Characterization of marine bacteria and the activity of their enzyme systems involved in degradation of the algal storage glucan laminarin

Article abstract of DOI:10.1111/j.1574-6941.2006.00219.x

The algal storage glucan laminarin is one of the most abundant carbon sources for marine prokaryotes. Its degradation was investigated in bacteria isolated during and after a spring phytoplankton bloom in the coastal North Sea. On average, 13% of prokaryotes detected by epifluorescence counts were able to grow in Most Probable Number dilution series on laminarin as sole carbon source. Several bacterial strains were isolated from different dilutions, and phylogenetic characterization revealed that they belonged to different phylogenetic groups. The activity of the laminarin-degrading enzyme systems was further characterized in three strains of Vibrio sp. that were able to grow on laminarin as sole carbon source. At least two types of activity were detected upon degradation of laminarin: release of glucose, and release of glucans larger than glucose. The expression of laminarinase activity was dependent on the presence of the substrate, and was repressed by the presence of glucose. In addition, low levels of activity were expressed under starvation conditions. Laminarinase enzymes showed minimal activity on substrates with similar glucosidic bonds to those of laminarin, but different sizes and secondary and/or tertiary structures. The characteristics found in these enzyme systems may help to elucidate factors hampering rapid carbohydrate degradation by prokaryotes.

Full text of DOI:10.1111/j.1574-6941.2006.00219.x

Characterization of marine bacteria and the activityoftheir enzyme
systems involved in degradation ofthe algal storage glucan
laminarin
Anne-Carlijn Alderkamp1, Marion van Rijssel1 & Henk Bolhuis2
1Departments of Marine Biology; and 2Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Haren, The
Netherlands
Correspondence: Henk Bolhuis, Microbial
Ecology, Centre for Ecology and Evolutionary
Studies, University of Groningen, P.O. Box 14,
9750 AA Haren, The Netherlands. Tel.: 131
50 3632191; Fax: 131 50 3632154;
e-mail: h.bolhuis@home.nl
Abstract
The algal storage glucan laminarin is one of the most abundant carbon sources for
marine prokaryotes. Its degradation was investigated in bacteria isolated during
and after a spring phytoplankton bloom in the coastal North Sea. On average, 13%
of prokaryotes detected by epifluorescence counts were able to grow in Most
Probable Number dilution series on laminarin as sole carbon source. Several
bacterial strains were isolated from different dilutions, and phylogenetic character-
ization revealed that they belonged to different phylogenetic groups. The activity of
the laminarin-degrading enzyme systems was further characterized in three strains
of Vibrio sp. that were able to grow on laminarin as sole carbon source. At least two
types of activity were detected upon degradation of laminarin: release of glucose,
and release of glucans larger than glucose. The expression of laminarinase activity
was dependent on the presence of the substrate, and was repressed by the presence
of glucose. In addition, low levels of activity were expressed under starvation
conditions. Laminarinase enzymes showed minimal activity on substrates with
similar glucosidic bonds to those of laminarin, but different sizes and secondary
and/or tertiary structures. The characteristics found in these enzyme systems may
help to elucidate factors hampering rapid carbohydrate degradation by prokar-
yotes.
Present address: Anne-Carlijn Alderkamp,
Dept of Geophysics, Stanford University,
Stanford, CA 94305-2215, USA
Received 21 March 2006; revised 2 July 2006;
accepted 1 August 2006.
First published online 24 October 2006.
DOI:10.1111/j.1574-6941.2006.00219.x
Editor: Riks Laanbroek
Keywords
carbohydrate; Vibrio sp.; marine; laminarin.
structural diversity as a consequence of the wide variety of
monosaccharides and the different glycosidic bonds between
Introduction
Microbial communities in marine ecosystems play a key role
in the cycling of organic carbon and nutrients. An estimated
50% of the primary production is cycled as dissolved organic
carbon (DOC) through the microbial loop to higher trophic
levels (Azam, 1998). Most of the bioavailable DOC is present
as high molecular weight (HMW) molecules (Amon &
Benner, 1994, 1996), and has to be cleaved by extracellular
enzymes prior to uptake, as bacteria can only transport
substrates with a maximum molecular mass of c. 600 Da
through their cytoplasmic membrane (Weiss et al., 1991). On
the basis of the location of the extracellular enzymes, two types
of extracellular enzymes can be distinguished: ‘free’ extracel-
lular enzymes, which occur dissolved in water or attached to
surfaces other than the cell that produced them; and ‘ecto-
enzymes’, which cross the cytoplasmic membrane and remain
them. The primary structure is determined by the types of
monosaccharide and their linkage, and leads to a secondary
structure, determining the shape of the polysaccharides (for
example, b-1,3-linked glucans form helices). Polysacchar-
ides may be linked to each other by hydrogen bridges,
determining a tertiary structure, e.g. a loose hydrogel, or a
tightly packed network structure similar to cellulose.
