potential significance of style enzymes from the mussel Choromytilus meridionalis in the
utilisation of living bacteria and the extent to which the activity of these enzymes could
supply the nitrogen requirements of the mussel.
Apart from the work of McHenery et al. (1979) and McHenery and Birkbeck (1979,
1982), there have been very few studies of the digestive enzymes which are required for the
utilisation of the bacterial component of such detrital diets.
The naturally occurring populations of marine bacteria are almost exclusively gram
negative. Although Repaske (1958) has shown that for lysis of Escherichia coli by
lysozyme, prior treatment with the chelating agent EDTA is required to destabilise the
cell wall, this does not appear to be the case in bacteriolysis within the stomach of marine
bivalves. McHenery et ai, (1979) described the occurrence of a lysozyme-like enzyme in
the bivalves Mytilus edulis, Modiolus modiolus, Chlamys opercularis and Mya arenaria
and suggested that its primary role is in the utilisation of bacteria. McHenery and
Birkbeck (1982) subsequently showed that the enzyme from M. edulis is a true N.
acetylmuramyl-hydrolase which is capable of degrading the cell walls of a variety of
bacteria including Micrococcus luteus, Escherichia coli and Bacillus suhtilis.
Clearly the occurrence and activity of such lysozyme-like enzymes is of importance in
estimating the significance of bacteria as a carbon, and above all, a nitrogen resource for
consumer organisms which are exploiting detrital diets with a high C:N ratio. This paper
examines the occurrence and role of lysozyme-like enzymes in the utilisation of bacteria
as a potential resource by the kelp bed mussel Choromytilus meridionalis whose carbon
balance on a detrital diet has been established (Seiderer et al., 1982).
MATERIALS AND METHODS
Sampling and Preparation of Style Extract
Specimens of the black mussel Choromytilus meridionalis (Krauss) were collected from a
rocky intertidal reef at Bloubergstrand, on the West Coast of South Africa, between
November 1982 and February 1983 and were transferred to the laboratory for immediate
extraction of the crystalline style. The styles of twenty specimens were removed, rinsed
and homogenised over ice with a glass tissue grinder in 12 ml 20 mM phosphate buffer,
pH 7,0, containing 150 mM NaCl (see also Langton, 1977 ; Seiderer and Newell, 1979 ;
Seiderer et ai, 1982, 1984). All glassware had been sterilised immediately prior to use. The
homogenate was centrifuged at 15,000 x g for 5 minutes and the supernatant diluted to 1.0
mg protein ml -1 (Groves et al., 1968) with phosphate buffer before lysozyme assays were
performed.
Isolation and Culture of Bacterial Strains
In order to isolate water column bacteria, samples of seawater were taken from Oudekraal, also on the west coast of South Africa. Subsamples were pipetted onto agar plates
made of 1.5% agar in seawater growth medium (Davis et al, 1983). Plates were then
incubated at 25 - 30°C for 24 - 48 hours. Twenty five strains were isolated and restreaked
at least five times before the clones were assigned to collections and stored on slants of
seawater agar at 4°C.
Bacterial isolates were also prepared from the gut contents of the mussel Choromytilus
meridionalis. The guts of four mussels were removed (excluding extraneous contamination and the contents cultured on seawater-agar plates at 20°C after treatment with 1 %
Trypsin for 30 minutes at 30°C. Cultures were also made from homogenised gut tissues
and twenty strains were isolated and stored on seawater agar at 4°C.
428
utilisation of living bacteria and the extent to which the activity of these enzymes could
supply the nitrogen requirements of the mussel.
Apart from the work of McHenery et al. (1979) and McHenery and Birkbeck (1979,
1982), there have been very few studies of the digestive enzymes which are required for the
utilisation of the bacterial component of such detrital diets.
The naturally occurring populations of marine bacteria are almost exclusively gram
negative. Although Repaske (1958) has shown that for lysis of Escherichia coli by
lysozyme, prior treatment with the chelating agent EDTA is required to destabilise the
cell wall, this does not appear to be the case in bacteriolysis within the stomach of marine
bivalves. McHenery et ai, (1979) described the occurrence of a lysozyme-like enzyme in
the bivalves Mytilus edulis, Modiolus modiolus, Chlamys opercularis and Mya arenaria
and suggested that its primary role is in the utilisation of bacteria. McHenery and
Birkbeck (1982) subsequently showed that the enzyme from M. edulis is a true N.
acetylmuramyl-hydrolase which is capable of degrading the cell walls of a variety of
bacteria including Micrococcus luteus, Escherichia coli and Bacillus suhtilis.
Clearly the occurrence and activity of such lysozyme-like enzymes is of importance in
estimating the significance of bacteria as a carbon, and above all, a nitrogen resource for
consumer organisms which are exploiting detrital diets with a high C:N ratio. This paper
examines the occurrence and role of lysozyme-like enzymes in the utilisation of bacteria
as a potential resource by the kelp bed mussel Choromytilus meridionalis whose carbon
balance on a detrital diet has been established (Seiderer et al., 1982).
MATERIALS AND METHODS
Sampling and Preparation of Style Extract
Specimens of the black mussel Choromytilus meridionalis (Krauss) were collected from a
rocky intertidal reef at Bloubergstrand, on the West Coast of South Africa, between
November 1982 and February 1983 and were transferred to the laboratory for immediate
extraction of the crystalline style. The styles of twenty specimens were removed, rinsed
and homogenised over ice with a glass tissue grinder in 12 ml 20 mM phosphate buffer,
pH 7,0, containing 150 mM NaCl (see also Langton, 1977 ; Seiderer and Newell, 1979 ;
Seiderer et ai, 1982, 1984). All glassware had been sterilised immediately prior to use. The
homogenate was centrifuged at 15,000 x g for 5 minutes and the supernatant diluted to 1.0
mg protein ml -1 (Groves et al., 1968) with phosphate buffer before lysozyme assays were
performed.
Isolation and Culture of Bacterial Strains
In order to isolate water column bacteria, samples of seawater were taken from Oudekraal, also on the west coast of South Africa. Subsamples were pipetted onto agar plates
made of 1.5% agar in seawater growth medium (Davis et al, 1983). Plates were then
incubated at 25 - 30°C for 24 - 48 hours. Twenty five strains were isolated and restreaked
at least five times before the clones were assigned to collections and stored on slants of
seawater agar at 4°C.
Bacterial isolates were also prepared from the gut contents of the mussel Choromytilus
meridionalis. The guts of four mussels were removed (excluding extraneous contamination and the contents cultured on seawater-agar plates at 20°C after treatment with 1 %
Trypsin for 30 minutes at 30°C. Cultures were also made from homogenised gut tissues
and twenty strains were isolated and stored on seawater agar at 4°C.
428