Download The patterns of extracellular protein formation by spontaneously

FEMS MicrobiologyLetters 70 (1990) 91-94
Published by Elsevier
91
FEMSLE 04048
The patterns of extracellular protein formation
by spontaneously-occurring rifampicin-resistant
mutants of Staphylococcus aureus
B. AI-Ani, M. A b o s h k i w a , R.E. Glass a n d G. C o l e m a n
Department of Biochemistry. Nottingham University Medical School. Queen's Medical Centre. Nottingham. U.K.
Received5 December 1989
Received28 February 1990
Accepted 15 March 1990
Key words: Staphylococcus aureus; Extracellular protein; Rifampicin-resistant mutants
1. S U M M A R Y
Spontaneous!y-occurring rifampicin-resistant
mutants of Sataphylococcus aureus were isolated
on 4 ~ ( w / v ) Tryptone Soya Agar containing 4
and 40 times the m.i.c, for rifampicin. A number
of colonies were selected at each rifampicin concentration and were grown aerobically in 3~ ( w / v )
Tryptone Soya Broth for 24 h at 37°C. In the case
of S. aureus RN4220 all the mutants grew to
bacterial densities up to approximately 1.7 times
more than the parent organism. The corresponding levels of extracellular protein secretion varied
over a 5-fold range, all the mutants being less
productive than the parent. By contrast, mutants
of the wild-type Wood 46 strain achieved bacterial
densities of only 4 5 - 8 3 ~ that of the parent whilst
exoprotein secretion showed a smaller 1.7-fold
variation. However, widely-differing patterns of
exoproteins were revealed by SDS-polyacryla-
Cc~,'~.spondence m: Dr. G. Coleman, Department of Biochem-
istry, Nottingham University Medical School, Clifton Bouleyard, Nottingham NG7 2UH, U.K.
mide gel electrophoresis of the parevt and mutant
organisms of both strains.
2. I N T R O D U C T I O N
Biochemical studies on the control of extracellular protein formation in the Gram-positive
organism Staphylococcus aureus showed a characteristic pattern of secretion in which massive
production of exoprotein occurred after the end of
exponential growth. This was accounted for in
terms of more resources being available for increased exoprotein formation when cell growth
decreased [1]. More recently, Recsei et al. [2] demonstrated the existence of a trans-active positive
control element which had a pleiotropic effect on
the production of a number of exoproteins. This
finding was confirmed by Jargon et al. [3] who
showed a similar effect exerted at the level of
transcription which was growth-rate dependent.
These data supported the conclusion that a key
factor in the regulation is the affinity of R N A
polymerase for specific promoters [1].
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
Glass and coworkers [4] have carried out a
detailed genetic analysis of the role of the/~-subunit of E. coli RNA polymerase in DNA transcription and they have demonstrated that single
amino acid substitutions can result in altered promoter selectivity [5]. They isolated spontaneouslyoccurring amber mutants which arose by single
base changes in a small defined region of the
RNA polymerase/i-subunit gene.
The isolation of spontaneously-occurring
rifampicin resistant mutants of S. aureus [6] provides a convenient means of generating organisms
with altered RNA polymerases. The effects of
these changes on the patterns and characteristics
of extracellular protein formation have been examined.
tone soya agar containing 10 /tg ml -~ chioramphenicol where growth was observed in all
cases.
Mutants of S. aureus (Wood 46) were isolated
in exactly the same manner the only difference
being the omission of chlorampbenicol.
3. MATERIALS AND METHODS
3. 4. Bacterial density determination
The method of Stormonth and Coleman [8] was
employed.
3.1. Organisnts
Staphylococcus aureus RN4220, a mutant
organism derived through a series of steps from
NTCC 8325, was obtained from dr. Richard
Novick. It was transformed by electroporation
with plasmid pCK1 which carries a chloramphenicol resistance gene [7]. This provided a
convenient "marker" to ensure that Rif-r mutants
were derived from the parent organism and not
from contaminants. The wild-type organism S.
aureus (Wood 46) (NCTC 7121) was also used.
