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Heterologous expression of Escherichia coli porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability
ELSEVIER
FEMS Microbiology Letters 133 ( 1995) 1OS-I I I
Heterologous expression of Escherichia coZi porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability In& Contreras b, Lorena Mutioz a, Cecilia S. Toro a, Guido C. Mora a,* J Unidad de Microbiologic,
Depurtumento
de Biologiu
Celular y Molec~ulor. Fwultud
Catcilicu de Chile, Cusilla 114-D. Suntiqo. h Departamento
de Bioquimico
y Biologicr Molecular.
Facultad Correo
de Cimcius
@imicas
1. Santiago,
Chik
de Ciencius Bioldgicus.
Pont$ciu
Uniwrsidad
Chile x FarmtrcPuticus.
Unirersidad
de Chile, Cusilla 233
Received 1 September 1995: acccepted8 September 1995
Abstract Electrophoretic analysis of outer membrane proteins showed that Sulmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherkhia co/i and S. vphimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing 15. co/i ompC-lucZ or ompF-1acZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. co/i expression was maximal at 0.7 osmM. ompF-1acZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37°C ompF-1ucZ expression was repressed in E. cob. while in S. typhi it was
induced. KeyMwds:
Salmonella
tvphi;
OmpC; OmpF; Regulation
1. Introduction The adaptation of Gram-negative bacteria to their surroundings includes changes in the composition of outer membrane proteins (OMPs). It has been reported that the relative amount of two of the most abundant OMPs of Escherichia coli and Salmonella fyphimurium, OmpF and OmpC pork, is regulated
^ Corresponding author. Fax: (562) 222 5515; E-mail: [email protected]. Federation of European Microbiological Societies SSDl 0378.1097(95)00345-2
by environmental conditions such as osmolarity, temperature, pH and oxygen availability [l-.5]. The reciprocal regulation of porins has been proposed to be an adaptive response of these bacteria to different habitats. In an external environment with poor nutrient contents and low temperature, the larger pore sized OmpF is preferentially expressed. the contrast, expression of the slightly smaller pore sized OmpC is favored when the bacteria colonize a nutrient-rich milieu, such as the intestine of the animal host, where conditions of high salinity, high temperature and low oxygen availability are encountered [6].
The human pathogen Salmonellu @phi produces two porin species with apparent molecular masses of 36 and 35 kDa, similar to S. typhimurium OmpC and OmpF porins, but it does not synthesize OmpD [7-91. In contrast to the situation in E. coli and S. t?,phimurium, relatively little is known about regulation of S. typhi porin expression by environmental conditions. Electrophoretic analysis suggested that OmpC expression is not regulated by medium osmolarity in aerobically grown bacteria [IO]. Concerning OmpF regulation, no studies have been reported. In the present work, we have used electrophoretic analysis and gene fusion technology to study porin regulation by medium osmolarity, temperature and oxygen availability in S. typhi.
2. Materials 2.1. Bacterial conditions
and methods strains, plasmids,
media and growth
Salmonella @phi Ty2 was obtained from the Institute of Public Health, Chile; E. coli JM 109 [ 111 and DHScu were donated by A. Venegas. Plasmid pKL542 (Amp’) containing E. coli ompC-IacZ cloned in pMLB524 was made available by M. Inouye; plasmid pMH622 (Amp’) containing E. coli ompF-1acZ cloned in pMLB524 was obtained from S. Maloy; plasmid pCR II (Invitrogen Corp.) containing the 1acZ gene and its promoter was provided by R. Vicuiia. All strains were routinely grown in Luria broth (1 .O% tryptone, 0.5% yeast extract, 0.5% sodium chloride). Medium 63 [ 121, supplemented with glucose 0.2%, tryptophan 25 mg 1-l and cysteine 25 mg II’ (M63-S), was used as the defined medium. When needed, media were supplemented with ampicillin 40 pug ml-’ . Media of differing osmolarity were prepared with M63-S diluted or supplemented with increasing amounts of NaCl. 0.06 and 0.16 osmM were obtained by 1:5 and I:2 dilution of M63-S, respectively. Media of 0.3, 0.5, 0.7, 0.9 and 1.l osmM were obtained by supplementing M63-S with 0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl, respectively. Media at all osmolarities contained glucose 0.210, cysteine 25 mg 1-l and tryptophan 25 mg 1-i. The generation time varied between 2.0 and 2.2 h in these media. Luria broth was also prepared at
differing osmolarities: Luria broth containing 0.0 1, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 M NaCl was 0.16, 0.23, 0.30, 0.33, 0.51, 0.68, 0.87, I.1 and 2.0 osmM, respectively. In some experiments, the osmolarity was shifted from 0.7 osmM to 0.16 osmM. To this end, bacteria were grown in 0.7 osmM M63-S for 60 min. centrifuged, washed and resuspended in the 0.16 osmM medium. Osmolarities were determined as described [ 131 or using an Advanced Osmometer Model 3W, Advanced Instruments. Aerobic growth was achieved by vigorous shaking of IO-ml cultures in 100-m] flasks plugged with cotton. Anaerobic cultures were grown without shaking in filled test tubes with the medium overlaid with mineral oil. Cultures were started with 0. I ml of an overnight inoculum grown in M63-S at 37°C. Bacteria were grown in media of different osmolarities at 30°C or 37°C aerobically or anaerobically, up to the mid-exponential phase. Subsequently, outer membrane fractions were obtained or ,@galactosidase assays were performed. 2.2. Outer membrane PAGE
protein preparation
and SDS-
The OMPs fraction was obtained as described [ 141. SDS-PAGE was performed in 12.5% polyacrylamide slabs [ 151. All samples were heated at 100°C in the sample buffer before electrophoresis. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpF and OmpC bands were obtained at 575 nm from ACD- IS Automatic Computing Densitometer, Gelman Sciences Inc. 2.3. @Galactosidase
assays
P-Galactosidase activity was determined from exponentially grown bacteria (ODhOOnm= 0.2) using the SDS-chloroform permeabilization method [161.
