persistence of bacterial proteolytic enzymes in lake ecosystems

R E S EA RCH AR T I C L E

Persistence of bacterial proteolytic enzymes in lake ecosystems

Bartosz Kiersztyn, Waldemar Siuda & Ryszard J. Chrost

Department of Microbial Ecology, Institute of Botany, University of Warsaw, Warsaw, Poland

Correspondence: Ryszard J. Chrost,

Microbial Ecology Department, Institute of

Botany, University of Warsaw, ul.

Miecznikowa 1, 02-096 Warsaw, Poland.

Tel./fax: +4822 554 1413; e-mail:

[email protected]

Received 26 May 2011; revised 19 November

2011; accepted 30 November 2011.

Final version published online 9 January

2012.

DOI: 10.1111/j.1574-6941.2011.01276.x

Editor: Riks Laanbroek

Keywords

extracellular enzymes; enzyme stability;

protein degradation; lakes.

Abstract

This study analyzes proteolytic enzyme persistence and the role of dead (or

metabolically inactive) aquatic bacteria in organic matter cycling. Samples from

four lakes of different trophic status were used. Irrespective of the trophic sta-

tus of the examined lakes, bacterial aminopeptidases remained active even 72 h

after the death of the bacteria that produced them. The total pool of proteo-

lytic enzymes in natural lake water samples was also stable. We found that the

rates of amino acid enzymatic release from proteinaceous matter added to pre-

served lake water sample were constant for at least 96 h (r2 = 0.99, n = 17,P 0.0001, Vmax = 84.6 nM h1). We also observed that proteases built intobacterial cell debris fragments remained active for a long time, even after the

total destruction of cells. Moreover, during 24 h of incubation time, about

20% of these enzymatically active fragments adsorbed onto natural seston par-

ticles, becoming a part of the attached enzymes system that is regarded as the

hot-spot of protein degradation in aquatic ecosystems.

Introduction

It is well documented that a majority of bacteria present

in natural aquatic ecosystems might be metabolically

inactive (dormant cells) or dead (Stevenson, 1978; Zweifel

& Hagstrom, 1995; Ouverney & Fuhrman, 1999; Luna

et al., 2002). Various methods for the separation of meta-

bolically active bacteria from metabolically inactive ones

have been developed (Table 1). Results obtained using

these methods showed that the share of dead (or inactive)

bacterial cells in both marine and freshwater environ-

ments ranged from a few to several dozen percent (Ber-

man et al., 2001; Adamczewski et al., 2010). In contrast

to the relatively high level of information concerning

many aspects of metabolic activity of living bacteria, and

their role in the microbial loop (Grossart et al., 2001;

Herndl et al., 2008), there is much less research focused

on the influence of dead bacteria debris on the active bac-

teria community. In particular, the stability of aquatic

bacterial enzymes and their possible role in the cycling of

organic matter is poorly elucidated. One of the few

exceptions is the early work of Vives-Rego et al. (1985),

who pointed to the possibility of the presence of catalyti-

cally active dead bacterial cell fragments in natural water.

Later studies focused on the measurement of enzyme

activity in preserved natural water samples suggested that

enzymes produced by bacteria remained active even after

their death (Haemejko & Chrost, 1986). However, up to

now, there have been no detailed analyses of enzymes

from dead bacteria treated as an important, active ele-

ment of the organic matter transformation system in

lakes.

In this study, we attempt to answer the following

questions: (1) how long do hydrolytic enzymes attached

to bacterial debris remain active after the death of the

bacteria, which have produced them? (2) Is the integ-

rity of the bacterial envelope necessary for the function-

ing of this group of enzymes? (3) Are active enzymes

built into fragments of bacterial walls and membranes

able to adsorb on natural seston surfaces? (4) What

kind of role may these types of enzymes play in aquatic

ecosystems? We chose extracellular leucine-aminopepti-

dase (LAP) as a model bacterial proteolytic enzyme.

There were three main reasons for this choice. First,

LAP is one of the best known and most studied

aquatic bacterial extracellular enzymes (Somville & Bil-

len, 1983; Fontigny et al., 1987; Chrost, 1991; Hoppe

et al., 1993; Martinez et al., 1996); second, it is pro-

2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 80 (2012) 124134Published by Blackwell Publishing Ltd. All rights reserved

MIC

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duced by almost all species of aquatic bacteria (Chrost,

1991); and third, in aquatic ecosystems, extracellular

aminopeptidase is produced mainly by heterotrophic

bacteria (Martinez et al., 1996).

