35.8
CHROM. DNA
4.8
3.7
3.4
2.6
Figure 2 : Agarose gel electrophoresis patterns for plasmids of manganese-oxidizing
parental Pseudomonas strain 57 (pZPl)
with its 9mDal plasmid band, the cured
derivative 57 without plasmid and E. coli
V517 as molecular weight standard. The
control E. coli V517 shows plasmid DNA
bands of 35.8, 4.8 (faint), 3.7, 3.4 and 2.6
(faint) MDal.
The reaction mixtures, tested in triplicate at 4°C, were of the composition described
above. The protein concentration of each of the cell-free extracts was diluted with sterile 3
% NaCl to 3.5 mg/ml (Lowry et ai, 1951), as modified by Henry (1965), and to this was
added 0.5 ml TRIS buffer, (THAM 0.5 M, pH 7.2), and 0.5 ml of a 10 mM MnSO
4
. H2O
solution. For inactivation, the cell- free extracts were boiled for 8 minutes or treated with
0.1 ml or a 1 mM HgCb solution. The amount of manganese oxide formed was estimated
at 24 h intervals over five days using o-tolidine reagent and the spot-test procedure.
Results of the measurement of manganese oxidation by cell-free extracts prepared from
Pseudomonas sp. 57 (pZPl) and its derivative are shown in Table 3. These experiments
demonstrated that oxidation of manganese as determined by the o-tolidine spot test by
parental strain 57 was greater than that of the cured variant. Manganese oxidation was
not observed to occur in reaction mixtures to which HgCL was added or in crude extracts
heated to boiling, thus, providing evidence for an enzymatic mechanism of manganese
oxidation. Results of additional experiments (Zelibor and Colwell, unpublished data)
indicated that cell membranes and periplasmic space components alone, or mixed together, did not catalyze measurable manganese oxidation.
Strain
Formation of Mn-oxide a
Incubation time (Days)
12
3
4
Pseudomonas sp. 57
Pseudomonas sp. 57
+
++
+++
+++
Table 3 : Oxidation of manganese by cell-free extracts prepared from
Pseudomonas strain 57 (pZPl) and cured derivative strain 57.
a oxidation of manganese was monitored using o-tolidine reagent in a
spot test. See Materials and Methods. Reaction intensities =+, weak, ++,
medium, +++, strong; controls without the cell-free extract gave no
reaction. Oxidation of manganese was inhibited completely by addition
of HgCh (ImM) or boiling the cell-free extract for 8 minutes.
337
CHROM. DNA
4.8
3.7
3.4
2.6
Figure 2 : Agarose gel electrophoresis patterns for plasmids of manganese-oxidizing
parental Pseudomonas strain 57 (pZPl)
with its 9mDal plasmid band, the cured
derivative 57 without plasmid and E. coli
V517 as molecular weight standard. The
control E. coli V517 shows plasmid DNA
bands of 35.8, 4.8 (faint), 3.7, 3.4 and 2.6
(faint) MDal.
The reaction mixtures, tested in triplicate at 4°C, were of the composition described
above. The protein concentration of each of the cell-free extracts was diluted with sterile 3
% NaCl to 3.5 mg/ml (Lowry et ai, 1951), as modified by Henry (1965), and to this was
added 0.5 ml TRIS buffer, (THAM 0.5 M, pH 7.2), and 0.5 ml of a 10 mM MnSO
4
. H2O
solution. For inactivation, the cell- free extracts were boiled for 8 minutes or treated with
0.1 ml or a 1 mM HgCb solution. The amount of manganese oxide formed was estimated
at 24 h intervals over five days using o-tolidine reagent and the spot-test procedure.
Results of the measurement of manganese oxidation by cell-free extracts prepared from
Pseudomonas sp. 57 (pZPl) and its derivative are shown in Table 3. These experiments
demonstrated that oxidation of manganese as determined by the o-tolidine spot test by
parental strain 57 was greater than that of the cured variant. Manganese oxidation was
not observed to occur in reaction mixtures to which HgCL was added or in crude extracts
heated to boiling, thus, providing evidence for an enzymatic mechanism of manganese
oxidation. Results of additional experiments (Zelibor and Colwell, unpublished data)
indicated that cell membranes and periplasmic space components alone, or mixed together, did not catalyze measurable manganese oxidation.
Strain
Formation of Mn-oxide a
Incubation time (Days)
12
3
4
Pseudomonas sp. 57
Pseudomonas sp. 57
+
++
+++
+++
Table 3 : Oxidation of manganese by cell-free extracts prepared from
Pseudomonas strain 57 (pZPl) and cured derivative strain 57.
a oxidation of manganese was monitored using o-tolidine reagent in a
spot test. See Materials and Methods. Reaction intensities =+, weak, ++,
medium, +++, strong; controls without the cell-free extract gave no
reaction. Oxidation of manganese was inhibited completely by addition
of HgCh (ImM) or boiling the cell-free extract for 8 minutes.
337