3. Results 3.1. Porin regulation in S. ephi
Ty2
The OMP profile of cells grown in media of different osmolarity was analysed. The amount of OmpC or OmpF was determined by densitometric
1. Contrems
et al. / FEMS Microhiolog~
Letters 133 (IYY5)
analysis of Coomassie brilliant blue-stained gels relative to the absorbance of OmpA, which is considered to be constitutively expressed [ 10,17,18]. This was done to reduce fluctuations in the levels of porins due to differences in the amount of sample loaded on the gel. Under aerobic growth conditions at 37°C in Luria broth, expression of OmpC increased with medium osmolarity up to 0.3 osmM and then remained constant up to 1.O osmM. At osmolarities higher than 1.0 osmM, OmpC expression was repressed (Fig. IA). Under anaerobiosis, maximal OmpC expression was observed at 0.3 osmM, being repressed at osmolarities higher than 0.3 osmM (Fig.
0.0
0.4
c,
26
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:
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0.4
g
0.3
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u
0.1
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ii p:
0.6
0.6
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(0smM)
0.6 30
2
0.2
107
II
Osmolarity
i ,~i _i
E d
’
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1
0.2
0.4
,
0.6
06
1.0
0.5 0.0
L
0.0
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-\;: 0.2
0.4
0.6
0.6
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Osmolarity
1.2
1.4
1.6
1.6
2.0
B
0.0
0.2
0.4
0.6
0.6
Osmolarity Omp
F
1.0
, ,j 1.2
1.4
1.6
1.6
2.0
(0smM) 0
Omp
F
(0smM) 0
Omp
c
Fig. 2. Electrophoretic analysis of S. @phi Ty2 OMPs. Proteins were obtained from bacteria grown at 37°C aerobically (A) or anaerobically (B) in minimal media M63-S of differing osmolarities. Samples were run in 12.5% acrylamide gels. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpC and OmpF bands were obtained. The amount of OmpF or OmpC present in the outer membrane is expressed relative to OmpA. This is a representative example of the analysis performed on several occasions.
(0smM)
$.‘------.._,
9
Ornp c
Fig. I. Electrophoretic analysis of S. @phi Ty2 OMPs. Proteins were obtained from bacteria grown at 37°C aerobically (A) or anaerobically (B) in Luria broth of differing osmolarities. Samples were run in 12.5% acrylamide gels. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpC and OmpF bands were obtained. The amount of OmpF or OmpC present in the outer membrane is expressed relative to OmpA. This is a representative example of the analysis performed on several occasions.
lB>. On the other hand, the amount of OmpF decreased as the osmolarity increased. OmpF was further repressed in anaerobically grown bacteria. When bacteria were grown in M63-S media under both aerobic and anaerobic conditions, the level of OmpC was reduced in a steady manner as the osmolarity increased, while OmpF expression was repressed (Fig. 2A, B). 3.2. Regulation
of E. coli ompC expression
in S.
typhi
S. ephi Ty2 was transformed with plasmid pKL542 which contains E. coli ompC-lacZ and
108
I. Conmxzs
ef al. /FEMS
Microbiology
Letters 133 (1995) 105-l I I
same results were obtained, regardless growth temperature was 30°C or 37°C. 3.3. Regulation typhi
0’
0.2
0.4
0.6
Osmolarity I3
-
1.2
(0smM) 100
r
E
200
b ._ .?, z
150
1
ll;Iz,,_,,,?+
/’
r
80
A
_’
2
I
5
60
s s
40
3 7ld
20
CL:
0: 0
I
0.0
0.2
0.4
0.6
0.6
Osmolarity
lacZ
/
I
10
1.2
1
0.0
0.2
-
and oxygen
Anaeroblosls
availability
0.4
0.6
Osmolarity
(osmbi)
Aeroblosls
Fig. 3. Effect of osmolarity or&-
1.0
in S.
4
250
2
0.6
of E. coli ompF expression
the
S. typhi Ty2/pMH622 bearing the E. co/i ompF-1acZ fusion was grown at 37°C at osmolarities ranging from 0.03 to 1.1 osmM. P-Galactosidase activity was reduced as the osmolarity increased. Under anaerobic conditions, expression of ompF
1
0.0
whether
0.6
1.0
1.2
(0smM)
w
on E. coli
expression. P-Galactosidase was measured in .S.