Materials and methods

Study area and sampling

Samples were collected from lakes of different trophic sta-

tus presented in Table 2. Subsamples (3 9 2 L) of pelagic

water were collected from the surface (0.5 m) and from

different depths (4, 8, 10 and 16 m) of the studied lakes.

The three subsamples, taken from each depth of the stud-

ied lakes, were mixed together (v/v) and transported in

the polyethylene containers to the laboratory in < 3 h.

Leucine-aminopeptidase activity

Maximal potential leucine-aminopeptidase activity (VmaxLAP) was measured fluorometrically (Chrost, 1990). To

3.9 mL of water samples, 0.1 mL of appropriate substrate

L-leucine-4-methyl-cumarinylamide hydrochloride (Leu-

MCA) solutions in ethanol was added, yielding final Leu-

MCA concentrations of 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0,

12.5, and 15.0 lM. Separate stock solutions for each Leu-MCA concentrations were prepared. The same final con-

centrations of ethanol were used for the preparation of

calibration standard solutions for fluorescence product

measurement (MCA). Fluorescence of the product

7-amino-4-methylcoumarin (AMC) was determined spec-

trofluorometrically (380 nm Ex. and 460 nm Em.) in a

Shimadzu RF 1501 spectrofluorometer at zero time and

after 0.51.0 h of sample incubation (temp. 20 C). Thetested enzyme-substrate system followed first-order

MichaelisMenten kinetics. The plot of the reactionvelocity (v) against substrate concentration [S] displayed

a rectangular hyperbola relationship, described by the

equation v = Vmax 9 [S]/(Km + [S]). Nonlinear regres-sion analysis was applied to calculate the kinetic parame-

ters of enzymatic reactions by means of PC software

Origin 6.1 (OriginLab Corporation, Northampton). Total

Table 1. The most commonly used methods to distinguish metabolically active bacteria from metabolically inactive (or dead) bacteria

Target Basis of measurement Reference

Electron transfer system Intracellular reduction of CTC

(5-cyano-2,3-ditolyl-tetrazolium chloride)

Rodriguez et al. (1992)

Membrane potential Anionic or cationic membrane-specific

dye staining (Sytox GreenR)

Suller & Lloyd (1999)

Membrane integrity LIVE&DEAD kit (Invitrogen, Molecular Probes) Luna et al. (2002)

Translation, transcription mRNA detection, rRNA detection Karner & Fuhrman (1997)

Active membrane transport

(substrate uptake)

Microautoradiography Ouverney & Fuhrman (1999)

Bacterial DNA synthesis [3H]methyl-thymidine incorporation rates Fuhrman & Azam (1982)

Presence of nucleoid DNA-DAPI staining followed by propanol washing Zweifel & Hagstrom (1995)

Table 2. Basic characteristics of the studied lakes

Lake GPS*

position Area (km2)

Depth (m)

Trophic Status TSI Secchi disc (m) Chlorophyll a (lg L1) Ptot(lmol P-PO34 L

1)Average Maximum

Kuc

5349N

2124E

0.99 8.0 28.8 Mesotrophic 38 2 4.5 0.2 3.3 1.4 0.3 0.1

Tatowisko

5352N

2134E

3.27 14.0 39.5 Hypereutrophic 70 4 0.5 0.1 87.3 7.2 2.8 0.4

Mikoajskie

5347N

2135E

4.98 11.2 25.9 Eutrophic 55 5 1.4 0.6 33.1 3.6 1.9 0.4

Zegrzynskie

5247N

2106E

33.00 3.0 15.0 Hypereutrophic 72 2 0.3 0.1 59.7 1.6 3.12 0.6

*Global Positioning System.Trophic State Index according to Carlson (1977).Total phosphorus.

FEMS Microbiol Ecol 80 (2012) 124134 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Persistence of extracellular enzymes in lakes 125

Vmax LAP was measured in nonfiltered samples (total

fraction). Free dissolved Vmax LAP activity (fraction

< 0.2 lm) was measured after filtration through 0.2-lmpolycarbonate (Nuclepore) filters. Vmax LAP of the free-

living bacteria fraction (0.21 lm) was calculated as thedifference between LAP activity measured after filtration

through 1.0-lm filters (polycarbonate, Nuclepore) andLAP activity in the 0.2-lm filtrate. Vmax LAP of attachedbacteria and enzymes trapped in biofilms surrounding

detritus (fraction > 1 lm) was calculated as the differencebetween total Vmax LAP activity and Vmax LAP activity in

the fraction < 1 lm.