Polysaccharides are degraded by glycoside hydrolases (EC
3.2.1.-), enzymes hydrolyzing the glycosidic bond between two
or more carbohydrate moieties. On the basis of the site of
cleavage, enzymes can be classified as exo-acting enzymes,
which remove one or more sugar units from the end of a
polysaccharide chain, and endo-acting enzymes, which ran-
domly hydrolyze bonds within the chains, thereby producing
more ends for the exoenzymes to act upon. Often a synergistic
action of these different hydrolases is necessary for efficient
degradation of polysaccharides (Driskill et al., 1999). There-
fore, degradation of a single substrate requires carefully
coordinated expression of the different enzymes, referred to
´
associated with the producing cell (Chrost, 1991).
Polysaccharides are important constituents of HMW
organic matter produced by algae (Biddanda & Benner,
1997; Biersmith & Benner, 1998). They display remarkable
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Degradation of the algal glucan laminarin by marine bacteria
109
as a system (Warren, 1996). Although carbohydrates are
usually considered to be labile substrates for prokaryotes, the
high concentration of carbohydrates in DOC (Benner et al.,
1992), marine sediments and sedimentary pore water (Cowie
& Hedges, 1984; Arnosti & Holmer, 1999) demonstrates that
carbohydrates are not always rapidly metabolized.
the Marsdiep, The Netherlands (5310001800N, 0414704200E)
from April through July 2002, during the phytoplankton
spring bloom. Samples were collected with a bucket, at high
tide, twice a week. For chlorophyll a analysis, water samples
were filtered though Whatman GF/F filters, extracted in
90% acetone, and subjected to fluorometric analysis. Phyto-
plankton abundance and species composition were deter-
mined on Lugol (nonacid) preserved samples (Utermohl,
1958) under a Zeiss inverted microscope, using 3-mL or 5-
mL counting chambers, under 50 ꢁ , 400 ꢁ and
1000 ꢁ magnification. Total bacterial numbers were counted
under an epifluorescence microscope after staining with
Hoechst dye no. 33258 (Paul, 1982) and by the Most
Probable Number (MPN) technique in liquid marine med-
ium and in laminarin medium (Clarke & Owens, 1983).
Marine medium consisted of artificial seawater supplemen-
ted with ‘minor salts’, trace elements, vitamins, Tris buffer
(pH 7.5) (Boehringer Mannheim), Na2HPO4 and NH4Cl as
in Janse et al. (1999), containing 0.01% yeast extract (w/v,
Becton Dickinson) and 0.01% casamino acids (w/v, Difco)
as carbon source. Laminarin medium contained no yeast
extract or casamino acids, but 2 mM glucose equivalents of
laminarin from Laminaria digitalis (Sigma) as carbon
source. As laminarin is a natural substrate with variable
polymer size, the substrate concentrations are expressed as
glucose equivalents. All medium components were sterilized
by autoclaving, except for the vitamins and the laminarin,
which were filter-sterilized (0.2 mm). The MPN counts were
performed in 200 mL of medium in 250-mL, 96-well micro-
plates, with seven replicates, incubated at 12 1C for at least
3 weeks. Positive growth was determined by visual turbidity.
The polysaccharide laminarin, the storage glucan found in
most algae and phytoplankton (Meeuse, 1962; Painter, 1983),
is one of the most abundant carbohydrates in the marine
ecosystem (Painter, 1983). It is a soluble b-1,3-D-glucose
polymer with some branching at positions C-2 and C-6, and is
also known as laminaran or leucosin. The size typically ranges
from 20 to 30 glucose residues, and some chains are terminated
by mannitol end-groups (Meeuse, 1962; Painter, 1983; Read
et al., 1996). These mannitol groups are absent in chrysolami-
naran, the type of laminarin that is the principal storage glucan
in diatoms and in the cosmopolitan genus Phaeocystis, which
are both important phytoplankton groups driving global
geochemical cycles (Nelson et al., 1995; Schoemann et al.,
2005). Photosynthesis by diatoms alone generates as much as
40% of the 45–50 billion metric tons of organic carbon
produced each year in the sea (Nelson et al., 1995). Glucan can
account for up to 80% of the organic carbon of diatoms and
Phaeocystis (Meeuse, 1962; Myklestad, 1974; Janse et al., 1996;
Granum et al., 2002; Alderkamp et al., 2006). Therefore, an
estimated 5–15 billion metric tons of laminarin are produced
annually. Laminarin is located intracellularly, in vacuoles
(Chiovitti et al., 2004). It may be released as DOC into the
marine environment after algal cell lysis (Brussaard et al., 1995),
or ‘sloppy feeding’ by copepods (Mꢀller et al., 2003), where it is
one of the most abundant substrates for marine bacteria.