100
-
typhi (A) and E. coli (B), each harboring
pKL542, which contains E. co/i on@lacZ gene fusion. Bacteria were grown aerobically or anaerobically, in minimal glucose media (M63-S) of differing osmolarities. P-Galactosidase activities are expressed relative to the activity (lOO’%o) measured at 1.1 osmM under aerobiosis at 37°C. Each assay was run in triplicate on four occasions. Values are means * standard deviation.
expression of this gene fusion was assayed in media of varying osmolarities at 37°C. As shown in Fig. 3A, P-galactosidase activity was maximal at 0.16 osmM under anaerobiosis and at 0.3 osmM under aerobiosis. At higher osmolarities, P-galactosidase was repressed. Endogenous OmpC was regulated in S. ryphi Ty2/pKL542 as it is in S. typhi Ty2 (results not shown). In E. coli JM109/pKL542 (Fig. 3B) grown anaerobically, maximal P-galactosidase expression was observed at 0.7 osmM, while in aerobiosis enzyme activity continuously increased throughout the range of osmolarities tested. The
01 0.0
1
0.2
0.4
0.6
Osmolarity -
Aeroblosls
0.6
1.0
1.2
(0smM) -
Anaeroblosls
Fig. 4. Effect of osmolarity and oxygen availability on E. coli ompF- lacZ expression. P-Galactosidase was measured in S. @phi (A) and E. coli (B), each harboring pMH622, which contains E. co/i ompF-lacZ gene fusion. Bacteria were grown aerobically or anaerobically, in minimal glucose media (M63-S) of differing osmolarities. P-Galactosidase activities are expressed relative to the activity (100‘7~) measured at 0.03 osmM under aerobiosis at 37°C. Each assay was run in triplicate on four occasions. Values are means + standard deviation.
109
was significantly lower than under aerobiosis at all osmolarities (Fig. 4A). Similar results were obtained in E. colt’ JM109/pMH622 (Fig. 4B). However, when these assays were performed at 30°C no repression of ompF by anaerobiosis occurred in S. ~phi/pMH622: P-galactosidase activities were similar to those in aerobiosis (data not shown). This was confirmed by experiments in which bacteria were grown at 30°C under aerobic or anaerobic conditions in 0.7 osmM medium and then shifted to 0.16 osmM.
3000 -
h
z 5 2500 0 cd 2000 ti -fj 1500 ._ : c, 1000 ii s M
500 -
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30
60
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;
1200
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1 120
I 150
I 160
(min)
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0
30
60
Time
90
120
150
160
(min)
-6000
:
2000
2
:"::ii
% 2
600
s ; M
400
‘-;-
-10000 , 0
I 30
1 60
Time
I 90
(min)
Fig. 6. Expression of onlpFP /acZ during a temperature shift from 30°C to 37°C. Bacteria were grown aerobically at 30°C during 60 min in M63-S medium, then shifted to 37°C. /3-Galactosidase was measured in S. npl?i (A) and E. co/i (B), each harboring pMH622. which contains E. co/i onyF- /wZ gene fusion. P-Galactosidase activities represent the difference between the activity measured at 30°C and the one measured at 37°C. Values are means k standard deviation.
B
;
\
-6000
-30
L
160
-4000
M ' -12000 .~ Qz
h
150
0
6
4 .d [
120
B
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90
ir;--‘-
~~~_~-~=--~~~..__~*~~..~ v
I:,,,,,, 0 -30 0
30
60
Time -
Aeroblosls
90
120
150
160
(min) -~~ Anaeroblcms
Fig. 5. Expression of ompF-/rcZ during a shift of osmolarity from 0.7 osmM to 0.16 osmM. Bacteria were grown aerobically or anaerobically at 30°C during 60 min in 0.7 osmM M63-S medium, then shifted to 0.16 osmM. P-Galactosidase was measured in S. fxphi (A) and E. di (B). each harboring pMH622, which contains E. co/i ompF- /acZ gene fusion. P-Galactosidase activities represent the difference between the activity measured at 0.7 osmM and the one measured at 0.16 osmM. Values are means, standard deviation.
In S. l[vphi/pMH622, induction of ompF was identical under both conditions of oxygen availability (Fig. SA). In contrast, in E. coli/pMH622 induction of otnpF was significantly increased under aerobic conditions (Fig. 5B). To further investigate the temperature regulation of OmpF. bacteria were grown at 30°C for 60 min, then the temperature was raised to 37°C. Remarkably, in S. @phi/pMH622 (Fig. 6A) P-galactosidase activity was induced during the temperature shift. As expected, in E. coli JMl09/pMH622, the opposite effect was observed (Fig. 6B).
I10
I. Contrerus
et (11./ FEMS Microbiology
4. Discussion In our study, electrophoretic analysis of major OMPs indicates that S. ~phi OmpC expression is not reciprocally regulated relative to OmpF as described for E. coli and S. typhimurium [6,19]. We found that both OmpC and OmpF are repressed as the osmolarity increases when bacteria are grown in minimal media. However, in Luria broth, expression of OmpC is slightly induced by osmolarity up to 0.3 osmM. Our results are different from those reported by Puente et al. [ 101, who suggested that S. typhi OmpC is not regulated by medium osmolarity. In their work, the electrophoretic analysis of porin expression was performed at only two different osmolarities, for this reason the authors may not have observed the osmolarity effect on OmpC expression. By using an E. coli ompC-lacZ fusion, we have demonstrated that the osmolarity which induces maximal OmpC expression in S. typhi (0.16 osmM) is consistently different from the one which induces maximal OmpC expression in E. coli (0.7 osmM). This might reflect an adaptation to the osmolarity prevailing in different environments inside the host. E. coli thrives in the intestine while S. typhi proliferates systemically. It is known that osmolarity, oxygen tension and temperature affect DNA topology [20], and that porin expression is sensitive to the level of DNA supercoiling [5,21]. Because the fusions used in our study are carried on a plasmid and the copy number of plasmids may be affected by DNA supercoiling, we have monitored expression of /acZ encoded in plasmid pCRI1 by measuring P-galactosidase activity versus medium osmolarity (0.16-I .O osmM). Our results indicate that an induction of enzyme expression occurs at osmolarities above 0.8 osmM (Fig. 3, insert). This suggests that our results are not influenced by such effects in the range of 0.06 to 0.8 osmM. As found in E. coli and S. typhimurium [S], S. typhi ompC is induced by anaerobiosis. However, in contrast to what has been described in E. coli (51, we found that OmpF expression is repressed by anaerobiosis both in E. coli and S. typhi. Finally, we have detected a different effect of temperature on the expression of ompF; when the growth temperature is shifted from 30°C to 37°C ompF-1acZ fusion is induced in S. typhi while the
Letten
133 f 19951 105% I I I
opposite effect is observed in E. coli. This is most interesting because the way porins are regulated in E. coli and S. typhimurium suggested that OmpC might be the only porin being expressed inside the host [6]. Our results suggest that this might not be the case for S. qphi. Although regulation of porins in S. gphi is similar in general terms to what has been described for E. coli, differences in the way environmental signals affect both OmpC and OmpF expression were found. The physiological significance of this differential regulation is at present not clear.
