FEMS Microbiology Letters 7 (1980) 157-162
0 Copyright Federation of European Microbiological Societies
Published by ElsevierlNorth-Holland Biomedical Press
157
A METHOD FOR IMPROVED LYSIS OF SOME GRAM-NEGATIVE BACTERIA
E.A. SCHWINGHAMER
CSIRO Division of Plant Industry, Canberra A.C.T. 2601, Australia
Received 10 November 1979
Accepted 30 November 1979
1. Introduction
Lysis of bacteria for isolation of DNA or other
cell constituents commonly involves the use of lysozyme to produce spheroplasts for “gentle lysis” or to
weaken the cell wall for subsequent lysis by detergents or other agents. However, some bacteria, notably
among Gram-negative species, are resistant to the
enzyme and require additional non-enzymatic treatments which may result in damage t o the molecule
to be isolated (e.g., nicking and loss of plasmic DNA
molecules). Susceptibility to lysis may also vary considerably between strains of a species and between
the phases of growth of a culture, with log phase cells
commonly being more susceptible than late-log or
stationary phase cells.
Several procedures for enhancing the action of
lysozyme on refractory bacteria have been reported.
One involves the use of dodecylamine [ I ] to sensitize
the cell wall to attack by the enzyme. Another procedure [2] uses mild osmotic shock in the presence of
EDTA to force lysozyme molecules through the outer
membrane (Gram-negative bacteria) and expose the
murein layer to rapid enzymatic degradation. I have
applied both of these methods to late-log phase cells
of Rhizobium spp. for isolation of plasmid DNA,
with only limited or variable improvement over
standard lysozyme methods. One likely reason for
failure of the osmotic shock procedure to facilitate
lysis of the rhizobia as effectively as with Escherichia
coli [2] appeared to be simple blockage of enzyme
uptake by the polysaccharide slime layer which is
characteristic of many rhizobia, especially those of
the fast-growing species. In attempts to circumvent
this problem I have used a modified form of the mild
osmotic shock procedure, including an essential preliminary wash of the cells in dilute detergent solution,
for rapid and more complete lysis of rhizobia. This
paper describes the detergent-wash/osmotic-shock
procedure as used for strains of fast-growing Rhizobium spp. as well as for some strains of other Gramnegative bacteria .
2. Materials and Methods
2.I . Strains and media
Strains of Gram-negative bacteria from six genera
are listed in the tables under Results. Escherichia and
Pseudomonas strains were obtained from Dr. J.
Langridge, CSIRO, Canberra; Klebsiella and Azotobacter strains were from Dr. C. Kennedy, ARC
Nitrogen Fixation Unit, Sussex, England. All other
strains were part of the author’s collection. Media
used were as follows: modified Bergersen’s medium
[ 3 ] for Agrobacterium, Pseudomonas, and all rhizobia
except strains CBl809 and CB756 (arabinose at 1 g/1
substituted for mannitol with these two strains);
Difco bactopeptone for Escherichia and Klebsiella;
modified Burk’s medium [4] for Azotobacter.
2.2. Lysis
Bacterial broth cultures grown to late-log stage
(except where indicated otherwise) in a shakerincubator, were adjusted to an absorbance ( A ~ O O )
of 1 .O to standardize comparison of lysis procedures.
158
Details for the main method of lysis, designated as
the “detergent-wash/osmotic-shock” procedure, are
outlined in Fig. 1. Other methods of lysis used for
comparison with this method are described briefly in
the text. All samples and solutions were kept cold
(0-5°C) through all stages until the final clearing by
Sarkosyl. Lysates were vortex-sheared for 5 sec to give
homogeneous, less viscous samples for comparison of
extent of lysis at A600.
The sucrose-Tris-EDTA solution used for the
osmotic shock treatment was a concentrated solution
of 1.6 M, 0.55 M and 0.10 M, respectively.
2.3. Isolation and assay of covalently closed circular
(CCC)DNA
Lysates were compared for CCC DNA yield by
cesium chloridelethidium bromide (CsClIEB) density
gradient centrifugation as described previously [5] .
Essential details concerning the gradients are the
following: CsCl49.35% w/w; EB 275 pg/ml; lysates
vortex-sheared 15 sec; centrifugation at 40 000 rev./
min for 40 h , 18’C, in type 6 5 Beckman rotor.
