Eur. J. Biochem. 209, 549-553 (1992) 0FEBS 1992
Effect of heat shock on expression of proteins not involved in the heat-shock regulon Anna FABISIEWICZ and Celina JANION Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Receivcd April 30, 1992) - EJB 92 0603
The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lac2 and their expression measured by the level of P-galactosidase. The results show that expression of umuC, recA andpolB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of adu, alkB and ulkA genes, after indqction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heatshocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins.
Exposure of cells to an abrupt increase of temperature leads to a transient dramatic increase in synthesis of the heatshock proteins. In Escherichia coli approximately 20 proteins belong to the heat-shock regulon, expression of their genes is positively regulated by the c~~~ factor. The quantity of heatshock proteins in the heat-shocked cells can increase from 2% up to 20% of the total cell protein [l, 21. In this study we attempted to estimate whether heat shock has any effect on gene expression, and synthesis of proteins, not involved in the heat-shock regulon. Among the genes tested were umuC, recA, polB ( d i d ) , belonging to the SOS regulon; ada, alkB and ulkA, belonging to the adaptive response and, lacZ of the lac operon. Gene expression, in each case, was monitored by the level of p-galactosidase transcribed from its own promoter or from the promoters in question fused to 1ucZ. MATERIALS AND METHODS Bacterial strains Bacterial strains and plasmids used are listed in Table 1. The majority of the bacterial strains were transformed with plasmid pSK1002 [ll], bearing umuC promoter fused to a lacZ devoid of its own promoter, but some were transformed with plasmid pDS2, bearing the rpoH gene controlled by the tac promoter, or plasmid pKK223-3, parental to pDS2 but without the rpoH gene [12]. All plasmids bear the ampicillin resistance (Ap) gene. Transformations were performed according to Maniatis [13]. Transformants were selected for ampicillin resistance and presence of the plasmid was checked by their phenotype (lacZ expression on induction of the SOS response), or by plasmid isolation. The transformants are indicated by the conjunction of the name of the bacterial strain and the name of the plasmid, for example AB1157/pSK1002 is the AB1157 strain bearing the plasmid pSK1002. The EC2406 - EC2410 strains were constructed by transformation Correspondence to C. Janion, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowiecka 36, PL-02-532 Warszawa, Poland Abbreviations. MC, mitomycin C ; MMNG, N-methyl-N-nitro-Nnitrosoguanidine; MMS, methyl methanesulfonate; IPTG, isopropylthio-P-u-galactopyranoside; LB, Luria-Bertani broth.
of the appropriate bacteria with Plvir [5], propagated in KB1717 (donor for recA: :lacZ, Ap; [14]), selected for ampicillin resistance and purified from P1 by subsequent replating of the transformants onto Ap-plates. Media The growth media were Luria-Bertani broth (LB), LB/ thymine (10 pg/ml) and A medium [5], enriched with 0.4% glucose, 0.4% casamino acids, 2 pg/ml thiamine and, when necessary, with 20 pg/ml thymine, 50 pg/ml ampicillin or 15 pg/ml tetracycline. Plates were solidified with Difco Bacto Agar (1.2%). Gene-expression assay Overnight cultures of bacteria in LB/thymine and ampicillin, when necessary, were diluted 1: 50 in enriched A medium and incubated to reach A600 0.3-0.6. The culture was then divided into 3-ml portions for the control and the tests and was treated as shown in Results. Inducers for gene expressions were mitomycin C (MC) (0.5 pg/ml) for promoters involved in the SOS regulon; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (1 pg/ml) and methyl methanesulfonate (MMS, 3 mM) with promoters involved in the adaptive response, and isopropyl-thio-p-D-galactopyranoside(IPTG, 1 mM) for lac2 expression of the lac operon. Times of incubation with the inducer were 60-90, 120 or 45 min, respectively. Then the aliquot was withdrawn and p-galactosidase activity assayed according to [5]. Heat shock was performed by immersion of the test tubes in a water bath and incubation for 10 min at 42°C for bacteria grown at 30°C or for 6 min at 50°C for bacteria grown at 37 "C. RESULTS Synthesis of P-galactosidase from umuC::lacZ fusion Heat-shocked, or non-shocked, cells bearing the pSK1002 plasmid were treated with MC and the level of 8-galactosidase in response to umuC derepression was measured (Table 2).