Acknowledgements We thank Drs. M. Inouye and S. Maloy for providing plasmids pKL542 and pMH622, respectively. This work was supported by grant FONDECYT 694-91 and by grants IDRC-porins and AIDProject 3-P-87-0124.
References [I] Van Alphen, W. and Lugtenberg, B. (1977) Influence of osmolarity and the growth medium on the outer membrane protein pattern of Escherichia co/i. J. Bacterial. 13 1, 623630. [2] Kawaji. H.. Mizuno, T. and Mizushima, S. (1979) Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane protein O-8 and O-9 of Escherichia co/i K-12. J. Bacterial. 140, 843-847. [3] Lundrigan, M.D. and Earhart, C.F. (1984) Gene encY of Eschrrichia cob K-12 affects thermoregulation of major porin expression. J. Bacterial. 157, 262-268. [4] Heyde. M. and Portalier, R. (1987) Regulation of major outer membrane porin proteins of Escherichia cob K12 by pH. Mol. Gen. Genet. 208, 51 l-5 17. [5] Ni Bhriain, N., Dorman, C.J. and Higgins, CF. (19891 An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression. Mol. Microbial. 3. 933-942. [6] Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial outer membrane permeability. Microbial. Rev. 49, l-32. [7] Calderbn, I., Lobes, S. and Mora, G. C. (1984) The hemolytic effect of Salmonella f&phi Ty2 porins. Eur. J. Biochem. 141, s79-583. [8] Labarca. P.. Lobos, S., Calderon, I. and Mora, G.C. (19861 Native and chemically modified porin channels from Salmonella tyhi Ty2 in planar lipid bilayers. FEBS Lett. 197. 21 I-216.
1. Contrerns
et al. / FEMS Microhiolog~
[9] Agiiero,
J., Mora, G.C., Mroczenski-Wildey. M.J.. Fernandez-Beros. M.E., Aron, L. and Cabello. F.C. (1987) Cloning, expression and characterization of the 36 KDal Salmonella typhi porin gene in E.dwrichia co/i. Microb. Pathog. 3. 399-407. [IO] Puente, J.L.. Verdugo-Rodriguez, A. and Calva. E. (1991) Expression of S&none//~~ typhi and Escherichiu co/i OmpC is influenced differently by medium osmolarity: dependence on E.schrrichic~ co/i OmpR. Mol. Microbial. S. 1205- 1210. [I I] Yanish-Perron. C.. Vieira, J. and Messing, J. (1985) Improved M 13 phage cloning vectors and host strains: nucleotide sequences of the M 13mpl8 Gene 33. 103-I 19.
and pUCI9
vectors.
[ 121 Eshoo. M.W. t 1988) Lac fusion analysis of the Eschrrid~icr
co/i: regulation
bet genes of by osmolarity, temperature, oxybetaine. J. Bacterial. 170. 5208-
gen, choline. and glycine 5215. [ 131 Tarterd, C. and Metcalf, E.S. (1993) Osmolarity and growth phase overlap in regulation of Su/monr/lrr nphi adherence to and invasion of human intestinal cells. Infect. Immun. 61, 3084-3089. [ 141 Toro. C.S., Lobos. S.R.. Calderbn, I., Rodriguez, M. and Mom, G.C. (I 990) Clinical isolate of a porin-less Snlmonrlki txphi resistant to high levels of chloramphenicol. Antimicrob. Agents Chemother. 34, 17 15- 17 19.
Letter.7 133 (IYYSI
[ 151 Lobos,
105-I
II
S.R. and Mord, G.C. (1991) Alteration
III
in the elec-
trophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electrophoresis 12. 448450. [16] Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY. [ 171 Tsui. P.. Helu, V. and Freundlich, M. (1988) Altered osmoregulation of oq~F in integration host factor mutants of Eschrricllirr co/i. J. Bacterial. 170, 4950-4953. [I81 Forst, S.. Delgado, J., Ramakrishnan. G. and Inouye, M. t 1988) Regulation of om& and ompF expression in EJdwrkhicr w/i in the absence of rnl,Z. J. Bacterial. 170, SO80-5085. 1191 Forst. S. and Inouye. M. (1988) Environmentally regulated gene expression for membrane proteins in E.cchrn’chicr co/i. Annu. Rev. Cell Biol. 4, 21-42. [20] Dorman. C.J. and Ni Bhriain. N. (1993) DNA topology and bacterial virulence gene regulation, Trends Microbial. 1. 92-98. 1211 Crdeme-Cook, K.A., May, G.. Bremer, E. and Higgins, C.F. (1989) Osmotic regulation of porin expression: a role for DNA supercoiling. Mol. Microbial. 3, 1287- 1294.