Number of bacteria and bacterial production

The number of bacteria (BN) was determined by direct

counting of cells on 0.2-lm, black polycarbonate mem-brane filters (Millipore) under epifluorescence microscope

according to Porter & Feig (1980). DAPI (4,6-diamidino-

2-phenylindole) in final concentration 1 lg L1 was usedfor bacteria staining (10 min, temp. 24 C). For bacterialcounting, a computer image analyzing system composed

of a Nikon epifluorescence E450 microscope, Nikon Digi-

tal Camera DXM 1200F and LUCIA 4.8 software (Labora-

tory Imaging Ltd, Czech Republic) was used. The bacteria

were counted from digital images of 1030 random fieldsof each membrane filters (from 50 to 100 bacteria per

field, 10003000 bacteria cells per each membrane filter,picture area: 5510 lm2, UV2A Nikon fluorescence filter Ex. 380420 nm, DM. 430 nm, Em. 435485 nm). Thetotal number of bacteria was determined after mild soni-

cation of the sample with addition of sodium pyrophos-

phate (56 s impulses, 300 W; sonication wavelength

amplitude: 22 lm; sodium pyrophosphate concentration,50 mM) according to Griebler et al. (2001). The number

of free-living bacteria was determined in the sample frac-

tion < 1 lm (filtered through a 1-lm polycarbonate fil-ter). The number of attached bacteria was calculated as

the difference between total number of bacteria and num-

ber of free-living bacteria.

Bacterial production was measured by means of tri-

tiated thymidine (TdR, 9097.5 Ci nmol1; NEN DuPont) incorporation according to Chrost & Rai (1993).

Metabolically active bacteria

To determine the share of bacteria with intact cell mem-

branes in total number of bacteria, LIVE/DEAD Bac-

LightTM Bacterial Viability Kit, Invitrogen Molecular

Probes, was used according to Invitrogen, Molecular

Probes standard procedure (Luna et al., 2002). The share

of bacteria with active oxidation chains was estimated by

CTC (5-cyano-2,3-ditolyl-tetrazolium chloride) reduction

test (Creach et al., 2003). The bacteria were stained in

unpreserved samples, two hours after the samples were

taken. After staining (both CTC test and LIVE/DEAD

test), the bacteria were collected on 0.2-lm black polycar-bonate membrane filters (Millipore), dried, and the filters

were mounted in BacLight mounting oil on microscopic

slides. CTC+ bacteria and LIVE/DEAD bacteria werestained on separate membrane filters. The bacteria were

counted immediately (CTC test) or after a 1-week storage

in 25 C (LIVE/DEAD test). We used the same count-ing methodology as for the DAPI-stained samples

described above. For counting CTC+ bacteria, we usedB3A Nikon filter (Ex. 430490 nm, DM. 505 nm, Em.> 520 nm) and for LIVE/DEAD bacteria, B2A Nikon fil-ter (Ex. 450490 nm, DM. 500 nm, Em. > 515 nm). Wecalculated the participation of CTC+ and MEM+ bacteriain the total DAPI visible bacteria number in the vertical

profile of the eutrophic Lake Mikoajskie, which we treat

as a model system of changes in the proportion of active

bacteria.

Concentration of amino acids and

oligopeptides

The summarized concentrations of free amino acids

(FAA) and short oligopeptides (SOP) were measured by

the o-phthaldialdehyde method (Roth, 1971), modified

by Siuda et al. (2007) as follows. Triplicate water samples

(3.9 mL) were supplemented with 0.05 mL of borate buf-

fer (0.4 M, pH = 9.0) and 32 lL of 2-mercaptoethanol,mixed and, after the addition of 0.05 mL of o-phthaldial-

dehyde (OPA) solution (1.25 mg mL1 in ethanol),immediately mixed again. The final concentrations of the

reagent in each replicate were 1.0 lM, 114.4 mM, and116.5 lM. The fluorescence of the samples was measuredin a Shimadzu RF 1501 spectrofluorometer (Ex. 330 nm,

Em. 455 nm) within 15 min. A calibration curve was

obtained by measurement of L-leucine standard concen-

trations (0.1, 0.5, 1.0, 1.5, 2.0, 2.5 lM). We did notobserve any significant influence of NH4 ions on themethod used.

