J. Genet. Vol. 67, No. 2, August 1988, pp. 67-73. 9 Printed in India.
Expression of drug resistance markers by sphereptlas~;s of
Mycobacterium smegmotis after fusion
C H A N C H A L S A D H U and K P G O P I N A T H A N
Department of Microbi.ology and Cell Biology, Indian Institute of Science, Bangalore
560 012, India.
MS received 22 April 1988
Abstract. Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several
hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl
sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion m~d
regeneration of the spl)eroplasts were attempted using drug resistant mutant strains of M.
smegnlalis. Recombinants were obtained from spheroplast fusion mediated by
polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of recombinm~t
properties was observed only after an initial lag in the isolated clones. This has been
explained as due to "chromosome inactiwltion'" in the fused product.
Keywords. Spheroplast fusion; mycobacteria; prophage activation; recombination;
chronaosonae inactivation.
hRroduc6on
G e n e r a t i o n of bacterial recombinants mediated by protoplast fusion has gained
{ i-n
mmOltance in recent years ( H o p w o o d et al 198o). This m e t h o d is particularly useful
~,'~r those species of bacteria where other means of genetic transfer are net
well-established. F r o m the genetic studies on m y c o b a c t e r i a by several groups of
workers it has generally e m e r g e d that even though the classical methods of gene
tiam~er, viz. conjugation, transformation and transcluction, operate in clifferenc
species of mycobacteria they are often inefficient and irreproducible ( G r a n g e
1982). F o r this reason the protoplast fusion system may be of potential advantage in
the s;. ~=ly of mycobacterial genetics.
We !lad reported earlier a rapid and efficient p r o c e d u r e for the isolation of
spbe
,sts from M. smegmatis, rising a c o m b i n a t i o n of lipase and lysozyme
(Sad!, and G o p i n a t h a n 1982). Rastogi et al (1983) using polyethylene glycol (PEG)
t?av,.
,rained spheroplast fusion in M. aurum yielding recombinants between
strains differing in pigment production. In the present report we present
~ bserva:~o;:s on the expression of drug resistance markers by spheroplasts of/14.
smegm~,, .... after fusion.
67
68
2.
ChaHchat Sadhu and K P Gopinathan
Materials arid method8
Materials: Lysozyme, lipase, polyethylene glycol (PEG) and dimethylsulfoxide
(DMSO) were from the Sigma Ct~emical Company, St. Louis, MO, USA. All other
chemicals were of analytical grade.
Bacterial strains: M. smegmatis SN2 and the novobiocin- and kanamycin-resistant
mutants of M. smegmatis SN2 were from our laboratory culture stocks.
Media (composition/litre): Minimal medium-(Karnik and Gopinathan 1980)
Asparagine, 5.0 g; KH2PO4, 5-9 g; K2SO4, 0.5 g; MgCOB, 0.5 g; citric acid, 1.5 g;
g!ycerol, 20 ml; pH adjusted to 7.4. Tween-80 (0.3% v/v) was included to minimize
clmnping of mycobacteria. Regeneration mediuln-peptone, 10.0 g; beef extract,
3.0 g; NaCI, 5.0 g; casamino acids, 0.1 g; glucose, 10.0 g; K2SO4, 0.25 g; CaCI2,
6H20, 0-22 g; MgCI2, 6H20, 0.20 g. LB medium-Bacto tryptone 10 g; yeast
extract 5 g; NaC1, !0 g.
Preparation of spheroplasts: Spheroplasts were prepared according to the procedure described earlicr (Sadhu and Gopinathan 1982). The method consists of two
steps: a) sensitization of the cells by exposure to glycine (0.2 M), and b) enzymatic
degradation of cell wall material. Glycine was added to early log phase cultures of
M. smegmatis and shaking was continued for 2 h at 37~ The cells were harvested
and resuspended in th e original volume of 10 mM Tris (pH 7.0) containing 15%
sucrose. Lipase and lysozyme were added to a final concentration of 500/zg/ml
each and the incubation continued. Nearly 100% conversion of bacillary forms to
spheroplasts was achieved within 2-3 h as monitored by phase contrast microscopy.