FEMS Microbiology Letters 133 ( 1995) 1OS-I I I
Heterologous expression of Escherichia coZi porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability In& Contreras b, Lorena Mutioz a, Cecilia S. Toro a, Guido C. Mora a,* J Unidad de Microbiologic,
Depurtumento
de Biologiu
Celular y Molec~ulor. Fwultud
Catcilicu de Chile, Cusilla 114-D. Suntiqo. h Departamento
de Bioquimico
y Biologicr Molecular.
Facultad Correo
de Cimcius
@imicas
1. Santiago,
Chik
de Ciencius Bioldgicus.
Pont$ciu
Uniwrsidad
Chile x FarmtrcPuticus.
Unirersidad
de Chile, Cusilla 233
Received 1 September 1995: acccepted8 September 1995
Abstract Electrophoretic analysis of outer membrane proteins showed that Sulmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherkhia co/i and S. vphimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing 15. co/i ompC-lucZ or ompF-1acZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. co/i expression was maximal at 0.7 osmM. ompF-1acZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37°C ompF-1ucZ expression was repressed in E. cob. while in S. typhi it was
induced. KeyMwds:
Salmonella
tvphi;
OmpC; OmpF; Regulation
1. Introduction The adaptation of Gram-negative bacteria to their surroundings includes changes in the composition of outer membrane proteins (OMPs). It has been reported that the relative amount of two of the most abundant OMPs of Escherichia coli and Salmonella fyphimurium, OmpF and OmpC pork, is regulated
^ Corresponding author. Fax: (562) 222 5515; E-mail: [email protected]. Federation of European Microbiological Societies SSDl 0378.1097(95)00345-2
by environmental conditions such as osmolarity, temperature, pH and oxygen availability [l-.5]. The reciprocal regulation of porins has been proposed to be an adaptive response of these bacteria to different habitats. In an external environment with poor nutrient contents and low temperature, the larger pore sized OmpF is preferentially expressed. the contrast, expression of the slightly smaller pore sized OmpC is favored when the bacteria colonize a nutrient-rich milieu, such as the intestine of the animal host, where conditions of high salinity, high temperature and low oxygen availability are encountered [6].
The human pathogen Salmonellu @phi produces two porin species with apparent molecular masses of 36 and 35 kDa, similar to S. typhimurium OmpC and OmpF porins, but it does not synthesize OmpD [7-91. In contrast to the situation in E. coli and S. t?,phimurium, relatively little is known about regulation of S. typhi porin expression by environmental conditions. Electrophoretic analysis suggested that OmpC expression is not regulated by medium osmolarity in aerobically grown bacteria [IO]. Concerning OmpF regulation, no studies have been reported. In the present work, we have used electrophoretic analysis and gene fusion technology to study porin regulation by medium osmolarity, temperature and oxygen availability in S. typhi.
2. Materials 2.1. Bacterial conditions
and methods strains, plasmids,
media and growth
Salmonella @phi Ty2 was obtained from the Institute of Public Health, Chile; E. coli JM 109 [ 111 and DHScu were donated by A. Venegas. Plasmid pKL542 (Amp’) containing E. coli ompC-IacZ cloned in pMLB524 was made available by M. Inouye; plasmid pMH622 (Amp’) containing E. coli ompF-1acZ cloned in pMLB524 was obtained from S. Maloy; plasmid pCR II (Invitrogen Corp.) containing the 1acZ gene and its promoter was provided by R. Vicuiia. All strains were routinely grown in Luria broth (1 .O% tryptone, 0.5% yeast extract, 0.5% sodium chloride). Medium 63 [ 121, supplemented with glucose 0.2%, tryptophan 25 mg 1-l and cysteine 25 mg II’ (M63-S), was used as the defined medium. When needed, media were supplemented with ampicillin 40 pug ml-’ . Media of differing osmolarity were prepared with M63-S diluted or supplemented with increasing amounts of NaCl. 0.06 and 0.16 osmM were obtained by 1:5 and I:2 dilution of M63-S, respectively. Media of 0.3, 0.5, 0.7, 0.9 and 1.l osmM were obtained by supplementing M63-S with 0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl, respectively. Media at all osmolarities contained glucose 0.210, cysteine 25 mg 1-l and tryptophan 25 mg 1-i. The generation time varied between 2.0 and 2.2 h in these media. Luria broth was also prepared at
differing osmolarities: Luria broth containing 0.0 1, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 M NaCl was 0.16, 0.23, 0.30, 0.33, 0.51, 0.68, 0.87, I.1 and 2.0 osmM, respectively. In some experiments, the osmolarity was shifted from 0.7 osmM to 0.16 osmM. To this end, bacteria were grown in 0.7 osmM M63-S for 60 min. centrifuged, washed and resuspended in the 0.16 osmM medium. Osmolarities were determined as described [ 131 or using an Advanced Osmometer Model 3W, Advanced Instruments. Aerobic growth was achieved by vigorous shaking of IO-ml cultures in 100-m] flasks plugged with cotton. Anaerobic cultures were grown without shaking in filled test tubes with the medium overlaid with mineral oil. Cultures were started with 0. I ml of an overnight inoculum grown in M63-S at 37°C. Bacteria were grown in media of different osmolarities at 30°C or 37°C aerobically or anaerobically, up to the mid-exponential phase. Subsequently, outer membrane fractions were obtained or ,@galactosidase assays were performed. 2.2. Outer membrane PAGE
protein preparation
and SDS-
The OMPs fraction was obtained as described [ 141. SDS-PAGE was performed in 12.5% polyacrylamide slabs [ 151. All samples were heated at 100°C in the sample buffer before electrophoresis. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpF and OmpC bands were obtained at 575 nm from ACD- IS Automatic Computing Densitometer, Gelman Sciences Inc. 2.3. @Galactosidase
assays
P-Galactosidase activity was determined from exponentially grown bacteria (ODhOOnm= 0.2) using the SDS-chloroform permeabilization method [161.