Analysis of enzyme stability

For the analysis of enzyme stability in preserved samples,

sodium azide (Formally, sodium azide is rather an inhibi-

tor that a total preservative. Nevertheless, sodium azide is

frequently used as a preservative in many reagents and

stock solutions that are utilized in science laboratories

and healthcare facilities. Although we are aware that the

term preservative is not fully precise, we decided to use

it for simplicity.) in final concentration 0.3% was used.

The following criteria motivated us to choose sodium


126 B. Kiersztyn et al.

azide as a fixing agent: (1) even in low (0.3%) concentra-

tions, it completely inhibits the metabolic activity of

aerobic microorganisms (Touminen et al., 1994; Arun-

mozi et al., 1997); (2) sodium azide does not damage cell

walls or cell membranes and therefore prevents the rapid

leakage of cellular content into the external environment

(Maselli et al., 2002); (3) sodium azide does not directly

affect the activity of proteolytic enzymes (Guellil et al.,

2001).

Analysis of LAP sorption and stability

Sorption of proteolytic enzymes bound to the fragments of

bacterial envelopes was conducted using a natural water

sample (2 L) taken, under nonsterile conditions, from the

surface layer (1 m depth) of highly eutrophied Lake

Zegrzynskie (Table 2). The sample was divided into two

subsamples (for the scheme of sample preparation see

Fig. 1). One of them was autoclaved twice (134 C,20 min) to completely inactivate bacterial enzymes and

then filtered through a 1-lm polycarbonate filter (Nucle-pore). Subsequently, thermally inactivated seston collected

on the 1-lm filter was resuspended in the autoclaved(enzymatically inactive), seston-free (filtered through

0.2 lm) lake water (AW lake water filtered through0.2 m membrane filter, autoclaved and filtered againthrough another 0.2 m membrane filter). By this proce-dure, a suspension of natural seston particles larger than

1 lm deprived of proteolytic activity was obtained (S enzymatically inactive seston particles larger than 1 msuspended in AW water). The second subsample was soni-

cated in ice (10 impulses, 30 s each, 0.8 kw, 40 lm) tocompletely disintegrate all microbial cells. After sonication,

two portions of the subsample were filtered through

0.2-lm and 1-lm polycarbonate filters to obtain filtratescontaining enzymatically active fragments of cells smaller

than 0.2 lm (E0.2 free proteolytic enzymes and enzymesin cell debris smaller than 0.2 m) and smaller than 1 lm(E1.0 free proteolytic enzymes and enzymes in cell debrissmaller than 1 m), respectively. Finally, six samples (vari-ants of the experiment) in three repetitions were prepared:

(I) lake water + AW (mixed v/v): control of enzymes activ-ity in natural lake water; (II) E0.2 + AW (mixed v/v): test-ing the activity of < 0.2 lm in size fraction of cellfragments containing active proteolytic enzymes; (III)

E1.0 + AW (mixed v/v): testing the activity of smaller than1.0 lm size fraction of cell fragments containing activeproteolytic enzymes; (IV) E0.2 + S (mixed v/v): testing thesorption efficiency of < 0.2 lm in size enzymatically activefragments of bacteria cells on larger inactive seston parti-

cles; (V) E1.0 + S (mixed v/v): testing the sorption effi-ciency of smaller than 1.0 lm enzymatically activefragments of bacteria cells on larger inactive seston parti-

cles; and (VI) S + AW (mixed v/v): control of activity ofenzymatically inactive seston particles. All samples were

preserved with sodium azide (final conc. 0.3%) and incu-

bated for 48 h at 20 C. After incubation, Vmax of LAP wasmeasured in samples I, II and III. Samples IV, V, VI were

filtered through 1.0-lm filters. The seston collected on thefilters was resuspended in AW. In the suspension, Vmax of

LAP bound to cell debris size fragments smaller than

0.2 lm (variant IV), and fragments < 1 lm in size (variantV), and adsorbed on the seston particles larger than 1 lm(initially lacking LAP activity) was measured.

Analyses of water samples in vertical profiles

The share of metabolically active bacteria in the whole

bacterial community was evaluated in samples taken from

the vertical profile (depth 0.5, 4, 8, 10, 16 m) of the

eutrophic Lake Mikoajskie during the summer stratifica-

tion period (July).

Analyses of LAP stability in lakes of different

trophic status

LAP stability was evaluated in the samples of surface

water taken in July from lakes of different trophic status

(Lake Kuc, Lake Mikoajskie, Lake Tatowisko; Table 2).

Vmax of LAP was measured repeatedly in the samples

fixed with 0.3% sodium azide over a period of 72 h.