Gradients were examined under ultraviolet light for
visual comparison of CCC DNA bands. With tritium-
labeled cell lysates the chromosome DNA band was
first removed from the top of the gradient with a
syringe, and the CCC DNA then harvested dropwise
from the bottom to allow comparison of both peak
and total radioactivity. Radioactivity was assayed by
liquid scintillation counting as described previously
PI.
3. Results
3.1. The lysis procedure
The detergent-wash/osmotic-shock procedure
found to be most effective for lysis of lysozymerefractory strains is shown in Fig. 1 as used for strains
of fast-growing Rhizobium spp. The volume and cell
concentration used (1 2 ml at A600= 1) and the
volume of lysate obtained (7 ml) would be suitable
for a 9 ml CsCl/EB gradient for plasmic DNA isolation, but these volumes can readily be varied depending on the strain and the intended use of the lysate.
Although Sarkosyl was the preferred detergent for
producing lysates for CCC DNA isolation, other
detergents like Triton X-100, Nonidet P-40, and
Bacterial culture grown to late log phase (1-2
. lo9 cells/ml)
1
Measure absorbance at 600 nm; use culture volume equivalent to 12 ml at A600 = 1.
1
Centrifuge (10 000 X g / l 5 min/O”C); resuspend cells in cold TS (Tris 0.05 M, NaClO.05
M, pH 8) buffer.
1
Detergent wash
Add Sarkosyl to 0.1% (v/v) final; vortex mix 15-30 sec; centrifuge as above and
decant closely (to minimize detergent carry over).
1
Resuspend cells in 0.4 ml TES (TS t EDTA 0.01 M); add 0.35 ml concentrated “sucrose
mix” (sucrose 1.60 M, Tris 0.55 M, EDTA 0.10 M); keep at 5°C for 10-20 min.
Mild osmotic shock
1
Add 0.15 ml lysozyme (5 mg/ml in Tris 0.05 M, pH 8), mix; add 3.6 ml cold EDTA
(0.01 M) or distilled water.
.1
Incubate 5-20 min (depending on the strain) at 5°C.
1
Add 2.5 ml of 2.5% Sarkosyl; mix slowly to clear.
Fig. 1. Procedure for lysis of bacteria made sensitive to lysozyme by detergent-wash/osmotic-shock treatment.
159
sodium dodecyl sulfate (SDS) were also used in preliminary trials and found to be satisfactory for
either the detergent wash or the final lysis. SDS
would, for example, be the detergent of choice if
post-lysis use involved removal of chromosomal DNA
at low temperatur: in high salt concentration [6].
The lysing detergent can also be made alkaline to
allow the clearing to occur under denaturation conditions, thus adapting the lysis method to a recently
developed procedure [7,8] for direct identification of
plasmids by agarose electrophoresis.
A significant additive effect of the detergent wash
and osmotic shock treatments was evident in experiments in which different steps in the lysis procedure
were individually omitted, as shown in Table 1. Use
of either treatment alone gave only a slight improvement in lysis (depending on the initial lysozyme
“resistance” of the particular strain), but a combination of both treatments enhanced clearing of cell
suspensions with all of the relatively lysis-resistant
strains tested. For example, strain L1 of R. Zeguminosarum is relatively resistant to lysis and required both
treatments to predispose the cells sufficiently for
maximum clearing. In contrast, a more lysis-sensitive
strain like Pseudomonas aeruginosa PA0 B(R19)
showed a considerable degree of lysis with the
individual treatments, although complete clearing still
required the full procedure shown in Fig. 1.
3.2. Test of lysis method on a range of bacteria
The applicability of the lysis method to rhizobia
(fast-growing and slow-growing) and to some other
Gram-negative bacteria was compared with a basic
lysozyme-only method (Table 2). Results very similar
to those shownwere obtained in a second experiment.
The strains varied greatly in their sensitivity to the
lysozyme-only method, with only two (P.putida
PpC379 and Azotobmter vinelandii UW) showing
good clearing.
