Lysis of Desulfovibrio vulgaris by

Vol. 96, No. 4
JOURNAL OF BACTERIOLCGY, Oct. 1968, p. 1427-1428
Copyright © 1968 American Society for Microbiology
P'inted in U.S.A.
Lysis of Desulfovibrio vulgaris by
Ethylenediaminetetraacetic
Acid and Lysozyme
JOHN E. FINDLEY AND J. M. AKAGI
Departmentt of Microbiology, The University of Kansas, Lawrence, Kanisas 66044
Received for publication 31 May 1968
The lysis of certain gram-negative bacteria by
ethylenediaminetetraacetic acid (EDTA) and
lysozyme was first reported by Repaske (7, 8).
Lysis by EDTA alone was reported for Pseudomonas aeruginosa (3, 8), Vibrio succinogenes (9),
and Alkaligenes faecalis (3). The mechanism for
lysis by EDTA (2) in conjunction with lysozyme
has been attributed to the interruption of the integrity of the cell surface resulting from interference with divalent cation bonding (4, 8). Hypotonicity of the surrounding medium and alkaline
conditions were shown to be requirements for the
lysis of V. succinogenes by EDTA or lysozyme (9).
Ochynski and Postgate (5) demonstrated that
Desulfovibrio vulgaris (6) cells were lysed by
EDTA and lysozyme. Under isotonic conditions,
these cells formed osmotically fragile vibrios
which rapidly lysed when transferred to an hypotonic environment.
As part of our program for determining the
various enzymatic activities associated with the
membrane fragments from D. vulgaris, the conditions for lysing these cells with EDTA and lysozyme were investigated.
D. vulgaris strain 8303
grown at 37 C for
24 hr, as previously described (1), in 300-ml
Florence flasks containing 200 ml of medium.
Cells were harvested by centrifugation at 7,000 X
g for 10 min. They were suspended in 5 ml of a
solution of 0.1 M EDTA in 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer, pH 9.0, and
allowed to remain at 0 C for 10 min. The suspension was centrifuged at 7,000 x g for 10 min and
the EDTA-treated cells were washed twice with
40 ml of 0.05 M Tris buffer, pH 7.0. The cells were
suspended in 2 ml of the same buffer and a sample
was removed such that, when it was added to 2.8
ml of 0.05 M Tris buffer, pH 9.0, the absorbancy
was approximately 0.8. After the initial absorbancy was recorded, 0.1 ml of a lysozyme solution
containing 150 ,ug was added to the cuvette, the
solution was mixed rapidly, and the change in
absorbancy was recorded. Absorbancy measurements were conducted at a wavelength of 620 nm
was
in 1-cm light path cuvettes with a Zeiss PMQ II
spectrophotometer connected to a Honeywell
Electronic 15 recorder.
Under the conditions of our assay, the maximal
rate of lysis occurred when lysozyme was added
to EDTA-pretreated cells. When EDTA-pretreated cells were exposed to lysozyme, a rapid
drop in absorbancy resulted, indicating lysis
(Fig. 1). Under identical conditions, untreated
cells plus lysozyme, EDTA-pretreated cells alone,
untreated cells plus EDTA, or untreated cells
incubated in the presence of both EDTA and
lysozyme did not show any significant drop in
absorbancy. In addition, when EDTA-pretreated
cells were incubated at pH 9.0 in the presence of
0.1 M EDTA, no lysis occurred. Phosphatc buffer
was capable of substituting for Tris buffer, thus
indicating that the latter buffer was not participating in the lysing phenomenon.
When the effect of EDTA concentration for
pretreating cells was investigated, the maximal
rate for lysis was observed with a concentration of
approximately 0.05 M. Pretreating cells with a
concentration of 0.01 M showed a decrease in
absorbancy corresponding to 60% of the 0.05 M
concentration within a 3-min interval. No lysis
was observed with 10-3 M EDTA during the same
time interval. Several divalent cations and organic
diamino and polyamino compounds were tested
for their ability to inhibit the lysis of D. vulgaris
by lysozyme. These compounds were added to
EDTA-pretreated cells just prior to the addition
of lysozyme (Table 1). Cadmium was the most
effective inhibitor and ferric or ferrous ions, in the
concentrations used, had no effect; higher concentrations of the iron compounds caused clumping of the cells and the degree of lysis could not be
measured. Sucrose in concentrations up to 0.04 M
did not stabilize the cells from lysing when tested
by the standard assay. The pH for obtaining rapid
lysis of these cells was found to be on the alkaline
side; lysis was considerably more rapid at pH 9.0
than under neutral conditions. Phase-contrast
microscopy of cells lysing under neutral condi-
1427
1 42'..8
NOTES
J. BACTERIOL.
rod-shaped forms which had lost their internal contents as indicated by their optically clear
appearance. Under both conditions, the medium
in which the cells were suspended became viscous,
and this viscosity was reduced by the addition of
small amounts of ribonuclease and deoxyribonuclease.