Leucine-aminopeptidase persistence in

bacterial size fractions

To compare LAP persistence in the fraction of free-living

bacteria (smaller than 1 lm) and the fraction predomi-nated by attached bacteria colonizing seston particles lar-

ger than 1 lm, samples of surface water from highlyeutrophied Lake Zegrzynskie were collected. Samples were

fixed with 0.3% sodium azide and then incubated for

8 days at 20 C. After fixation, maximal potential LAPactivity in the fraction of free-living and attached bacteria

was measured several times during the period of incuba-

tion (8 days). As controls, we used unfixed samples trea-

ted in the same way.

Stability of proteases in lake water

To determine the stability of various (not only LAP) pro-

teases present in the lake water, a methodology developed

by the authors and described in detail by Siuda et al.

(2007) was used. Three 2-liter (in three repetitions) sam-

ples taken nonsterilely from the surface water (0.5 m

depth) of the eutrophic Lake Mikoajskie were preserved

with sodium azide (0.3%), enriched with High Powder



Azure (HPA, Sigma) to the final concentration of

0.23 mg HPA mL1 and incubated for 91 h at 20 C.HPA (Hide Powder Azure, also known as Remazol Bril-

liant Blue RHide) is a hide powder (mainly scleropro-teins) covalently linked to Remazol Brilliant Blue dye (for

spectrophotometry). It is a general substrate for proteases,

including trypsin, commonly used as a substrate for a

wide spectrum of bacterial extracellular exo- and endo-

proteases. We have used HPA from Sigma, catalog num-

ber 89435. The samples were supplemented with HPA to

avoid the reduction of enzymatic hydrolysis rate that

would have been caused by a decrease in natural protein

concentration during the incubation period. In subsam-

ples taken during incubation, the increase in the concen-

tration of amino acids and SOP enzymatically liberated

from HPA was measured by OPA method modified by

Siuda et al. (2007). Autoclaved samples supplemented

with HPA and treated the same way as the experimental

samples were used as a control for no enzymatic release

of amino acids.

All measurements and determinations were done with

triplicate subsamples.

Results

Metabolically active bacteria in the water

column

The analysis of the samples from the vertical profile of

the eutrophic Lake Mikoajskie showed that only about

Fig. 1. Scheme of the samples preparation for analysis of LAP sorption and stability.



12% (12.7% LIVE/DEAD test, 12.1% CTC test) ofthe bacteria present in the surface water and from 3% to

4% (4.01% LIVE/DEAD test, 3.05% CTC test) of the bac-

teria present in the hypolimnion were metabolically active

(Fig. 2). Vertical changes in the number of potentially

active bacteria obtained by the two independent methods

(CTC and LIVE/DEAD test) were strongly and positively

correlated (r2 = 0.97, P 0.005). The total number ofbacteria was relatively constant (depth: 0.5 m 6.6 0.3*106 mL1, 4 m 6.8 0.24*106 mL1, 6 m 7.0 0.2*106 mL1, 10 m 7.2 0.3*106 mL1, 16 m 7.2 0.3*106 mL1). The maximal potential aminopepti-dase activity was relatively constant throughout the whole

vertical profile of the lake (from 199 39.5 at the depthof 0.5 m to 158.53 29.8 nM h1 at the depth of 16 m)and did not follow the rapid decrease in the bacteria CTC

+ and MEM+ (intact cell membrane) abundance (Fig. 2).This suggested that aminopeptidase activity might not be

related only to living, metabolically active bacterial cells

but also to dead and/or inactive bacteria.

Persistence of aminopeptidase activity in lake

water

This interpretation was confirmed by the model experi-

ments. We found that, irrespective of the trophic status

of the examined lakes, aminopeptidase was active even

72 h after the death of the bacteria that produced it (It is

widely accepted that the major pool of extracellular pro-

teases able to act in outer cell aquatic conditions is pro-

duced by bacteria (Chrost, 1991; Martinez et al., 1996).