3. Results 3.1. Porin regulation in S. ephi
Ty2
The OMP profile of cells grown in media of different osmolarity was analysed. The amount of OmpC or OmpF was determined by densitometric
1. Contrems
et al. / FEMS Microhiolog~
Letters 133 (IYY5)
analysis of Coomassie brilliant blue-stained gels relative to the absorbance of OmpA, which is considered to be constitutively expressed [ 10,17,18]. This was done to reduce fluctuations in the levels of porins due to differences in the amount of sample loaded on the gel. Under aerobic growth conditions at 37°C in Luria broth, expression of OmpC increased with medium osmolarity up to 0.3 osmM and then remained constant up to 1.O osmM. At osmolarities higher than 1.0 osmM, OmpC expression was repressed (Fig. IA). Under anaerobiosis, maximal OmpC expression was observed at 0.3 osmM, being repressed at osmolarities higher than 0.3 osmM (Fig.
0.0
0.4
c,
26
2 0
2.0 15
:
2 2
0.5
0.4
g
0.3
3 1 al
0.2
u
0.1
1.0
ii p:
0.6
0.6
1.0
(0smM)
0.6 30
2
0.2
107
II
Osmolarity
i ,~i _i
E d
’
105-I
1
0.2
0.4
,
0.6
06
1.0
0.5 0.0
L
0.0
Osmolarity
-\;: 0.2
0.4
0.6
0.6
1.0
Osmolarity
1.2
1.4
1.6
1.6
2.0
B
0.0
0.2
0.4
0.6
0.6
Osmolarity Omp
F
1.0
, ,j 1.2
1.4
1.6
1.6
2.0
(0smM) 0
Omp
F
(0smM) 0
Omp
c
Fig. 2. Electrophoretic analysis of S. @phi Ty2 OMPs. Proteins were obtained from bacteria grown at 37°C aerobically (A) or anaerobically (B) in minimal media M63-S of differing osmolarities. Samples were run in 12.5% acrylamide gels. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpC and OmpF bands were obtained. The amount of OmpF or OmpC present in the outer membrane is expressed relative to OmpA. This is a representative example of the analysis performed on several occasions.
(0smM)
$.‘------.._,
9
Ornp c
Fig. I. Electrophoretic analysis of S. @phi Ty2 OMPs. Proteins were obtained from bacteria grown at 37°C aerobically (A) or anaerobically (B) in Luria broth of differing osmolarities. Samples were run in 12.5% acrylamide gels. Densitometric measurements of the Coomassie brilliant blue-stained OmpA, OmpC and OmpF bands were obtained. The amount of OmpF or OmpC present in the outer membrane is expressed relative to OmpA. This is a representative example of the analysis performed on several occasions.
lB>. On the other hand, the amount of OmpF decreased as the osmolarity increased. OmpF was further repressed in anaerobically grown bacteria. When bacteria were grown in M63-S media under both aerobic and anaerobic conditions, the level of OmpC was reduced in a steady manner as the osmolarity increased, while OmpF expression was repressed (Fig. 2A, B). 3.2. Regulation
of E. coli ompC expression
in S.
typhi
S. ephi Ty2 was transformed with plasmid pKL542 which contains E. coli ompC-lacZ and
108
I. Conmxzs
ef al. /FEMS
Microbiology
Letters 133 (1995) 105-l I I
same results were obtained, regardless growth temperature was 30°C or 37°C. 3.3. Regulation typhi
0’
0.2
0.4
0.6
Osmolarity I3
-
1.2
(0smM) 100
r
E
200
b ._ .?, z
150
1
ll;Iz,,_,,,?+
/’
r
80
A
_’
2
I
5
60
s s
40
3 7ld
20
CL:
0: 0
I
0.0
0.2
0.4
0.6
0.6
Osmolarity
lacZ
/
I
10
1.2
1
0.0
0.2
-
and oxygen
Anaeroblosls
availability
0.4
0.6
Osmolarity
(osmbi)
Aeroblosls
Fig. 3. Effect of osmolarity or&-
1.0
in S.
4
250
2
0.6
of E. coli ompF expression
the
S. typhi Ty2/pMH622 bearing the E. co/i ompF-1acZ fusion was grown at 37°C at osmolarities ranging from 0.03 to 1.1 osmM. P-Galactosidase activity was reduced as the osmolarity increased. Under anaerobic conditions, expression of ompF
1
0.0
whether
0.6
1.0
1.2
(0smM)
w
on E. coli
expression. P-Galactosidase was measured in .S.