3.2. Isolation of mutants
3~ (w/v) Tryptone Soya Broth (Oxoid) containing 10/tg m l - i chloramphenicol (Sigma) was
inoculated with the parent organism (S. aureus
RN4220+pCK1) and incubated overnight at
37°C. Aliquots of the culture, 0.1 ml containing
approximately l0 s bacteria, were spread evenly on
4~ (w/v) Tryptone Soya Agar (Oxoid) containing
0.08 or 0.8 /tg ml -~ rifampicin (Lepetit, Milan,
Italy), that is, 4 and 40 times, respectively, the
m.i.c. After incubation for 24 h at 37°C, a small
number of mutant colonies appeared on each plate
which were purified on the same rifampicin-containing medium and a single colony selected in
each case. All the isolated mutants were checked
for chloramphenicol resistance on 4% (w/v) Tryp-
3. 3. Growth of the organisms
3~ (w/v) Tryptone Soya Broth, 50 ml batches
in 250 ml conical flasks, was inoculated with the
rifampicin-resistant mutants and the parent
organisms and incubated at 37 ° C in a Gyrotary
incubator-shaker (New Brunswick Scientific Co.
Inc., New Jersey, U.S.A.). After 24 h samples were
taken for assay of bacteria density, total exoprotein and for SDS-polyacrylamide gel electrophoresis.
3.5. Extracellular protein estimation
The protein content of culture supernatant
fractions was estimated by the method of Scdmak
and Grossberg [9] as described by Coleman et al.
[101.
3.6. SDS-polyacrylamide gel electrophoresis
This was carried out as described by Laemmli
[11]. Samples were prepared, in each case, by
precipitating the exoprotein from 1 ml aliquots of
culture supernatant fraction with 0.1 mi 100~
(w/v) trichloroacetic acid. The precipitates were
dissolved in 95 /tl sample buffer plus 5 /tl of a
saturated solution of Tris (Sigma) and 15/tl was
introduced into each sample well.
4. RESULTS AND DISCUSSION
Three rifampicin-resistant mutants isolated on
4% (w/v) Tryptone soya agar containing 4 times
the m.i.c, and three isolated in the presence of 40
times the m.i.c, for rifampicin were chosen at
random, in each case. They were incubated aerobically in 3% (w/v) Tryptone soya broth for 24 h at
37°C after which time growth had ceased. The
bacterial densities and extracelhilar protein levels
in the culture media were determined in each case
and compared with the values obtained for the
parent organisms.
It can be seen in Table 1 that the parent
organism, S. aureus RN4220 + pCK1 achieved the
highest level of exoprotein from the lowest
bacterial density. The exoprotein formed per mg
of bacterial dry weight was 127/~g, which was 1.6
times higher than that of mutant 2 which produced more exoprotein than any of the other
mutants from the lowest mutant bacterial density.
Mutant 1 achieved the highest bacterial density
and, within the limits of experimental error, the
equal lowest amount of exoprotein. However, the
other mutants did not all conform to the same
pattern.
Mutants 3 and 5 which were isolated in the
presence of different rifampicin concentrations
achieved the same bacterial density and the same
exoprotein levels. Further, they produced the same
SDS-polyacrylamide gel electrophoresis pattern
(Fig. 1). This suggested that they were identical or
that the mutation in each was in a region which
produced the same effect. The parent produced a
characteristic SDS-polyacrylamide gel electro-
Table 1
Bacterial densitiesand extracellular protein levelsin a culture
of Staphylococcus aureu~ RN4220, transformed with plasmid
pCKI, and a number of rifampicin-resistantmutants after 24 h
growth at 37°C in 3% (w/v) Tryptone soya broth
Rifamplein-resistammutants 1-3 wereisolatedon 40 times the
m.i.c, and mutants 4-6 on 4 times the m.i.c, of rifampicin.All
values were the mean of dupUcate assays with a standard
deviation of leas than 5:5%
Oqpmism
Parent
Mutant I
Mutant 2
Mutant 3
Mutant 4
Mutant 5
Mutant 6
Bacterial
density
(ms dry wt.
ml- l )
2.01
3.37
2.30
2.83
2.47
2.86
2.69
Exoprotein Exoprotein
formed
per
mg bacteria
(~g)
(~gml - t )
253
56
180
102
54
106
69
! 27
17
78
36
22
37
26
w
W w
M
P
1
2
N
3
4
.....