Fusion of spherophlsts: Spheroplast fusion was carried out by the following
modification of the procedure for streptomyces described by Hopwood et al (1977).
Equal amounts of sPheroplasts of the strains to be fused were mixed and
centrifuged at 5000 • g for 10 rain. The pellet was resuspended in 0.1 ml of
PEG-1000 and 0.14 ml of the lninimal medium (without Tween-80) containing
DIVISO(15%). After 1 rain, 0.1 lnl PEG and 0.3 ml medium were added, which was
followed after 3 min by the addition of 1.2 ml of medium. Samples of this
suspension were layered on regeneration plates and incubated at 37~ for 3-4 days.
Recombinants were screened by serially transferring the individual colonies on
Lg-agar plates containing novobiocin (25 /xg/ml) and/or kanalnycin (100/xg/ml).
3.
Results and d~seussien
Formation of sl)heropIasts of M. smegmatis SN2: The treatment of M. srnegmatis
cells presensitized by growth in presence of glycine, with a combination of lipase
and lysozyme yielded spheroplasts (figure 1). The presence of lipase was most
essential for the production of spheroplasts. Due to the presence of large amounts
and multiple layers of lipopolysaccharides, mycobacterial cell walls are resistant to
lysozyme alone, which is widely used to prepare spheroplasts from gram-positive
bacteria. The yield of spheroplasts was > 95% efficient and was achieved within
2-3 h.
Spheroplast fusioJl in mycobact'eria
69
Spheroplasts were stable for several hours at room temperature. They were lysed
by treatment with 0.1% sodium dodecyl sulfate (SDS), in contrast to the parental
cells.
Regeneratiot~ and filsion of st)heroplasts: The mycobacterial spheroplasts regenerated with fair efficiency on a suitable regeneration medium. They could be
distinguished from the parental cells by separately scoring for the total regenerants
and the osmotically resistant (after SDS treatment) cells plating on the regular
medium. Thus, in a population of 5 • 10~ cells, after spheroplasting 8.5 • 103 cells
remained resistant to SDS-lysis which correspond to the parental cell population.
o n the regeneration medium, however, 1-8 x 105 colonies contributed both by the
parental cells as well as the regenerated spheroplasts, were obtained. Therefore,
95% of the colonies on the regeneration medium arise from spheroplast
regeneration.
Temperature is one of the important parameters which affects spheroplast
regeneratio n (Hopwood 1981). To determine the optimum temperature of
regeneration, M. smegmatis spheroplasts prepared by the above method were
spread on regeneration medium and incubated at different temperatures. At 37~
the regeneration of spheroplasts was better (1-5-2.0 times) than at the other
temperatures tested.
Fusion of protoplasts has been reported in a large number of bacterial species but
the frequencies vary considerably. Fodor and Alfoldi (1976) have reported a
frequency of 10-5 to 5 x 10.3 in the case of Bacillus megaterium protoplasts. On the
other hand, Frehel et al (1979) have observed a fusion frequency as high as 65% of
the input protoplasts in B. subtilis. To check whether effective fusion is occurring
under our conditions we have fused wild-type M. smegmatis spheroplasts with the
spheroplast s prepared from M. smegmatis lysogenized with its temperate phage 13
and monitored prophage activation by plating the fused spheroplasts in the
presence of indicator bacteria. In the plates which received the fused spheroplasts,
the number of plaques were 25-30 times greater than that of the control plates
which received the spheroplasts of the lysogen only, indicating effective fusion.
Gelle#'atio~l qf recombinant by spheroplast.fiesion : Spheroplasts were produced from
novobiocin- and kanamycin-resistant cells of M. smegmatis and the spheroplasts
from these two strains were fused with PEG. Recombinant.s were screened among
tile regenerated bacilli. The flow chart of tile screening proceclure is given in
figure 2. The number of colonies appearing at each step is given in parentheses. As
indicated, no recombinant appeared when the fused spheroptasts were directly
plated on the regeneration medium containing both novobiocin and kanamycin.