100
-
typhi (A) and E. coli (B), each harboring
pKL542, which contains E. co/i on@lacZ gene fusion. Bacteria were grown aerobically or anaerobically, in minimal glucose media (M63-S) of differing osmolarities. P-Galactosidase activities are expressed relative to the activity (lOO’%o) measured at 1.1 osmM under aerobiosis at 37°C. Each assay was run in triplicate on four occasions. Values are means * standard deviation.
expression of this gene fusion was assayed in media of varying osmolarities at 37°C. As shown in Fig. 3A, P-galactosidase activity was maximal at 0.16 osmM under anaerobiosis and at 0.3 osmM under aerobiosis. At higher osmolarities, P-galactosidase was repressed. Endogenous OmpC was regulated in S. ryphi Ty2/pKL542 as it is in S. typhi Ty2 (results not shown). In E. coli JM109/pKL542 (Fig. 3B) grown anaerobically, maximal P-galactosidase expression was observed at 0.7 osmM, while in aerobiosis enzyme activity continuously increased throughout the range of osmolarities tested. The
01 0.0
1
0.2
0.4
0.6
Osmolarity -
Aeroblosls
0.6
1.0
1.2
(0smM) -
Anaeroblosls
Fig. 4. Effect of osmolarity and oxygen availability on E. coli ompF- lacZ expression. P-Galactosidase was measured in S. @phi (A) and E. coli (B), each harboring pMH622, which contains E. co/i ompF-lacZ gene fusion. Bacteria were grown aerobically or anaerobically, in minimal glucose media (M63-S) of differing osmolarities. P-Galactosidase activities are expressed relative to the activity (100‘7~) measured at 0.03 osmM under aerobiosis at 37°C. Each assay was run in triplicate on four occasions. Values are means + standard deviation.
109
was significantly lower than under aerobiosis at all osmolarities (Fig. 4A). Similar results were obtained in E. colt’ JM109/pMH622 (Fig. 4B). However, when these assays were performed at 30°C no repression of ompF by anaerobiosis occurred in S. ~phi/pMH622: P-galactosidase activities were similar to those in aerobiosis (data not shown). This was confirmed by experiments in which bacteria were grown at 30°C under aerobic or anaerobic conditions in 0.7 osmM medium and then shifted to 0.16 osmM.
3000 -
h
z 5 2500 0 cd 2000 ti -fj 1500 ._ : c, 1000 ii s M
500 -
I
Q
'130
0
30
60
Time
h 2000
5
-
s0
$ $ :
1600 -
;
1200
: -z ._ :: cl 0
;;ii_ry
2 6
-2000 I-I
I
1 120
I 150
I 160
(min)
-30
0
30
60
Time
90
120
150
160
(min)
-6000
:
2000
2
:"::ii
% 2
600
s ; M
400
‘-;-
-10000 , 0
I 30
1 60
Time
I 90
(min)
Fig. 6. Expression of onlpFP /acZ during a temperature shift from 30°C to 37°C. Bacteria were grown aerobically at 30°C during 60 min in M63-S medium, then shifted to 37°C. /3-Galactosidase was measured in S. npl?i (A) and E. co/i (B), each harboring pMH622. which contains E. co/i onyF- /wZ gene fusion. P-Galactosidase activities represent the difference between the activity measured at 30°C and the one measured at 37°C. Values are means k standard deviation.
B
;
\
-6000
-30
L
160
-4000
M ' -12000 .~ Qz
h
150
0
6
4 .d [
120
B
\
L ? ,._
90
ir;--‘-
~~~_~-~=--~~~..__~*~~..~ v
I:,,,,,, 0 -30 0
30
60
Time -
Aeroblosls
90
120
150
160
(min) -~~ Anaeroblcms
Fig. 5. Expression of ompF-/rcZ during a shift of osmolarity from 0.7 osmM to 0.16 osmM. Bacteria were grown aerobically or anaerobically at 30°C during 60 min in 0.7 osmM M63-S medium, then shifted to 0.16 osmM. P-Galactosidase was measured in S. fxphi (A) and E. di (B). each harboring pMH622, which contains E. co/i ompF- /acZ gene fusion. P-Galactosidase activities represent the difference between the activity measured at 0.7 osmM and the one measured at 0.16 osmM. Values are means, standard deviation.
In S. l[vphi/pMH622, induction of ompF was identical under both conditions of oxygen availability (Fig. SA). In contrast, in E. coli/pMH622 induction of otnpF was significantly increased under aerobic conditions (Fig. 5B). To further investigate the temperature regulation of OmpF. bacteria were grown at 30°C for 60 min, then the temperature was raised to 37°C. Remarkably, in S. @phi/pMH622 (Fig. 6A) P-galactosidase activity was induced during the temperature shift. As expected, in E. coli JMl09/pMH622, the opposite effect was observed (Fig. 6B).
I10
I. Contrerus
et (11./ FEMS Microbiology
4. Discussion In our study, electrophoretic analysis of major OMPs indicates that S. ~phi OmpC expression is not reciprocally regulated relative to OmpF as described for E. coli and S. typhimurium [6,19]. We found that both OmpC and OmpF are repressed as the osmolarity increases when bacteria are grown in minimal media. However, in Luria broth, expression of OmpC is slightly induced by osmolarity up to 0.3 osmM. Our results are different from those reported by Puente et al. [ 101, who suggested that S. typhi OmpC is not regulated by medium osmolarity. In their work, the electrophoretic analysis of porin expression was performed at only two different osmolarities, for this reason the authors may not have observed the osmolarity effect on OmpC expression. By using an E. coli ompC-lacZ fusion, we have demonstrated that the osmolarity which induces maximal OmpC expression in S. typhi (0.16 osmM) is consistently different from the one which induces maximal OmpC expression in E. coli (0.7 osmM). This might reflect an adaptation to the osmolarity prevailing in different environments inside the host. E. coli thrives in the intestine while S. typhi proliferates systemically. It is known that osmolarity, oxygen tension and temperature affect DNA topology [20], and that porin expression is sensitive to the level of DNA supercoiling [5,21]. Because the fusions used in our study are carried on a plasmid and the copy number of plasmids may be affected by DNA supercoiling, we have monitored expression of /acZ encoded in plasmid pCRI1 by measuring P-galactosidase activity versus medium osmolarity (0.16-I .O osmM). Our results indicate that an induction of enzyme expression occurs at osmolarities above 0.8 osmM (Fig. 3, insert). This suggests that our results are not influenced by such effects in the range of 0.06 to 0.8 osmM. As found in E. coli and S. typhimurium [S], S. typhi ompC is induced by anaerobiosis. However, in contrast to what has been described in E. coli (51, we found that OmpF expression is repressed by anaerobiosis both in E. coli and S. typhi. Finally, we have detected a different effect of temperature on the expression of ompF; when the growth temperature is shifted from 30°C to 37°C ompF-1acZ fusion is induced in S. typhi while the
Letten
133 f 19951 105% I I I
opposite effect is observed in E. coli. This is most interesting because the way porins are regulated in E. coli and S. typhimurium suggested that OmpC might be the only porin being expressed inside the host [6]. Our results suggest that this might not be the case for S. qphi. Although regulation of porins in S. gphi is similar in general terms to what has been described for E. coli, differences in the way environmental signals affect both OmpC and OmpF expression were found. The physiological significance of this differential regulation is at present not clear.