Microscopic observation of the samples (data not shown)

revealed that there were no other potential sources of

extracellular proteases, like fungi or numerous cyanobac-

teria cells, in the studied lakes.). The largest decrease of

LAP Vmax was observed in the samples from the eutro-

phied Lake Mikoajskie and the smallest (to 74.5% of ini-

tial activity) in the samples from the mesotrophic Lake

Kuc (Fig. 3). We assumed that the greater decrease in

potential proteolytic activity in the highly eutrophied

lakes in comparison with the mesotrophic lake might

have been a consequence of a decrease in the activity of

enzymes associated with seston particles and attached

bacteria. We tested the latter hypothesis by comparing

aminopeptidase persistence in a fraction of free-living

bacteria and in a fraction containing bacteria and

enzymes attached to seston particles larger than 1 lm.We found that LAP was more stable in the fraction

< 1 lm than in the fraction larger than 1 lm. After8 days of incubation of the samples preserved with

sodium azide (0.3% final conc.), the maximal potential

LAP activity decreased from 350 to about 50 nM h1 inthe fraction of attached bacteria. In the fraction of free-

living bacteria, Vmax of LAP remained almost unchanged

during the whole time of incubation after death of the

microorganisms (Fig. 4). We did not observe any activity

of free, dissolved LAP (in the fraction < 0.2 lm), inwater samples from the examined lakes. To ascertain that

persistence is not only a specific attribute of aminopepti-

dase, we tested the stability of the whole pool of proteo-

lytic enzymes in natural lake water. We found that the

rates of amino acid enzymatic release from proteinaceous

matter (HPA model substrate) added to the preserved

lake water sample was constant for at least 96 h

(r2 = 0.99, n = 17, P 0.0001, Vmax = 84.6 nM h1),suggesting the substantial stability of this kind of

enzymes.

To confirm that the integrity of bacterial envelopes is

not a prerequisite for the activity of the enzymes, we

applied sonication prior to measuring potential amino-

peptidase activity. The preparation of enzymes and sam-

ple is described in detail in the methodology chapter

(Analysis of LAP sorption and stability) and on Fig. 1.

We observed no changes in Vmax LAP in the fraction

smaller than 1 lm (E1.0, see Fig.1) and an about 75%decrease in LAP activity in the fraction smaller than

0.2 lm of sonicated samples (E0.2, see Fig. 1) comparedto the nonsonicated samples (Fig. 4a). After 24-h incuba-

tion of proteolytically active fragments of bacterial cells in

Fig. 2. Percentage of contribution of bacterial cells with active

oxidation chain (CTC+) and cells with intact cell membranes (MEM+)

in the total bacteria number (bars) and maximal potential

aminopeptidase activity Vmax LAP (curve with dots) in the vertical

profile of eutrophic Lake Mikoajskie.



the presence of natural seston particles larger than 1 lm(artificially deprived of proteolytic activity, see Fig. 1), we

found that 17% and 20% of the LAP activity in the soni-

cated fraction smaller than 0.2 (variant IV) and 1 lm(variant V), respectively, was transferred onto the fraction

of particles larger than 1 lm (Fig. 5b). This was becauseof the adsorption of active LAP bound to small bacterial

cell debris on the surface of natural seston particles.

In our analyses of water samples taken from the surface

layer of three Mazurian lakes (Lake Mikoajskie, Kuc, and

Tatowisko) during different vegetation periods (Table 2),

we did not find any correlation between the number of

bacteria and aminopeptidase activity (LAP Vmax), either

in the fraction of free-living (r2 = 0.19, n = 12, P = 0.14)or that of attached bacteria (r2 = 0.22, n = 12, P = 0.12).Neither did we observe any correlation between the total

number of bacteria and bacterial production (r2 = 0.23,n = 9, P = 0.19). However, we found a strong positivelinear relationship between FAA + SOP concentrationand LAP activity in the fraction of attached bacteria

(r2 = 0.72, n = 12, P 0.008).

Discussion

The results of the experiments described in this article

suggest that the extracellular proteolytic enzymes present

in natural lake water are relatively stable and a large pool

of them remain active for a long time. They performed

their function for several days after the death of the bac-

teria that produced them. This was not very surprising

because, as opposed to the intracellular enzymes which

do not act on substrate effectively in outer cell space

(Hoffman & Decho, 2000), extracellular enzymes are

Fig. 4. Time course changes of maximal potential aminopeptidase

activity (Vmax LAP) in the fraction of free-living bacteria (< 1 m) and

fraction of attached bacteria (> 1 m) in lake water samples fixed

with 0.3% sodium azide sample from highly eutrophic Lake

Zegrzynskie.

Fig. 5. Comparison of Vmax LAP in the natural, non-sonicated,

samples of surface lake water from Lake Zegrzynskie (I) and Vmax LAP

in size fragments of cell debris < 0.2 m (II) and smaller than 1.0 m

(III) after destruction of microorganisms by sonication (a), and (b)

comparison of Vmax LAP of size fragments of bacterial cell debris

< 0.2 m (variant IV) and smaller than 1.0 m (variant V) attached

and unattached to seston particles larger than 1.0 m in size after

24 h of incubation. The percentages refer to share of initial LAP

activity transferred to the surface of seston larger than 1.0 m after

24 h incubation time. Variant VI represents the control of seston

enzymatic activity without cell debris addition.

