to compare the responses of the three main bacterial groups in our samples (Vibrio,
Pseudomonas and Flavobacterium) to starvation survival. Physiological adaptations of
mutated Vibrio strains are examined in relation to their mannitol utilisation phenotype,
in order to establish the mechanisms of selection which might be operating on these
populations.
MATERIALS AND METHODS
Isolation of bacterial strains
Water column bacteria were isolated as described in Seiderer et al., 1984. Kelp associated
bacteria were obtained by incubating 10 ml of sterile seawater (S W) with 2cm x 2cm pieces
of kelp (Ecklonia maxima) that had been handled aseptically. Bacteria were plated out
after 18hr incubation, and subsequently purified and maintained as described in Davis et
al., 1983.
Mannitol uptake and utilisation
Cells were grown overnight in one of 3 media made up with S W, a) 1 % casamino acid, b)
SW broth (0.5% peptone; 0.1% yeast extract) and c) 1/10 SW broth. Mannitol
( 1 % w/v) was added as an inducer where indicated. Bacteria were harvested, washed and
resuspended in SM salts + 0.01 M Tris, pH 7.8 (Novitsky and Morita, 1976). The OD
600
was adjusted to 0.2.
Mannitol uptake was measured after adding C 14 mannitol (59 mCi/ mmol, 12µM) to the
cells. Subsamples (50 /il) were taken at appropriate times, filtered onto 0.45 µm Gelman
filters, (0.22 pm Millipore filters for starved cells) and washed with 1 ml SW.
Scintillation fluid was added to the samples and they were counted in a Packard 460 liquid
scintillation counter.
Mutagenesis
A Vibrio strain was mutated using N-methyl—N’-nitro-N—nitrosoguanidine (NTG) and
then grown in SWB. Counter-selection against wild type cells was carried out using
growth in 1 % mannitol and 15 µg.ml -1 of ampicillin. Mutants were detected by plating
onto mannitol tetrazolium plates (Lee et ai, 1983).
Starvation studies
Bacteria were grown overnight in 1/10 SWB, washed and resuspended in SM salts
containing no nitrates or phosphates (Novitsky and Morita, 1976). The cells were kept in
a 10°C water bath.
Viable cell counts were determined by plating onto SW agar (SWB + 1.5 %agar). Uptake
and respiration of the following radiolabelled sugars and amino acids (Amersham) were
measured during the starvation period : C 14 mannitol (59 mCi/ mmol) ( 11.3 µM), C 14 glucose (28 mCi/mmol) (2.9 µM), C 14 glutamate (280 mCi/mmol) (1.2 µM), C 14 alanine
(165 mCi/mmol) (1.9 µM) and C 14 aspartic acid (224 mCi/mmol) (1.5 µM).
RESULTS AND DISCUSSION
Figure 1 shows the survival of Vibrio, Pseudomonas and Flavobacterium strains during a
starvation survival experiment under conditions similar to those employed by Novitsky
and Morita (1977). These strains represent the three major groups of kelp associated
186
Pseudomonas and Flavobacterium) to starvation survival. Physiological adaptations of
mutated Vibrio strains are examined in relation to their mannitol utilisation phenotype,
in order to establish the mechanisms of selection which might be operating on these
populations.
MATERIALS AND METHODS
Isolation of bacterial strains
Water column bacteria were isolated as described in Seiderer et al., 1984. Kelp associated
bacteria were obtained by incubating 10 ml of sterile seawater (S W) with 2cm x 2cm pieces
of kelp (Ecklonia maxima) that had been handled aseptically. Bacteria were plated out
after 18hr incubation, and subsequently purified and maintained as described in Davis et
al., 1983.
Mannitol uptake and utilisation
Cells were grown overnight in one of 3 media made up with S W, a) 1 % casamino acid, b)
SW broth (0.5% peptone; 0.1% yeast extract) and c) 1/10 SW broth. Mannitol
( 1 % w/v) was added as an inducer where indicated. Bacteria were harvested, washed and
resuspended in SM salts + 0.01 M Tris, pH 7.8 (Novitsky and Morita, 1976). The OD
600
was adjusted to 0.2.
Mannitol uptake was measured after adding C 14 mannitol (59 mCi/ mmol, 12µM) to the
cells. Subsamples (50 /il) were taken at appropriate times, filtered onto 0.45 µm Gelman
filters, (0.22 pm Millipore filters for starved cells) and washed with 1 ml SW.
Scintillation fluid was added to the samples and they were counted in a Packard 460 liquid
scintillation counter.
Mutagenesis
A Vibrio strain was mutated using N-methyl—N’-nitro-N—nitrosoguanidine (NTG) and
then grown in SWB. Counter-selection against wild type cells was carried out using
growth in 1 % mannitol and 15 µg.ml -1 of ampicillin. Mutants were detected by plating
onto mannitol tetrazolium plates (Lee et ai, 1983).
Starvation studies
Bacteria were grown overnight in 1/10 SWB, washed and resuspended in SM salts
containing no nitrates or phosphates (Novitsky and Morita, 1976). The cells were kept in
a 10°C water bath.
Viable cell counts were determined by plating onto SW agar (SWB + 1.5 %agar). Uptake
and respiration of the following radiolabelled sugars and amino acids (Amersham) were
measured during the starvation period : C 14 mannitol (59 mCi/ mmol) ( 11.3 µM), C 14 glucose (28 mCi/mmol) (2.9 µM), C 14 glutamate (280 mCi/mmol) (1.2 µM), C 14 alanine
(165 mCi/mmol) (1.9 µM) and C 14 aspartic acid (224 mCi/mmol) (1.5 µM).
RESULTS AND DISCUSSION
Figure 1 shows the survival of Vibrio, Pseudomonas and Flavobacterium strains during a
starvation survival experiment under conditions similar to those employed by Novitsky
and Morita (1977). These strains represent the three major groups of kelp associated
186