However, 836 colonies appearing oll nonselective mediuln (plates having no drug)
when individually transferred to drug containing plates, o!Hy 3-4~ of them grew
on either of the plates, contrary to expectation. In fact, the entire population
should have grown on plates containing either novobiocin or kananlycin due to the
parental phenotypes. This observation suggested that during the process of
spheroplast formation or fusion, there was a loss of drug resistance properties in
most of the cells.
The colonies which had grown oll novobiocin were transferred to mediunl
containing kanamycin or novobiocin, or both. All of then1 grew oil novobiocin,
some (3 out of 14) grew oil kanalnycin, and only one (out of 14) grew oil mediunl
70
dT~
Chanchal Sadhu at~d K P Gopinathan
A
Spheroplast fusion in mycobacteria
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72
Chanchal Sadhu arm K P Gopinathan
I .ovo. sP...o,LAsTs I
[ ~-' S,...O,L~STS i
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Figure 2. Flow chart for screening recolnbinants.
containing both novobiocin and kanamycin. Evidently, the loss of antibiotic
resistance noted previously was only transient because resistance to kanamycin
reappeared. Resistance to both the drugs, however, was found only in one colony.
Subsequent transfer of the colonies from kanamycin plates o n - t o medium
containing kanamycin or novobiocin showed that all were now resistant to both
antibiotics. After this period, the colonies appeared stable in terms of their
antibiotic resistance and further loss or gain of resistance was not noted. A similar
pattern was obtained for the colonies which were grown on kanamycin containing
plates after transferring from the nonselective plates (Transfer-l).
St)herol)last ,filsion in m y c o b a c t e r i a
73
T h e s e o b s e r v a t i o n s s u g g e s t e d that there was a lag in the e x p r e s s i o n of
r e c o m b i n a n t c h a r a c t e r s in the fused spherophtsts of M. s m e g m a t i s . In B.
m e g a t e r i u m a t r a n s i t o r y s e g r e g a t i o n of m a r k e r s was o b s e r v e d after fusion of
a u x o t r o p h i c p r o t o p l a s t s ( F o d o r and Alfoldi 1976). S u b s e q u e n t l y , H o t c h k i s s and
G a b o r (1983) have discussed different features of c h r o m o s o m e interaction and
e x p r e s s i o n after fusion of Bacillus p r o t o p l a s t s and p r e d i c t e d the o c c u r r e n c e of
' c h r o m o s o l n e i n a c t i v a t i o n ' in the fused p r o d u c t . In o u r case, most of the clones
resulting from s p h e r o p l a s t f o r m a t i o n , l'usion and r e g e n e r a t i o n initially e x p r e s s e d
n e i t h e r of the a n t i b i o t i c resistances of the p a r e n t s . This can be e x p l a i n e d by a
p o s s i b l e r e p r e s s i o n of o n e g e n o m e by the o t h e r before r e c o m b i n a t i o n as seen in the
Bacillus system. On s u b s e q u e n t replica plating both the c h a r a c t e r s were I:ound to
be s i m u l t a n e o u s l y and stably e x p r e s s e d . This may r e p r e s e n t true r e c o m b i n a n t s .
In conclusion, we have d e m o n s t r a t e d that (a) c]rug resistance m a r k e r s can be
i n t r o d u c e d into M. s m e g m a t i s cells via spherotslast fusion, and (b) there is a
c o n s i d e r a b l e lag b e t w e e n the fusion step and a p p e a r a n c e of r e c o m b i n a n t s .
H o w e v e r , to m a k e full use of the s p h e r o p l a s t fusion system for gene m a p p i n g in
m y c o b a c t e r i a , further studies on the events s u b s e q u e n t to fusion are requi~ed.
Acknowledgements
W e t h a n k Ms R e k h a R a m a s w a m y for technical assistance and D r V R
S u b r a m a n y a m , R e g i o n a l M e d i c a l R e s e a r c h C e n t r e , B h u b a n e s h w a r , for c a r r y i n g
o u t the s p h e r o p l a s t viability assays. This w o r k was s u p p o r t e d by grants from the
D e p a r t l n e n t of Science and T e c h n o l o g y , and the D e p a r t m e n t of A t o m i c E n e r g y ,
G o v e r n m e n t of India.
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