Fig. 3. Time course changes of maximal potential aminopeptidase

activity (Vmax LAP) in lake water samples fixed with 0.3% sodium

azide in the studied lakes: Kuc, Mikoajskie, and Tatowisko.



adapted to outer membrane conditions such as pH,

salinity, redox potential and the presence of various reac-

tion activators and inhibitors dissolved in water. From a

biochemical point of view, all proteolytic enzymes are

proteins. Therefore, the lifetime of each protease in aqua-

tic environments depends mainly on the rate of its enzy-

matic degradation by other proteolytic enzymes, which in

turn depends on the probability of contact between the

digesting and digested enzymes. It is well documented

that attached bacteria usually express very high potential

maximal aminopeptidase activity, much higher than free-

living bacteria (Becquevort et al., 1998; Unanue et al.,

1998). Because of the higher probability of enzyme-

enzyme contact, at least theoretically, seston-attached

enzymes should be degraded faster than the enzymes of

free-living bacteria. This assumption was confirmed by

the results of our experiments. We found that proteases

attached to the seston particles colonized by bacteria were

less stable than those produced by free-living microorgan-

isms (Fig. 4). Moreover, enzyme stability in the highly

eutrophic lake (where the number of attached bacteria is

high) was usually much lower than in less eutrophic lakes

where free-living bacteria predominated (Fig. 3).

The idea of cross-hydrolysis as the main cause of fall-

ing protease activity was also supported by the result of

the HPA degradation experiment. The addition of HPA

to the sample containing nonliving bacteria led to an

increase in the stability of proteolytic enzymes (the rate

of enzymatic release of the amino acids from HPA was

constant for at least 91 h). We concluded that the high

HPA concentration created conditions for proteolytic

enzyme saturation, reducing the probability of physical

contact between two protease molecules and in that way

prevented their mutual destruction. It is worth noting

that the particulate substrate (HPA) was hydrolyzed with-

out the active colonization of HPA by living bacteria.

This suggests that passive adsorption of dead bacteria

with still active enzymes is sufficient for the effective

degradation of HPA. The findings of Haemejko & Chrost

(1986), who demonstrated the enzymatic degradation of

particulate substrate in preserved water samples, indirectly

point to the same conclusions. We also found that the

integrity of bacterial envelopes was not a necessary condi-

tion for the functioning of proteases. Proteases bound to

small fragments of bacterial cell debris remained active.

These kinds of enzymes may play an important ecological

role during protein degradation after, for example, phage

lysis of bacteria. Lysis, leading to the fragmentation of

bacterial envelopes (Shibata et al., 1997), is assumed to

be one of the main reasons of bacterial mortality in aqua-

tic ecosystems (Wommack & Colwell, 2000).

The tendency of small particles to aggregate in larger

structures (McCave, 1984; Armstrong & Barlocher, 1989)

may have important consequences in the context of the

enzymatic activity of dead bacterial cells. Considering that

the average size of an aquatic bacterial cell is about

0.6 lm (length) 9 0.3 lm (width) and bacterial concen-tration is about 109 cells L1, their total surface areaexceeds 1000 mm2 L1. Assuming that (i) the percentageof dead bacteria in lake water is usually quite high and

(ii) proteolytic enzymes are relatively stable, dead and

metabolically inactive bacteria may constitute a huge

enzymatically active platform for protein degradation

processes in lake water.

Owing to the methodological difficulties associated

with distinguishing between enzymes attached to the sur-

face of alive bacteria and enzymes attached to dead bacte-

ria, it is hard to show directly which part of the total

proteolytic activity in natural lake water is associated with

dead bacterial cells or their fragments. Nevertheless, there

are reasons to assume the widespread presence of such an

activity in aquatic environments. These reasons are

related to the fact that enzymes attached to dead bacteria

and their fragments hydrolyze proteins and polypeptides

without the coupled assimilation of amino acids (end

products of catalysis).