The data presented show that EDTA and lysozyme are required for lysing D. vulgaris cells. The
relatively high concentration of EDTA required
for this phenomenon may possibly be attributed
to the large amount of ferrous sulfide precipitate
surrounding their outer layers. Because EDTA
TIME, MINUTES
can partially inhibit lysozyme activity (9), it was
FIG. 1. Requirement for ED TA anid lysozyme. Cells necessary to remove excess EDTA before lysotreated with EDTA were washed (as described) and zyme treatment. The inhibition of lysis of D.
incubated at pH 9.0 with and without lysozyme. Cells vulgaris by cadmium was surprising since it does
not treated with EDTA, but subjected to the same wash- not seem reasonable to assume that this metal is
ing procedure, were incubated with EDTA alone (0.1 normally present in
the outer layers of this orM), lysozyme alone, or with EDTA (0.1 M) plus lysozyme. Curves A and B (identical plots) represent un- ganism. Because both Cd++ and Zn++ inhibited
treated cells, incubated in the presence of EDTA, and lysis by lysozyme, the possibility exists that some
EDTA-pretreated cells respectively; curve C, untreated thiol complex, present on the cell surface, may be
cells plus lysozyme; curve D, untreated cells pluis EDTA involved during lysis by lysozyme. When the
and lysozyme; curve E, EDTA-pretreated cells plus sulfhydryl groups are bound by Cd++ or Zn++,
lysozyme. Wavelength, 620 nm.
perhaps this complex renders the cell resistant to
lysozyme action.
TABLE 1. Effect of divalenzt cations and diamines
on lysis of ED TA-treated Desulfovibrio vulga-is
This study was supported by Public Health Service
by lysozymea
Additions
Concn (M)
None
CdCI2 ............
CaCl2 ............
MgCI ............
MnC12 ...........
CoCl2 ............
ZnCI 2 ............
Spermidine .
Spermine ........
Putrescine .......
Cadaverine . ....
FeCI3 ............
FeCl2 ............
3
3
3
3
3
3
3
3
3
3
3
3
3
3
X 10-4
X 10-5
X 10-3
X 10-4
X 10-3
X 10-4
X 10-4
X 10-3
X 10-4
X 10-3
X 10-4
X 10-3
X 10-3
X 10-3
3 X 10-3
3 X 10-5
3 X 10-5
(3Am2i
Relative
0.47
0.0
0.38
0.20
0.33
0.25
0.39
0.24
0.07
0.08
0.01
0.35
0.31
100
0.0
82
42
70
54
84
52
14
16
1.9
74
66
56
79
68
100
97
0.26
0.35
0.32
0.51
0.45
lys1s
a Cells were
pretreated with 0.1 M EDTA at
pH 9.0, and the respective compounds were added
just prior to the addition of 150 pg of lysozyme.
tions showed a predominance of spherical forms
which were optically dense. These cells slowly
disintegrated into cell fragments which, presumably, corresponded to a decrease in absorbancy.
Cells lysing at pH 9.0 appeared to be swollen,
research grant Al 04672 from the National Institute of
Allergy and Infectious Diseases and by Public Health
Service training grant GM-703. J. M. Akagi was the
recipient of Public Health Service Research Career
Development Award I-K3-GM30, 262.
LITERATURE CITED
1. Akagi, J. M., and L. L. Campbell. 1962. Studies on
thermophilic sulfate-reducing bacteria. III.
Adenosine triphosphate-sulfurylase of Clostridium migrificans and Desulfovibrio desulfuricans.
J. Bacteriol. 84:1194-1201.
2. Carson, K. J., and R. G. Eagon. 1966. Am. J.
Microbiol. 12:105-108.
3. Gray, G. W., and S. D. Wilkinson. 1965. J. Appl.
Bacteriol. 28:153-164.
4. Leive, L. 1965. Proc. Natl. Acad. Sci. U.S. 53:
745-750.
5. Ochynski, F. W., and J. R. Postgate. 1963. p. 426441. In C. H. Oppenheimer (ed.), Symposium on
marine microbiology. Charles C Thomas, Publisher, Springfield, Ill.
6. Postgate, J. R., and L. L. Campbell. 1966. Classification of Desulfovibrio species, the nonsporulating sulfate-reducing bacteria. Bacteriol. Rev.
30:732-738.
7. Repaske, R. 1956. Biochim. Biophys. Acta 22:189191.
8. Repaske, R. 1958. Biochim. Biophys. Acta 30:225232.
9. Wolin, M. J. 1966. Lysis of Vibrio suiccinogenies
by ethylenediaminetetraacetic acid or lysozyme.
J. Bacteriol. 91:1781-1786.

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