Laminarin seems to be rapidly degraded in the pelagic system
(Keith & Arnosti, 2001; Arnosti et al., 2005).
Isolation of bacterial strains
Very few studies have characterized the enzyme systems of
marine bacteria degrading substrates that are relevant in marine
systems. Hydrolyzing activity in the marine environment has
mainly been determined using small substrate proxies, consist-
ing of a monomer such as glucose linked to a fluorophore such
as methylumbelliferyl (MUF), the fluorescence of which in-
creases upon hydrolysis (Martinez et al., 1996; Arrieta &
Herndl, 2002). Because they lack the structural properties of
real substrates, these substrate proxies will probably detect
mainly exo-type activities. Therefore, in this study laminarin
was used as a relevant carbohydrate substrate to study the
enzyme systems of marine bacteria that are abundant during a
phytoplankton bloom in the coastal North Sea.
Bacterial strains were isolated from the lowest and the
highest positive MPN dilutions on the laminarin medium
of the 29 June sample and from the highest positive dilution
of the 15 July sample, by plating on the marine medium
described above solidified with 2% agar (w/v, granulated,
Becton Dickinson), and incubating at 12 1C. Bacterial
cultures were grown in cotton-plugged Erlenmeyer flasks
(culture volume o 20% of the maximum Erlenmeyer
volume), under continuous aeration (200 r.p.m.), in the
medium described above, at 25 1C.
Sequencing of 16S rRNA gene
Single colonies from plates were resuspended in sterile
MilliQ water and used as templates in a PCR reaction using
the universal 16S rRNA gene primers B8F (50-AGAGTTTG
ATCCTGGCTCAG-30) and U1406R (50-GACGGGCG
GTGTGTRCA-30) (Sambrook et al., 1989). The amplified
16S rRNA gene was sequenced on an ABI automated DNA
sequencer (PE Applied Biosystems) with primer U1406R.
Materials and methods
Sampling
Surface water samples from the coastal North Sea were
collected from the ‘Royal NIOZ jetty’ in the tidal inlet of
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110
A.-C. Alderkamp et al.
Sequence similarities for at least 500 bp of the 16S rRNA gene
sequence were determined by BLAST analysis (Altschul et al.,
1997) of the National Center for Biotechnology Information
database. Phylogenetic analysis of the obtained sequences
and their close relatives was performed using the neighbor-
joining method with 1000 bootstrap replicates using MEGA
version 3.0 software (Kumar et al., 2004).
method was originally developed to determine carbohydrate
concentrations in natural seawater, with detection levels as
low as 2.5 mM glucose equivalents. It involves hydrolysis of
polysaccharides prior to colorimetric detection of each
reducing sugar molecule by the alkaline ferricyanide reac-
tion. For the purpose of this study, hydrolysis of polymers
was omitted, and the enzymatic release of reducing sugars
was determined. The release of glucans larger than glucose
was determined by subtraction of the glucose concentration
from the total reducing ends. The protein concentration was
measured using the Bradford method (Bradford, 1976). To
test the effect of the buffer, incubations were also carried out
using GF/P-filtered and autoclaved natural seawater that was
buffered with Tris (50 mM final concentration; pH 7.5).
Preparation of extracellular and crude enzyme
extracts
Laminarin-degrading activity was examined in pure cultures
of bacteria grown on 2 mM laminarin as sole carbon source.
Two hundred milliliters of culture was harvested in mid-
exponential growth phase, by centrifugation at 3500 g for
30 min at 4 1C. To obtain extracellular enzymes, the super-
natant from the harvested cultures was transferred to clean
tubes and centrifuged again at 3500 g for 30 min at 4 1C. The
supernatant was stored on ice until the activity was assayed
on the same day. To obtain ‘free’ cellular enzymes, including
ectoenzymes, cell pellets were washed twice with ice-cold
artificial seawater buffered with Tris (pH 7.5), and resus-
pended in 50 mM sodium phosphate buffer (pH 7.5). Cells
were disrupted by French press (9000 bar) and debris was
removed by centrifugation at 20 000 g for 10 min at 4 1C. As
the cell debris interfered with the laminarinase assay, and the
supernatant contained more than 90% of the laminarin-
degrading activity, the supernatant was used as a crude
extract of cellular and ectoenzymes. Extracts were stored on
ice until the activity was assayed on the same day.