The large share of such enzymes in natural lakes can

easily explain the following observations: (1) the lack of a

strong correlation between bacterial numbers and proteo-

lytic activity; (2) the presence of free dissolved amino

acids, even in bacteria-abundant environments, where at

least theoretically, potential bacterial demand for carbon

and nitrogen should be high (Kiersztyn, 2005); (3) the

lack of a strong correlation between bacterial production

and protease activity; and (4) the release of FAA from

organic particles colonized by bacteria observed by Hoppe

(1991) and Smith et al. (1992). It is the latter observa-

tion, confirmed by the results of our 2-year investigations,

which we consider especially interesting. We found a

strong positive correlation (r2 = 0.70, P 0.008)between amino acid concentration and aminopeptidase

activity in the seston particles fraction. This result sug-

gests that amino acids are liberated from particulate mat-

ter into the surrounding water, as was proposed by

Hoppe (1991). Such a loss of FAA is disadvantageous for

attached bacteria producing proteolytic enzymes, because

they spend energy on enzyme synthesis but do not gain

the full benefits of monomer possession, as the latter are

consumed only partially. This paradoxical loss of mono-

mers after hydrolysis is inconsistent with the idea of a

hydrolysis-uptake coupling system (Fuhrman, 1987),

which assumes that all monomers enzymatically liberated

by bacteria are immediately assimilated by them. If we

suppose that the biofilm surrounding the particles func-

tions as a trap for dead but still enzymatically active

bacteria and bacterial cell debris, the release of amino



acids from the seston particles into the surrounding envi-

ronment can be easily explained.

The results of our experiments lead us to propose a

division of the dead bacterial proteolytic enzyme pool

into two fractions. The first pool slow but stable prote-

ases includes the enzymes of free-living bacteria and

those connected with small (

We would like to stress that proteolytic enzymes

attached to dead bacteria and their fragments seem to be

an important element of the ecosystem effectively enrich-

ing aquatic environments with easily utilizable FAA by a

variety of aquatic microorganisms, not only heterotrophic

bacteria. Amino acids liberated by these enzymes may not

only stimulate bacterial growth but also, after their extra-

cellular deamination, serve as an ammonium nitrogen

source for autotrophic phytoplankton (Berman & Bronk,

2003). We propose the conceptual model of the hypothet-

ical role of dead bacteria in the hydrolysis of proteins in

lake ecosystems (Fig. 6). Processes such as autolysis and

viral lysis, sloppy feeding of zooplankton, lethal mutation,

solubilization of fecal material or active protein excretion

enrich the aquatic environment with labile particulate

combined amino acids (PCAA) and dissolved combined

amino acids (DCAA). PCAA and DCAA aggregate or

adsorb on the surface of large particles, becoming a com-

ponent of an effective particle-associated protein degrada-

tion system. Consistent with the coupling system idea,

live, active bacteria assimilate all the amino acids released

through DCAA or PCAA hydrolysis. However, according

to the results of our research, enzymes associated with

the surfaces of dead bacteria can also hydrolyze particu-

late organic matter (POM) or their fragments trapped in

the biofilm matrix surrounding organic matter particles.

Such enzymes (together with the trapped free extracellu-

lar enzymes) hydrolyze proteins without the parallel

assimilation of amino acids. These amino acids, after

their partial release from the particles, stimulate the

growth of free-living bacteria community. A similar

mechanism, although with less intensity, may apply to

free-living bacteria proteases, which were also found to be

relatively stable. As a conclusion, we propose to treat

dead bacteria not only as a source of nutrients but also as

an active part of the microbial food web, enriching the

aquatic environment in the monomeric, labile fraction of

dissolved organic matter. Thus, dead bacteria may be

treated, not only as a source of detritus but also as an

active catalytic component of the biogenic transformation

system, a component which can hydrolyze polymers with-

out parallel assimilation of monomeric products.

Conclusions

Proteolytic enzymes attached to seston (dead bacteria

and their fragments) seem to be an important element

of the ecosystem, which very effectively catalyze hydroly-

sis of proteinaceous matter in lake water. Persistent

enzyme activity associated with dead bacteria might be

also considered a form of seemingly altruistic behavior.

Dead, but still long-lasting enzymatically active bacteria

may provide an easily utilizable pool of monomers, pro-

viding an easy start for the next bacterial generations

(Fig. 6).

Acknowledgements

We would like to thank professor Aleksander Swiatecki

and dr. Dorota Gorniak from the University of Warmia

and Mazury for helping with the CTC experiments. The

critical comments of professor Zdzisaw Markiewicz that

helped to improve our manuscript are greatly appreci-

ated. The authors would also like to thank the anony-

mous reviewers for their valuable comments. This study

was supported by the Polish Ministry of Science and

Higher Education projects: N304 023237 awarded to B.K.

and Nr N304 017540 awarded to R.J.C.

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