5 50 Table 1. E. coli K-12 bacterial strains and plasmids. Strain/plasmid
Genotype/fusion and phenotype
AB1157
thr-1 ara-14 leuB6 A(gpt-proA)62 lacy1 ts-33 supE44 galK2 hisG4 rfbDl mgl-51 rpsL31 kdgK5l xyl-5 mtl-1 argE3 thi-1 wt F galE TetR as CG2245 but groEL as CG2245 but groES ara A(lac-pro) thi trp(Am) supF(Ts) rpoH(Am) rpsL A(gpt-lac)S as CSH26 but with fusion ada' : :lacZ on plasmid as CSH26 but with fusion alkA' : :lacZ on plasmid as ABI 157 but rfa550 alkA51: :Mu-dl(ApR,lac) as AB1157 but rfa550 alkA.71: :pSGl as AB1157 but rfa5.50 alkB52: :Mu-dl(ApR,lac) lac (U169) recA441 s f A l 1 dinAl : :Mud as ABI 157 but Id(recA : :lacZ, ApR) as CG2245 but Ld(recA : :IacZ, ApR) as CAG1951 but Ld(recA: :lacZ, ApR) as CG2241 but Id(recA: :IacZ, ApR) as CG2244 but Id(recA : :lacZ, ApR) umuC : :IacZ, ApR ptac: :rpoH, ApR without fusion, parental to pDS2
CG2245 CG2241 CG2244 CSH26 CAG1951 CSH26/ pYM3 CSH26/ pMCPlOOO MV1571 MV1902 MV1601 GWlOlO EC2406 EC2407 EC2408 EC2409 EC2410 pSK1002 pDS2 pKK223-3
Source or reference 3 4 C. Georgopoulos C . Georgopoulos C. A. Gross, 5 6
-f
8 9 9 9 10 A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion 11 12 P harmacia
Table 2. Effect of beat shock on B-galactosidase expression from urnuDC::lacZfusion. AB1157/pSK1002 were grown at 37°C and heat shocked (HS) at 50°C. The remaining bacteria were grown at 30°C and heat shocked at 42°C. Time of incubation with MC (0.5 pg/ml) at 37°C or 3 0 T , respectively, was 90 min. Heat shock to control sample has no influence on level of P-galactosidase. Values for control were subtracted from results with MC, HS and MC. Strain
Genotype
n
P-galactosidase expression in control
+ MC
HS
697 f 528 591 f 235 741 258 782 k 330 970 & 416 804 & 216
1050 f 746 1019 & 406 1002 f 228 776 2x7 996 f 190 699 f 156
+ MC
+
HS MC/+ MC
U AB1157/pSK1002 CG2245/pSK 1002 CSH26/pSK1002 CG2244lpSKI 002 CG2241/pSK1002 CAGl951/pSKlOO2
Hsp' Hsp' Hsp' groES groEL rpoH
6 3 2 3 2 5
110 41 200 & 78 91 f 21 231 22 293 1 68 10
+
There is a low but distinct (
Effect of heat shock on expression of proteins not involved in the heat-shock regulon Anna FABISIEWICZ and Celina JANION Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Receivcd April 30, 1992) - EJB 92 0603
The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lac2 and their expression measured by the level of P-galactosidase. The results show that expression of umuC, recA andpolB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of adu, alkB and ulkA genes, after indqction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heatshocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins.
Exposure of cells to an abrupt increase of temperature leads to a transient dramatic increase in synthesis of the heatshock proteins. In Escherichia coli approximately 20 proteins belong to the heat-shock regulon, expression of their genes is positively regulated by the c~~~ factor. The quantity of heatshock proteins in the heat-shocked cells can increase from 2% up to 20% of the total cell protein [l, 21. In this study we attempted to estimate whether heat shock has any effect on gene expression, and synthesis of proteins, not involved in the heat-shock regulon. Among the genes tested were umuC, recA, polB ( d i d ) , belonging to the SOS regulon; ada, alkB and ulkA, belonging to the adaptive response and, lacZ of the lac operon. Gene expression, in each case, was monitored by the level of p-galactosidase transcribed from its own promoter or from the promoters in question fused to 1ucZ. MATERIALS AND METHODS Bacterial strains Bacterial strains and plasmids used are listed in Table 1. The majority of the bacterial strains were transformed with plasmid pSK1002 [ll], bearing umuC promoter fused to a lacZ devoid of its own promoter, but some were transformed with plasmid pDS2, bearing the rpoH gene controlled by the tac promoter, or plasmid pKK223-3, parental to pDS2 but without the rpoH gene [12]. All plasmids bear the ampicillin resistance (Ap) gene. Transformations were performed according to Maniatis [13]. Transformants were selected for ampicillin resistance and presence of the plasmid was checked by their phenotype (lacZ expression on induction of the SOS response), or by plasmid isolation. The transformants are indicated by the conjunction of the name of the bacterial strain and the name of the plasmid, for example AB1157/pSK1002 is the AB1157 strain bearing the plasmid pSK1002. The EC2406 - EC2410 strains were constructed by transformation Correspondence to C. Janion, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowiecka 36, PL-02-532 Warszawa, Poland Abbreviations. MC, mitomycin C ; MMNG, N-methyl-N-nitro-Nnitrosoguanidine; MMS, methyl methanesulfonate; IPTG, isopropylthio-P-u-galactopyranoside; LB, Luria-Bertani broth.