In contrast to the lysozyme-only procedure, the
new method gave good clearing with 11 of the 16
strains. Lysates of R. leguminosarum L1 and the two
Agrobacterium tumefaciens strains were mostly
cleared, while to two slow-growing strains of Rhizobium were the most resistant to lysis. Easily lysed
strains like P. putida PpG379 began to clear during
incubation with lysozyme, before the final addition
of Sarkosyl. With such sensitive bacteria the pretreatments can be omitted;if the full procedure is used,
however, the cell concentration can be markedly
increased (culfure/lysate volume ratio >2) to give
very concentrated lysates. Conversely, with lysisresistant bacteria like strains CB1809 and CB756 the
cell concentration must be reduced or other lysis
conditions made more stringent, e.g., longer exposure
period and higher temperature during lysozyme treatment, two successive detergent washes, increased
osmotic drive (higher ratio of dilution with EDTA or
water), or increased volume and concentration of
Sarkosyl in the final step.
It should be noted that the absorbance readings
given in Tables 1 and 2 were taken on undiluted lysate
samples; dilution was avoided bacause of the added
variable of some dilution-induced lysis during absorbance determination. The readings for the partly cleared
lysates are therefore underestimates of the true
absorbance, due to non-linearity of absorbance in
TABLE 1
Absorbance ( A ~ o oof) lysates obtained by the complete detergent-wash/osmotic-shock procedure and by abbreviated procedures
with one or more steps omitted a
Strain
Cells in TS only
(control for no
lysis)
Lysozyme
omitted
Deterg. wash
and osmotic
shock omitted
Deterg. wash
omitted
Osmotic
shock omitted
Complete
procedure
L1 (R. leguminosarum)
PA08 (R19) (P. aeruginosa)
0.98
0.96
0.78
0.48
0.69
0.56
0.59
0.20
0.47
0.12
0.11
0.03
a
The complete procedure was as shown in Fig. 1 . Volumes were adjusted as required (e.g., where lysozyme or osmotic shock
treatments were omitted), with TES, to give the same volume (4.5 ml) before lysing with 2.5 ml Sarkosyl. A 6 0 0 readings are
means of duplicate lysates.
160
TABLE 2
Lysis of some Gram-negative bacteria by lysozyrne, with or without detergent-wash/osmotic-shock treatment
Species
Strain
A600 of lysatea
No pre-treatment of cells
Rhizobium Ieguminosarum
R h izobium trifolii
R hizobium meliloti
Rhizobium japonicum
Agrobacteriurn tumefaciens
Pseudomonas putida
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumoniae
Azotobacter vinelandii
L1
L25
T1
T2 3
u45
su47
CB1089
CB756
B6S3
C5 8
PpG379
P A 0 8 (R19)
1230 (R68.45)
SB1801 (pRD1)
5023
uw
(a)
Detergen t-wash/osmo tic-shock
treatment of cells
(b)
0.74
0.85
0.92
0.81
0.91
0.89
0.94
0.85
0.93
0.68
0.04
0.33
0.80
0.3 3
0.89
0.08
0.14
0.04
0.10
0.08
0.10
0.10
0.49
0.22
0.18
0.17
0.02
0.02
0.03
0.02
0.05
0.05
a The procedure for method (b) was as given in Fig. 1 except that the culture/lysate volume ratio was 1.5. With method (a) the
detergent was omitted in t h e second centrifugation and the pellet was resuspended directly in 4.35 ml TES for lysozyme treatment, without osmotic shocking. Approximate visual ratings of lysate turbidity relative to absorbance readings were as follows:
clear, <0.08; slightly turbid, 0.08-0.20; moderately turbid, 0.20-0.50; turbid (little or no lysis), >0.50. As a “control” for
non-lysis, the absorbance of cells (strains L1 and P. putida PpC379) from the first centrifugation, resuspended in TS buffer,
gave a n A 6 o o (samples undiluted) of 0.98 and 0.97.
relatively turbid samples, but the measurements
nonetheless allowed direct, reproducible evaluation
of the major treatment/strain differences. Absorbance
measurements of the starting cell cultures as used for
adjusting initial sample volumes were, however, taken
on cell suspensions diluted in TS buffer.
3.3. CCC DNA yield as a function of method of lysis
The yield of plasmid DNA in lysates was used as
another criterion (apart from absorbance) of effective lysis and minimum damage to a cellular constituent. Lysates of two plasmid-carrying strains (a
third strain, P. putida PpC379, did not incorporate
sufficient tritium label to allow quantitation), prepared
by three different lysis procedures, were compared
for CCC DNA (tritium labeled) content by CsCl/EB
density gradient analysis (Table 3). The carbenicillin
method for weakening the cell wall prior to adding
lysozyme was included in these experiments because
it has been used successfully for plasmid DNA isolation from Rhizobium spp. and Agrobacterium spp.