Kinetic analyses: apparent K m and Vmax
determinations
Apparent Km and Vmax were determined for crude extracts
from each strain. Total activity and glucose release were
determined at different substrate concentrations (0.1–20 mM
glucose equivalents). At least eight substrate–activity data
pairs were fitted according to Michaelis–Menten kinetics
using the nonlinear regression program TABLECURVE (Jandel
Scientific, AISN Software).
Substrate specificity
The substrate specificity of crude extracts was determined
using the enzyme activity assay described above using 0.5%
(w/v) of the following substrates: curdlan from Alcaligenes
faecalis (Sigma), b-glucan, dietary fiber control (Sigma),
b-D-glucan from barley (Sigma), lichenan from Cetraria
islandica (Sigma), b-1,3-glucan from Euglena gracilis
(Fluka), and 20 mM glucose equivalents of pullulan
from Aureobasidium pullulans (Sigma). To determine the
solubility of these substrates, solutions were incubated at
room temperature for 1 h and mixed several times, before
centrifugation (14 000 g, 10 min). Total carbohydrate con-
centration was determined in the supernatant by the phe-
nol–sulfuric acid method (Liu et al., 1973).
Laminarinase assays
Extracellular enzymes and crude cell extracts were tested for
their capacity to hydrolyze laminarin. To the supernatant
containing the extracellular enzymes, 10 mM glucose
equivalents of laminarin (final concentration) was added,
and triplicate samples were incubated at 25 1C. Crude cell
extracts containing cellular and ectoenzymes were diluted
1 : 10 in 50 mM sodium phosphate buffer (pH 7.5), and
20 mM glucose equivalents of laminarin (final concentra-
tion) was added. Two controls were incubated: 20 mM
glucose equivalents of laminarin in 50 mM sodium phos-
phate buffer, and crude cell extract in 50 mM sodium
phosphate buffer. Triplicate samples were incubated at
25 1C. After 3 h and after overnight incubation, a sample
was taken, heat inactivated (3 min at 80 1C) and stored at
ꢂ 20 1C. The release of glucose was measured using the
Boehringer D-glucose test combination (Boehringer, Man-
nheim). This method selectively measures the D-glucose
concentration, with a lower detection limit of 18 mM. The
release of reducing sugars was measured using the alkaline
ferricyanide reaction, using the reagent 2,4,6-tripyridyl-S-
triazine (Sigma) (Myklestad et al., 1997). This sensitive
Results and discussion
Isolation of bacterial strains from MPN dilution
series growing on laminarin as a sole carbon
source
The phytoplankton bloom of 2002 was dominated by the
colony-forming haptophyte Phaeocystis globosa from 6 June
until 27 June. Prokaryote numbers varied between 2.3 ꢁ 109
and 3.3 ꢁ 109 cells Lꢂ1 over the period April through July
(Fig. 1). On average, 13% of the prokaryotes detected by
epifluorescence microscopy were able to grow in the MPN
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Degradation of the algal glucan laminarin by marine bacteria
111
dilution series, both on marine medium and on laminarin
medium. The percentage of prokaryotes that were able to
grow on the marine medium increased from 5–6% during
the wax of the P. globosa bloom to 37% during the wane of
the bloom (29 June). The percentage of prokaryotes able to
grow on laminarin was highest (33%) on 5 June and varied
between 2% and 14% in the other samples.
ing laminarin as a sole carbon source. This suggests that a
high fraction of prokaryotes had laminarin-degrading en-
zymes. Laminarin degradation seems to be a common
feature in marine microbial communities, as it was degraded
in all of the various marine microbial communities tested
(Keith & Arnosti, 2001; Arnosti et al., 2005). As laminarin is
the principal storage glucan of the haptophyte Phaeocystis
globosa, which dominated the phytoplankton bloom, and
also of the diatoms that were abundant prior to the P.
globosa bloom, it is not surprising that a large fraction of the
prokaryotes could use laminarin as a carbon source during
the investigated period.