5
6
Fig. 1. SDS-polyacrylamidegel electrophoresispatterns of the
extracellular proteins produced in a 24 h culture of Staphylococcus aureus RN4220. transformedwith plasmid pCKI (lane
P) and a number of rifampicin-resistantmutants (lanes 1-6).
as in Table i, g-own in 3% (w/v) Tryptone soya broth at
37°C. Exoproteins from the same volume of culture supernatant fractions were loaded in adjacent lanes. Molecular
weight marker proteins (MW-SDS-70L;Sigma)wereseparated
in lane M. from top to bottom. 66. 45. 36. 29. 24. 20.1 and
14.2 kDa.
phoresis pattern which was the same in the presence and absence of plasrmd p C K I . The similarity
between the patterns of exoproteins produced by
mutants 3 and 5, as shown in Fig. 1, appeared to
be extended to mutant 6 which, however, produced considerably less total exoprotein. The other
rifampicin-resistant organisms were quite different
from each other and from the parent organism.
These differences cannot be ascribed to proteolytic degradation since the patterns and amounts
of protein in the supernatant fractions remain
unchanged even after prolonged incubation.
in order to establish that the changes reported
were due to spontaneous mutations resulting in
rifampicin resistance and independent of the
organism's origin a parallel study was carried out
with a wild-type bacterium from a completely
different source, namely, S . a u r e u s (Wood 46).
This further study provided results which led to
the same conclusions as those obtained with the
mutant RN4220 strain.
On the basis of the frequency of the occurrence
of the spontaneously-occurring rifampicin-resistant mutants, the mutations would be expected
to have arisen by single base changes resulting in
single amino acid substitutions in the fl-subunit
[5]. These substitutions have been shown to cause
dramatic changes in the characteristics of secretion and patterns of formation of extracellular
proteins by $. aureus.
In E. coil single amino acid substitutions in the
/~-subunit of R N A polymerase can result in altered promoter selectivity [5]. However, whilst our
data suggest a role for the transcriptional apparatus in the regulation of expression of particular
exoproteins whether this involvement is direct or
indirect and where the regulatory target is located
remains the subject of an ongoing investigation.
S . aure~;s P.~,220 was chosen for this study
due to its ability to be readily transformed to
chloramphenicol resistance by plasmid pCK1. This
choice seemed justified since its characteristics of
total extracellular protein secretion were very similar to those of the widely-studied wild-type
organism S. aureus (Wood 46). Thus, the parent
organisms produced 127 and 139 lag exoprotein
per mg bacteria, res~.ectiwly, after 24 h growth in
3% ( w / v ) Tryptone soya broth.
It is interesting to note that Peng et al. [12]
reported that S. aureus RN4220 was low in exoprotein gene expression (exp-) which is not in
agreement with the similarity, quoted above, between RN4220 and Wood 46. The reason for this
disagreement is not known, although, unexplained
differences of this type have been recorded in the
RN4220-related 8325-4 strain (Novick, personal
communication).
In the present case the apparent disagreement
could be associated with differences in composition of the growth media. Thus, Peng et al. [12]
used a 1% casamino a c i d / l % yeast extract/0.5%
glucose-containing medium whereas in this work
the orsanisms were grown in 3~ Tryptone soya
broth which was shown by Coleman and Abbas-Ali
[13] to support a lower growth rate but a higher
exoprotein productivity than a richer casein hydrolysate/yeast extract medium. More recently,
Coleman et al. [14] showed that the presence of
glucose had a marked effect on exoprotein production by S. aureus strain VS. The addition of 1~
glucose to a washed suspension of the bacteria,
from an overnight culture, in 3~0 Tryptone soya
broth resulted in a 10-fold reduction of exoprotein
formation to a level closer to that observed in an
e x p - mutant.
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Fchrenbach, F., Freer, J. and Jcljaszewicz,J., eds.), pp.
99-106, Academic Press, London.
[21 Recse/, P., Kreiswirth, B., O'Reilly, M., Schlieven, P.,
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[3] Janzon, L., Lofdahl, S. and Arvidson, S. (1986) FEMS
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[9] Sedmak, J.J. arid Grossberg, S.E. (1977) Anal. Biochcm.
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[12] Peng, H.oL,, N0vick, R.P., Kn:iswirth, q., Kornblum, J.
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