A buffered Mn (IV) stabilizing medium with artifical sea water (Modified K broth)
amended with Cu 2+ or Mn 2+ was developed to measure the effect of those metals on viable
counts and manganese oxidation rates. This medium was composed of (g/l) proteosepeptone (Difco) 2.0 ; yeast extract (Difco) 0.5 ; FeSO
4
. 7H2O (Fisher) 0.00001 ; Hepes
(Calbiochem) 11.9 (final concentration (50 mM) ; Na-pyrophosphate (Fisher) 4.4 (final
concentration 0.02 M) ; p H adjusted to 7.0. After sterilization, the medium was amended
with MnSO 4 . H 2 O (0.2 M) 10 mis or CuSO
4
.5H 2 O (16 mM) 7.0 mis using asceptic
technique.
Growth of Pseudomonas 57 (pZPl) and its cured derivative was measured according to
methods described by Koch (1981). Cultures were grown in 100 ml of the Modified K
broth and incubated during the duration of the experiment in 250 ml Erlenmeyer flasks at
25°C, in duplicate, on a rotory shaker at 80 rpm. At specified time intervals, aliquots were
serially diluted in ASW and plated on YP-agar. Plates were incubated at room temperature and counts made after incubation for 4 days and also after 8 days. Only those plates
containing between 30 and 300 colony-forming units (CFU) were counted. Rates of
manganese oxidation were determined by measuring residual Mn 2+ after filtration
(0.2/^m Millipore) and centrifugation (3 000 rpm for 10 minutes at 4°C) by atomic
absorption (Perkin-Elmer 5 000 ; air- acetylene flame, impact bead nebulizer).
The results indicate that, under the experimental conditions tested, no significant difference in growth rate exists between strains whether with or without heavy metal amendment. However, loss of pZP1 results in a slight lag in the medium prepared without heavy
metals (Fig. 1a). The viable cell counts in the medium amended with Cu 2+ were less for
both strains, relative to their respective controls (Fig. lb). The effect of Cu 2+ may be one
of nutrient limitation for these strains at concentrations below toxic levels (70 µg/ml).
The effect of Mn 2+ on growth differs from that of copper for both strans, as shown in Fig.
lc. Both strains exhibited an increased lag growth phase when exposed to 170 µg/ml
Mn 2 *. Loss of the plasmid, pZPl, resulted in a 10 hour increase in lag time, relative to the
parental strain.
The loss of Mn
2+ from the culture medium during growth of strain 57 and strain 57 (pZPl)
is shown in Figure 3. Both strains oxidized manganese at the same rate, suggesting the
oxidation mechanism is not at all plasmid encoded. However, manganese oxidation was
correlated with growth for each strain, suggesting that the manganese oxidation mechanism is coupled to cell respiration. Results of other experiments (Zelibor and Colwell,
unpublished data) indicate that manganese oxidation does not occur in both strains when
the total organic nutrient concentration is below 125 µg/ml.
In conclusion, these studies confirm the presence of plasmid DNA in manganeseoxidizing strains of deep sea bacteria, most of which had been stored for up to 8 years at
4°C prior to examination for plasmids. Spontaneous loss of the ability to produce
manganese oxide was not observed. Twenty-two of 47 strains of the genera Alcaligenes,
Pseudomonas and Vibrio, that had been isolated from samples of seawater collected at
the sediment-water interface and from manganese nodules of the pelagic Central Pacific
Ocean and from seawater samples collected in the Mediterranean Sea harbored plasmids.
Six manganese-oxidizing strains of Pseudomonas spp. isolated from samples collected in
the Central Pacific Ocean and one unidentified strain from the Mediterranean Sea
selected for curing experiments revealed only Pseudomonas strain 57, isolated from a
manganese nodule, to be cured successfully. The plasmid was 9 mDal and designated
pZPl.
In studies carried out using a marine Pseudomonas strain 57 (pZPl), manganese oxida339
amended with Cu 2+ or Mn 2+ was developed to measure the effect of those metals on viable
counts and manganese oxidation rates. This medium was composed of (g/l) proteosepeptone (Difco) 2.0 ; yeast extract (Difco) 0.5 ; FeSO
4
. 7H2O (Fisher) 0.00001 ; Hepes
(Calbiochem) 11.9 (final concentration (50 mM) ; Na-pyrophosphate (Fisher) 4.4 (final
concentration 0.02 M) ; p H adjusted to 7.0. After sterilization, the medium was amended
with MnSO 4 . H 2 O (0.2 M) 10 mis or CuSO
4
.5H 2 O (16 mM) 7.0 mis using asceptic
technique.
Growth of Pseudomonas 57 (pZPl) and its cured derivative was measured according to
methods described by Koch (1981). Cultures were grown in 100 ml of the Modified K
broth and incubated during the duration of the experiment in 250 ml Erlenmeyer flasks at
25°C, in duplicate, on a rotory shaker at 80 rpm. At specified time intervals, aliquots were
serially diluted in ASW and plated on YP-agar. Plates were incubated at room temperature and counts made after incubation for 4 days and also after 8 days. Only those plates
containing between 30 and 300 colony-forming units (CFU) were counted. Rates of
manganese oxidation were determined by measuring residual Mn 2+ after filtration
(0.2/^m Millipore) and centrifugation (3 000 rpm for 10 minutes at 4°C) by atomic
absorption (Perkin-Elmer 5 000 ; air- acetylene flame, impact bead nebulizer).
The results indicate that, under the experimental conditions tested, no significant difference in growth rate exists between strains whether with or without heavy metal amendment. However, loss of pZP1 results in a slight lag in the medium prepared without heavy
metals (Fig. 1a). The viable cell counts in the medium amended with Cu 2+ were less for
both strains, relative to their respective controls (Fig. lb). The effect of Cu 2+ may be one
of nutrient limitation for these strains at concentrations below toxic levels (70 µg/ml).
The effect of Mn 2+ on growth differs from that of copper for both strans, as shown in Fig.
lc. Both strains exhibited an increased lag growth phase when exposed to 170 µg/ml
Mn 2 *. Loss of the plasmid, pZPl, resulted in a 10 hour increase in lag time, relative to the
parental strain.
The loss of Mn
2+ from the culture medium during growth of strain 57 and strain 57 (pZPl)
is shown in Figure 3. Both strains oxidized manganese at the same rate, suggesting the
oxidation mechanism is not at all plasmid encoded. However, manganese oxidation was
correlated with growth for each strain, suggesting that the manganese oxidation mechanism is coupled to cell respiration. Results of other experiments (Zelibor and Colwell,
unpublished data) indicate that manganese oxidation does not occur in both strains when
the total organic nutrient concentration is below 125 µg/ml.
In conclusion, these studies confirm the presence of plasmid DNA in manganeseoxidizing strains of deep sea bacteria, most of which had been stored for up to 8 years at
4°C prior to examination for plasmids. Spontaneous loss of the ability to produce
manganese oxide was not observed. Twenty-two of 47 strains of the genera Alcaligenes,
Pseudomonas and Vibrio, that had been isolated from samples of seawater collected at
the sediment-water interface and from manganese nodules of the pelagic Central Pacific
Ocean and from seawater samples collected in the Mediterranean Sea harbored plasmids.
Six manganese-oxidizing strains of Pseudomonas spp. isolated from samples collected in
the Central Pacific Ocean and one unidentified strain from the Mediterranean Sea
selected for curing experiments revealed only Pseudomonas strain 57, isolated from a
manganese nodule, to be cured successfully. The plasmid was 9 mDal and designated
pZPl.
In studies carried out using a marine Pseudomonas strain 57 (pZPl), manganese oxida339