The fraction of culturable prokaryotes was high in
comparison to other studies (Ferguson et al., 1984; Button
et al., 1993; Eilers et al., 2000), especially following the
P. globosa bloom. A similar result was obtained by Noord-
kamp et al. (2000), following the same MPN procedure used
in this study. The liquid, marine medium with relatively low
carbon concentrations (Janse et al., 1999) seems suitable for
culturing a high fraction of the marine prokaryotes present
in the coastal North Sea. Over the whole study period, there
was no difference between the fraction of culturable prokar-
yotes on marine medium and that on the medium contain-
Nineteen different bacterial strains were isolated from
several dilutions of the MPN series on laminarin as a sole
carbon source and subjected to phylogenetic analysis (Table
1, Fig. 2). Strains isolated from the same dilution with
identical 16S rRNA gene sequences were considered to be
the same (numbers in parentheses in Table 1). These strains
were able to grow on either laminarin as a sole carbon
source, or on byproducts of laminarin hydrolysis by other
strains. The strains that were isolated from the highest
dilutions were the most abundant of the isolates. An
estimate of their abundance in the original sample is given
in Table 1.
40
30
20
10
(a)
The isolates belonged to different phylogenetic groups
that are known to be abundant in coastal waters, such as
Roseobacter, Bacteroidetes, Pseudoalteromonas, and Vibrio
(Eilers et al., 2000; Pinhassi et al., 2004). Members of
Roseobacter and Bacteroidetes have previously been isolated
from the coastal North Sea, and culture-independent analy-
sis showed their abundance in marine systems (Eilers et al.,
2000a, b). In addition, they were detected during and after a
Phaeocystis bloom in a mesocosm (Brussaard et al., 2005),
and in stable microbial enrichments degrading Phaeocystis
carbohydrates (Janse et al., 2000). Gammaproteobacteria
such as Pseudoalteromonas and Vibrio have also frequently
been isolated, but usually comprise o 1% of the total
prokaryote population (Eilers et al., 2000a, b). However,
bacteria from the genus Vibrio are ubiquitous and have long
served as models for heterotrophic processes. They play an
important role in coastal seas and estuaries, owing to their
widespread abundance and high metabolic activities. They
are present both as free-living bacteria and attached to
particles, algae, copepods and fish (Huq et al., 1990; Heidel-
berg et al., 2002). They are capable of growing rapidly under
nutrient-rich conditions, and surviving prolonged periods
0
1e+10
(b)
1e+9
1e+8
1e+7
Apr
May
Jun
Jul
Aug
Date (2002)
Fig. 1. (a) Temporal dynamics of chlorophyll a during spring and
summer 2002. (b) Total prokaryote numbers counted by epifluorescence
microscopy (black bars) and the Most Probable Number (MPN) technique
on marine medium (light gray bars) and medium containing laminarin as
sole carbon source (dark gray bars). Data for MPN counts on marine
medium on 15 July are missing due to an infection. Error bars indicate a
standard deviation of at least 10 counted fields (microscopy), or a 95%
confidence interval of seven replicates (MPN).
¨
of starvation (Oliver et al., 1991; Nystrom et al., 1992;
McDougald et al., 2002), and are known to grow on complex
substrates such as chitin (Li & Roseman, 2004; Meibom
et al., 2004). As members of the genus Vibrio were isolated
both from enrichments and from the highest dilution, we
chose three Vibrio strains for further characterization of
enzymes involved in the degradation of laminarin.
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112
A.-C. Alderkamp et al.
Table 1. Bacterial strains isolated from the MPN dilution series on laminarin as sole carbon source inoculated with surface samples from the Marsdiep,
The Netherlands
Presence in original
Ã
Strain
Sample date
Sample dilution
sample (cells Lꢂ1
)
Closest phylogenetic matchw
B1A
B1B (4)z
29 June
29 June
29 June
29 June
29 June
29 June
15 July
15 July
15 July
15 July
1 : 10
5 ꢁ 104
5 ꢁ 104
5 ꢁ 107
5 ꢁ 107
5 ꢁ 107
5 ꢁ 107
5 ꢁ 106
5 ꢁ 106
5 ꢁ 106
5 ꢁ 106
Vibrio splendidus
1 : 10
Cobetia marina
B4C
1 : 104
1 : 104
1 : 104
1 : 104
1 : 103
1 : 103
1 : 103
1 : 103
Vibrio splendidus
C4B
Vibrio sp. PMV19
C4C
Vibrio sp. PMV19
C4E
Vibrio splendidus
ABE3A (2)
ABC3C (2)
ABC3A (5)
AB F3A
Pseudoalteromonas tetradonis
Pseudoalteromonas tetradonis
Sulfitobacter pontiacus
Uncultured member of Bacteroidetes
Ã
These are conservative estimates, based on the presence of a single cell from the isolate in the dilution.
wAll isolates were more than 99% similar to their closest match.
zNumbers in parentheses are the number of strains isolated from the same dilution sample with identical 16S rRNA gene sequences.