[9]. The data show that the new method of lysis not
only improves clearing of the cell suspensions but also
gives a better yield of intact DNA, relative to that
released by the standard lysozyme procedure or even
the carbenicillin procedure. Comparable results based
only on visual observation of DNA band size have
also been obtained in other experiments with R. trifolii T1, R. meliloti U45,and E. coli strains.
4. Discussion
The lysis procedure (Fig. 1) was designed mainly
to facilitate isolation of plasmid DNA from lysozyme-
161
TABLE 3
Effect of method of cell lysis on yield of CCC DNA a
Strain
Method of lysis b
Radioactivity
Total count of
lysate in gradient
(cpm . lo3)
Count in CCC
DNA band
(cpm)
Percent of total
count present in
CCC DNA band
L1
(R. legurninasarum)
Lysozyme only: no pre-treatment
Carbenicillin pre-treatment
Detergent-wash, osmotic-shock
pre-treatment
1266
1412
1742
4 260
18 380
50 720
0.34
1.30
2.88
B6S3
Lysozyme only; no pre-treatment
Carbenicillin pre-treatment
Detergent-wash, osmo tic-shock
pre-treatment
463
9 84
1115
2 070
19 210
33710
0.44
1.95
3.02
(A. tumefaciens)
a DNA isolated by CsCl/EB density gradient centrifugation (see Methods). The DNA was tritium-labeled by growing the bacteria
H]
in nutrient broth containing 2 jtCi/ml of [ m ~ e t h y l - ~thymidine.
h All three methods included lysozyme; culture ( A ~ o oand
) final lysate volumes were the same for all treatments. The detergentwash/osmotic-shock method was used as in Fig. 1. The “lysozyme~nly”procedure was identical except for omission of the
detergent in the second centrifugation and omission of the shockdilution treatment. The carbenicillin method differed from the
“lysozyme-only” procedure in the prelysozyme exposure of the cells to carbenicillin. Cells from the first centrifugation were
resuspended and incubated for 120 min at 29OC in nutrient broth containing 500 pg/ml carbenicillin, then centrifuged and
resuspended in TES for lysozyme treatment.
resistant bacteria but should also be adaptable to isolation of enzymes, ribosomes, cytoplasmic membranes,
or other cellular components. A non-ionic detergent
can be used for the final lysis, or the detergent can be
omitted and the cells subjected to mild mechanical
disruption after enzyme treatment. Alternatively the
cells could be harvested as spheroplasts by following
the lysis procedure through the osmotic shock step
(to induce lysozyme penetration), then adding sucrose
and resuspending the cells, after centrifugation, in a
small volume of sucrose or other hypertonic solution.
The spheroplasts could then be lysed without the use
of a detergent, by suitable dilution with a hypotonic
medium.
The effectiveness of the lysis procedure depends
largely Qn two key steps: (a) predisposing the cell
surface to lysozyme penetration by washing in dilute
detergent, and (b) osmotically shocking the cells by
quick dilution from sucrose into water or 10 m M
EDTA. The effect and probable mechanism of the
second step have been described by Witholt et al. [ 2 ]
as a destabilization of the lipopolysaccharide-containing cytoplasmic membrane by the high concen-
tration of Tris-EDTA, followed by an osmotically
driven influx of lysozyme molecules through membrane pores to the murein layer. The mechanism of
the detergent wash is more obscure. Comparison of
the total carbohydrate content (anthrone reaction) of
cells from four strains ( L l , T1, CB1809, B6S3)
before and after the detergent wash failed to detect
any significant removal of polysaccharide. It is possible, however, that removal of even a small, quantitatively insignificant amount of capsular gum could
expose a sufficient number of membrane openings to
allow for enzyme penetration. Alternatively, the
detergent may affect the permeability of the outer
membrane [ 101 itself by altering the lipopolysaccharide, phospholipid, or lipoprotein structure of the
membrane.
Acknowledgments
I thank Dr. W.F. Dudman, Division of Plant
Industry, CSIRO, Canberra, for the anthrone tests.
162
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