotypes decreased together with the diversity indices,
which
was explained mainly by the proliferation of UP-PCR groups A+,
B
, and C
(Fig.
5). Hence, a pronounced shift in the population
structure of
Pseudomonas cannot explain the detection of the
AprX-like
proteinase in casein-amended microcosms. We suggest
that the casein
amendment stimulated the expression of the
AprX-like
Pseudomonas proteinase, eventually for strains
with high growth potential
in the microcosms, e.g., A+. A more detailed
evaluation of the
origin of the AprX-like proteinase is not feasible at
present
due to the broad specificity of the antibody, narrow size range
of AprX-like proteins, and high diversity of
Pseudomonas isolates.
The proliferation of nonproteolytic phenotypes in the casein-amended
microcosms is in accordance with the hypothesis that
casein
hydrolysates were available to bacteria independent of
their proteinase
expression. All
Pseudomonas isolates from our
study tested
positive for LAP activity (Table
1), and alanine-aminopeptidase
is also
a general function for gram-negative bacteria (
36).
Hence,
proteolytic as well as nonproteolytic pseudomonads were
likely members
of the bacterial consortium that metabolized protein
in the lake water.
Future studies will be important to address
long-term effects of
protein amendment. Also, it is important
to compare responses of
bacteria in different habitats, e.g.,
associated with organic particles
versus freely suspended in the
water.
| |
ACKNOWLEDGMENTS |
We thank Lene Nielsen for excellent technical assistance, A. C. Magee and D. A. McDowell for providing the proteinase antibody, Peter Stephensen Lübeck for recommendations on the UP-PCR
protocol, and Morten Søndergaard for useful comments on the manuscript.
The work was supported by the Danish National Research Council (grant
no. 9601319).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Genetics and Microbiology, Department of Ecology, The Royal Veterinary
and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Phone: 45 3538 2645. Fax: 45 3528 2606. E-mail:
jaw{at}kvl.dk.
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Applied and Environmental Microbiology, November 2001, p. 4955-4962, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.4955-4962.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