of the appropriate bacteria with Plvir [5], propagated in KB1717 (donor for recA: :lacZ, Ap; [14]), selected for ampicillin resistance and purified from P1 by subsequent replating of the transformants onto Ap-plates. Media The growth media were Luria-Bertani broth (LB), LB/ thymine (10 pg/ml) and A medium [5], enriched with 0.4% glucose, 0.4% casamino acids, 2 pg/ml thiamine and, when necessary, with 20 pg/ml thymine, 50 pg/ml ampicillin or 15 pg/ml tetracycline. Plates were solidified with Difco Bacto Agar (1.2%). Gene-expression assay Overnight cultures of bacteria in LB/thymine and ampicillin, when necessary, were diluted 1: 50 in enriched A medium and incubated to reach A600 0.3-0.6. The culture was then divided into 3-ml portions for the control and the tests and was treated as shown in Results. Inducers for gene expressions were mitomycin C (MC) (0.5 pg/ml) for promoters involved in the SOS regulon; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (1 pg/ml) and methyl methanesulfonate (MMS, 3 mM) with promoters involved in the adaptive response, and isopropyl-thio-p-D-galactopyranoside(IPTG, 1 mM) for lac2 expression of the lac operon. Times of incubation with the inducer were 60-90, 120 or 45 min, respectively. Then the aliquot was withdrawn and p-galactosidase activity assayed according to [5]. Heat shock was performed by immersion of the test tubes in a water bath and incubation for 10 min at 42°C for bacteria grown at 30°C or for 6 min at 50°C for bacteria grown at 37 "C. RESULTS Synthesis of P-galactosidase from umuC::lacZ fusion Heat-shocked, or non-shocked, cells bearing the pSK1002 plasmid were treated with MC and the level of 8-galactosidase in response to umuC derepression was measured (Table 2).
5 50 Table 1. E. coli K-12 bacterial strains and plasmids. Strain/plasmid
Genotype/fusion and phenotype
AB1157
thr-1 ara-14 leuB6 A(gpt-proA)62 lacy1 ts-33 supE44 galK2 hisG4 rfbDl mgl-51 rpsL31 kdgK5l xyl-5 mtl-1 argE3 thi-1 wt F galE TetR as CG2245 but groEL as CG2245 but groES ara A(lac-pro) thi trp(Am) supF(Ts) rpoH(Am) rpsL A(gpt-lac)S as CSH26 but with fusion ada' : :lacZ on plasmid as CSH26 but with fusion alkA' : :lacZ on plasmid as ABI 157 but rfa550 alkA51: :Mu-dl(ApR,lac) as AB1157 but rfa550 alkA.71: :pSGl as AB1157 but rfa5.50 alkB52: :Mu-dl(ApR,lac) lac (U169) recA441 s f A l 1 dinAl : :Mud as ABI 157 but Id(recA : :lacZ, ApR) as CG2245 but Ld(recA : :IacZ, ApR) as CAG1951 but Ld(recA: :lacZ, ApR) as CG2241 but Id(recA: :IacZ, ApR) as CG2244 but Id(recA : :lacZ, ApR) umuC : :IacZ, ApR ptac: :rpoH, ApR without fusion, parental to pDS2
CG2245 CG2241 CG2244 CSH26 CAG1951 CSH26/ pYM3 CSH26/ pMCPlOOO MV1571 MV1902 MV1601 GWlOlO EC2406 EC2407 EC2408 EC2409 EC2410 pSK1002 pDS2 pKK223-3
Source or reference 3 4 C. Georgopoulos C . Georgopoulos C. A. Gross, 5 6
-f
8 9 9 9 10 A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion A. Fabisiewicz and C. Janion 11 12 P harmacia
Table 2. Effect of beat shock on B-galactosidase expression from urnuDC::lacZfusion. AB1157/pSK1002 were grown at 37°C and heat shocked (HS) at 50°C. The remaining bacteria were grown at 30°C and heat shocked at 42°C. Time of incubation with MC (0.5 pg/ml) at 37°C or 3 0 T , respectively, was 90 min. Heat shock to control sample has no influence on level of P-galactosidase. Values for control were subtracted from results with MC, HS and MC. Strain
Genotype
n
P-galactosidase expression in control
+ MC
HS
697 f 528 591 f 235 741 258 782 k 330 970 & 416 804 & 216
1050 f 746 1019 & 406 1002 f 228 776 2x7 996 f 190 699 f 156
+ MC
+
HS MC/+ MC
U AB1157/pSK1002 CG2245/pSK 1002 CSH26/pSK1002 CG2244lpSKI 002 CG2241/pSK1002 CAGl951/pSKlOO2
Hsp' Hsp' Hsp' groES groEL rpoH
6 3 2 3 2 5
110 41 200 & 78 91 f 21 231 22 293 1 68 10
+
There is a low but distinct (