Fig. 2. Neighbor-joining tree based on partial
16S rRNA gene sequences derived from bacterial
isolates and close relatives (identified via a BLAST
search). The scale bar indicates 2% of sequence
variation.
Laminarinase activity in three strains of
Vibrio sp.
plasmatic enzymes and/or ectoenzymes. The total laminar-
inase activity closely followed Michaelis–Menten kinetics in
each of the three strains (r2 = 0.978, 0.943 and 0.944, respec-
tively). At least two types of activity were detected: release of
glucose, and release of glucans larger than glucose (Table 2).
These activities are consistent with the presence of a gene
encoding an endo-b-glucanase of glycoside hydrolase family
16 (http://afmb.cnrs-mrs.fr/CAZY/fam/acc_GH.html) (Hen-
rissat, 1991) (EC 3.2.1.39) and genes encoding exo-b-1,3
glycosidases of family 17 (EC 3.2.1.58) in the genome
sequence of Vibrio vulnificus (Kim et al., 2003). As substrate
cleavage by an endo-b-1,3-glucanase yields a new free end
The laminarinase activity was not affected by the use of either
Tris-buffered, filtered seawater or sodium phosphate buffer;
therefore, the sodium phosphate buffer was used in the
assays. Laminarinase activity was detected both in the med-
ium and in crude cell extracts of the three Vibrio strains,
indicating the presence of both extracellular enzymes and
ectoenzymes. As more than 90% of the activity could be
detected in the crude cell extract, this was used for further
characterization of the enzyme systems degrading laminarin.
These extracts probably include intracellular enzymes, peri-
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Degradation of the algal glucan laminarin by marine bacteria
113
Table 2. Apparent kinetic parameters for laminarinase activity of crude cell extracts of Vibrio sp. strains B1A, B4C, and C4B. One unit of activity is
expressed as mmol reducing ends released per hour per gram protein at pH 7.5 and 25 1C. The standard deviation (SD) for at least eight independent
measurements is given in parentheses
Total activity,
max (U)
Total activity,
m (mM)
V
max/Km values
Glucose-releasing
Strain
V
K
for total activity
activity, Vmax (U)
B1A
B4C
C4B
34.17 (1.84)
10.26 (0.67)
8.53 (0.36)
4.50 (0.73)
0.78 (0.16)
0.57 (0.12)
7.6
13.1
15.0
0.83 (0.03)
0.90 (0.02)
0.81 (0.03)
that the exo-b-1,3 glycosidase can act upon, the synergistic
interaction of these enzymes is likely to be responsible for
efficient degradation of laminarin. Upon prolonged incuba-
tion, laminarin was degraded by more than 95% to glucose by
the crude extracts of each of the three strains.
(Table 2). The higher rate of glucan release than of glucose
release resulted in the accumulation of glucan intermediates
during degradation at high substrate concentrations
(Table 3). At lower substrate concentrations, however, the
proportion of reducing ends released as glucose increased,
suggesting a lower Km value for the glucose-releasing activity
than for the total activity. Release of glucans was also
reported during microbial degradation of high concentra-
tions (2% w/v) of complex carbohydrates in Laminaria
thallus (Uchida, 1995). In the sea, bacterial hydrolysis of
polymers of aggregates and uptake of low molecular weight
compounds are often uncoupled processes, resulting in
release of free polymers from particles into the surrounding
water mass (Cho & Azam, 1988; Smith et al., 1992, 1995;
Both the total laminarinase activity (Vmax) and the
affinity constant (Km) were highest in strain B1A and similar
in B4C and C4B (Table 2). The Vmax/Km ratio, which
represents the slope of the Michaelis–Menten equation at
low substrate concentrations, is an indicator of the ability of
the strain to achieve high hydrolysis rates at low substrate
concentrations (Healey, 1980). This ratio was higher for
strain B1A than for strains B4C and C4B. With similar rates
of uptake and metabolism of the reaction products, strains
B4C and C4B would be better competitors at low substrate
concentrations, whereas strain B1A would be a better
competitor at higher substrate concentrations.