Acknowledgements We thank Drs. M. Inouye and S. Maloy for providing plasmids pKL542 and pMH622, respectively. This work was supported by grant FONDECYT 694-91 and by grants IDRC-porins and AIDProject 3-P-87-0124.
References [I] Van Alphen, W. and Lugtenberg, B. (1977) Influence of osmolarity and the growth medium on the outer membrane protein pattern of Escherichia co/i. J. Bacterial. 13 1, 623630. [2] Kawaji. H.. Mizuno, T. and Mizushima, S. (1979) Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane protein O-8 and O-9 of Escherichia co/i K-12. J. Bacterial. 140, 843-847. [3] Lundrigan, M.D. and Earhart, C.F. (1984) Gene encY of Eschrrichia cob K-12 affects thermoregulation of major porin expression. J. Bacterial. 157, 262-268. [4] Heyde. M. and Portalier, R. (1987) Regulation of major outer membrane porin proteins of Escherichia cob K12 by pH. Mol. Gen. Genet. 208, 51 l-5 17. [5] Ni Bhriain, N., Dorman, C.J. and Higgins, CF. (19891 An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression. Mol. Microbial. 3. 933-942. [6] Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial outer membrane permeability. Microbial. Rev. 49, l-32. [7] Calderbn, I., Lobes, S. and Mora, G. C. (1984) The hemolytic effect of Salmonella f&phi Ty2 porins. Eur. J. Biochem. 141, s79-583. [8] Labarca. P.. Lobos, S., Calderon, I. and Mora, G.C. (19861 Native and chemically modified porin channels from Salmonella tyhi Ty2 in planar lipid bilayers. FEBS Lett. 197. 21 I-216.
1. Contrerns
et al. / FEMS Microhiolog~
[9] Agiiero,
J., Mora, G.C., Mroczenski-Wildey. M.J.. Fernandez-Beros. M.E., Aron, L. and Cabello. F.C. (1987) Cloning, expression and characterization of the 36 KDal Salmonella typhi porin gene in E.dwrichia co/i. Microb. Pathog. 3. 399-407. [IO] Puente, J.L.. Verdugo-Rodriguez, A. and Calva. E. (1991) Expression of S&none//~~ typhi and Escherichiu co/i OmpC is influenced differently by medium osmolarity: dependence on E.schrrichic~ co/i OmpR. Mol. Microbial. S. 1205- 1210. [I I] Yanish-Perron. C.. Vieira, J. and Messing, J. (1985) Improved M 13 phage cloning vectors and host strains: nucleotide sequences of the M 13mpl8 Gene 33. 103-I 19.
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[ 121 Eshoo. M.W. t 1988) Lac fusion analysis of the Eschrrid~icr
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gen, choline. and glycine 5215. [ 131 Tarterd, C. and Metcalf, E.S. (1993) Osmolarity and growth phase overlap in regulation of Su/monr/lrr nphi adherence to and invasion of human intestinal cells. Infect. Immun. 61, 3084-3089. [ 141 Toro. C.S., Lobos. S.R.. Calderbn, I., Rodriguez, M. and Mom, G.C. (I 990) Clinical isolate of a porin-less Snlmonrlki txphi resistant to high levels of chloramphenicol. Antimicrob. Agents Chemother. 34, 17 15- 17 19.
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[ 151 Lobos,
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S.R. and Mord, G.C. (1991) Alteration
III
in the elec-
trophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electrophoresis 12. 448450. [16] Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY. [ 171 Tsui. P.. Helu, V. and Freundlich, M. (1988) Altered osmoregulation of oq~F in integration host factor mutants of Eschrricllirr co/i. J. Bacterial. 170, 4950-4953. [I81 Forst, S.. Delgado, J., Ramakrishnan. G. and Inouye, M. t 1988) Regulation of om& and ompF expression in EJdwrkhicr w/i in the absence of rnl,Z. J. Bacterial. 170, SO80-5085. 1191 Forst. S. and Inouye. M. (1988) Environmentally regulated gene expression for membrane proteins in E.cchrn’chicr co/i. Annu. Rev. Cell Biol. 4, 21-42. [20] Dorman. C.J. and Ni Bhriain. N. (1993) DNA topology and bacterial virulence gene regulation, Trends Microbial. 1. 92-98. 1211 Crdeme-Cook, K.A., May, G.. Bremer, E. and Higgins, C.F. (1989) Osmotic regulation of porin expression: a role for DNA supercoiling. Mol. Microbial. 3, 1287- 1294.