´
Unanue et al., 1998; Azua et al., 2003). If the differences in
kinetic properties between the release of glucan and glucose
found in this study are general to other marine endohydro-
lase and exohydrolase activities, this may explain part of the
mechanism. In aggregates, the carbohydrate concentrations
The glucose-releasing activity was c. 10% of the total
laminarinase activity at saturating substrate concentrations
´
are high (Azua et al., 2003), leading to high substrate
Table 3. The ratio of glucose formation to glucan formation after
overnight incubation of crude cell extracts of Vibrio sp. strains B1A, B4C
and C4B with different concentrations of laminarin
concentrations for glycosidases. The higher Vmax of endohy-
drolases than of exohydrolases will thus lead to the accumu-
lation of polymer and/or oligomer intermediates. If these
poly/oligomers are too large to be taken up by the prokar-
yotes, they will be released into the surrounding water.
Accumulation of intermediates is unlikely to occur in the
environment outside aggregates, where substrate concentra-
tions are much lower, because the glucose/glucan release
ratio is higher at lower substrate concentrations (Table 3).
Laminarin concentration
Strain
1 mM
5 mM
10 mM
B1A
B4C
C4B
0.85
0.85
0.67
0.18
0.15
0.18
0.11
0.14
0.11
Table 4. Relative laminarinase activity normalized to Vmax rates of crude extracts of cells grown on laminarin and harvested at mid-exponential phase
Growth phase
Exponential
Carbon source
B1A (%)
B4C (%)
C4B (%)
Laminarin
100
ND
ND
22
ND
78
3
100
ND
ND
15
ND
132
3
100
ND
ND
11
ND
104
2
Pyruvate
Glucose
Pyruvate1laminarin
Glucose1laminarin
Laminarin
Stationary
Pyruvate
Glucose
9
3
2
Pyruvate1laminarin
Glucose1laminarin
21
12
29
32
44
34
ND, no activity could be detected, the detection limit being 0.5% of the activity on laminarin.
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FEMS Microbiol Ecol 59 (2007) 108–117
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

114
A.-C. Alderkamp et al.
To compare the kinetic parameters with those of different
1991; Li & Roseman, 2004). The explanation put forward by
Li & Roseman (2004) is that secreted chitinase from starving
cells comes into contact with the insoluble chitin in the
microenvironment of the organism and generates a disac-
charide and/or oligomer gradient. The organism senses the
soluble oligomer intermediates and swims up the gradient
towards the chitin. In addition, oligomers induce the
expression of the full chitin degradation system. Although
laminarin is a soluble substrate, and may therefore directly
be sensed by the organism, we speculate that expression of
the laminarin degradation system may be regulated in a
similar fashion. Thus, expression upon carbon starvation of
different extracellular hydrolase enzymes may be a mechan-
ism for the sensing of potential substrates in the Vibrio
microenvironment.
b-glucosidases present during and after a bloom of P. globosa
in the coastal North Sea, determined using the fluorogenic
substrate analogue MUF–b-D-glucoside (Arrieta & Herndl,
2002), we express the Km values per mol of substrate, using
an average size for the laminarin molecule of 25 glucose
units. This leads to Km values for the total activity of 180,
31.2 and 22.8 mmol Lꢂ1 laminarin for strains B1A, B4C, and
C4B, respectively. These values are in the range of 12.1 to
over 282.7 mmol Lꢂ1 MUF–glucose detected using MUF-b-
D-glucoside (Arrieta & Herndl, 2002).
Expression of laminarinase activity in three
strains of Vibrio sp.
When cultures were grown to exponential phase on glucose
or pyruvate as a sole carbon source, no laminarinase activity
was detected in crude extracts (Table 4). When cultures were
grown on a mixture of laminarin and pyruvate as carbon
sources, low levels of laminarinase activity were detected.
Therefore, we conclude that during the exponential growth
phase of the Vibrio strains, expression of laminarinase
activity was dependent on the availability of laminarin.
However, when cultures were grown on a mixture of
laminarin and glucose, no laminarinase activity was de-
tected. This suggests that synthesis of laminarinase is
repressed in the presence of glucose. In the stationary
growth phase, laminarinase activity was detected in all
cultures. Stationary cultures grown on either pyruvate or
glucose expressed low levels of laminarinase activity,
whereas cultures grown on a mixture of laminarin and
glucose, or laminarin and pyruvate, expressed intermediate
activity.
Substrate specificity of the laminarinase
enzymes
The activity of the laminarinase enzymes in the crude cell
extracts of the Vibrio sp. strains grown on laminarin until
mid-exponential phase was tested on several glucose poly-
mers that differ from laminarin in size, solubility and
structure (Table 5). Curdlan and glucan from E. gracilis are
both b-1,3-glucans and thus have a similar primary and
secondary structure to laminarin, but are much larger in size
and are insoluble polymers. There was low activity on
curdlan, but no activity was detected on the glucan from E.
gracilis (Table 6). Barley glucan and lichenan have b-1,3-
glucosidic bonds, connecting stretches of b-1,4-linked glu-
cose, and consequently differ in secondary and tertiary
structure from laminarin. Low activity was detected on both
substrates. Pullulan is a repeating structure of three a-1,4-
linked glucoses (maltotriose) connected by a-1,6-glucosidic
bonds; it is a soluble substrate differing from laminarin in its
primary, secondary and tertiary structure. No activity was
detected with it. Each of the Vibrio strains, however, was able
to grow on pullulan as sole carbon source (results not
shown). The absence of pullulanase activity in strains grown
on laminarin as sole carbon source shows that expression of
Enzyme synthesis triggered by the presence of a suitable
substrate and inhibition by monomeric compounds is a
common feature of b-glucosidases in marine bacteria
´
(Chrost, 1991; Middelboe et al., 1995; Chin et al., 1998).
The expression of low levels of activity upon carbon starva-
tion resembles the expression of extracellular chitinase
activity upon starvation in Vibrio furnisii (Bassler et al.,
Table 5. Relevant information on the substrates used to determine the substrate specificity of crude cell extracts of Vibrio sp. strains B1A, B4C and C4B
grown on laminarin to mid-exponential phase
Substrate
Backbone
Branches
Size
Solubility (%) Source
Laminarin
b-1,3-Glucose
b-1,3 Cellotriose and No
b-1,6
3.9 kDa
49 MDa
100
Food reserve in most algae
Barley glucan
21.6
Cell wall constituent in barley and other higher plants
cellotetrose
Lichenan
b-1,3–1,4 Glucose
No information
b-1,3-Glucose
No
No information 15.7
Cell wall constituent of Irish moss
Dietary glucan
Curdlan
No information No information
5.5
No
No
No
100 kDa
500 kDa
200 kDa
0.15
0.12
Extracellular bacterial glucan
Euglena glucan b-1,3-Glucose
Pullulan a-1,6-Maltotriose
Food reserve in yeast
100
Extracellular polysaccharide in yeast, containing
similar linkage types as amylopectin
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2006 Federation of European Microbiological Societies
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FEMS Microbiol Ecol 59 (2007) 108–117

Degradation of the algal glucan laminarin by marine bacteria
115
Table 6. Relative activity (%) of crude cell extracts of Vibrio sp. strains
B1A, B4C and C4B normalized to Vmax rates of crude extracts at mid-
exponential phase grown on laminarin
Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller
W & Lipman DJ (1997) Gapped BLASTand PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
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Barley
Dietary
Euglena
Strain glucan Lichenan glucan
Curdlan glucan
Pullulan
Amon RMW & Benner R (1994) Rapid cycling of high-
molecular-weight dissolved organic-matter in the ocean.
Nature 369: 549–552.
B1A
B4C
C4B
1.9
1.7
0.7
2.4
1.1
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ND
ND
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1.2
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ND
ND
ND
ND
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Amon RMW & Benner R (1996) Bacterial utilization of different
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ND, no activity could be detected, the detection limit being 0.5% of the
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pullulanase activity is likely to be dependent on the presence
of pullulan; in a similar way, expression of laminarinase
activity was dependent on the presence of laminarin.
The minimal activity of laminarinase enzymes on sub-
strates similar to laminarin with respect to their primary and
secondary structure may have important implications for
polymer degradation in the marine environment. Polymers
derived from algae are known to assemble spontaneously
into hydrogels (Chin et al., 1998), which may be the
precursors of larger particles, such as transparent exopoly-
meric particles or marine snow (Verdugo et al., 2004).
Although particles are regarded as ‘hot spots’ of microbial
abundance and activity (Azam, 1998), assemblage may
influence the secondary structure of the polymers, analo-
gous to the difference between laminarin and curdlan. If the
difference in degradation potential between laminarin and
curdlan is representative of the difference in degradation
potential between ‘free’ polymers and polymers embedded
in a gel structure, turnover times may be increased from
days to years. This may be an additional explanation of why
carbohydrates are usually regarded as labile substrates for
marine microorganisms, but nevertheless form an impor-
tant fraction of the DOC in the marine environment
(Benner et al., 1992), in marine sediments, and in sedimen-
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We thank J. M. Arrieta and G. J. Herndl for their stimulating
discussions and their hospitality at the Royal NIOZ during
the sampling in the spring of 2002. J. M. van Iperen is
acknowledged for the chlorophyll a analysis and Phaeocystis
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