Antonie van Leeuwenhoek 61: 339--342, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Short communication
Bacteriophage O105clz induces the GroEL-homologue protein in Bacillus subtilis Robin R. Staples ~, Brian S. Miller & Uldis N. Streips*
Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, Kentucky 40292, USA; (;present address: Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA; * requests for offprints) Received 9 August 1991; accepted 18 November 1991
Key words: Bacillus subtilis, GroEL, heat shock, phage induction Abstract
Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue of Bacillus subtilis was shown to be induced upon infection with 0105clz, a clear plaque mutant of the temperate bacteriophage O105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homoiogue when cells were infected with 0105clz.
Introduction
In Escherichia coli there are at least 17 sigma-32 dependent heat shock proteins (HSPs) induced, when cells are subjected to a sudden increase in temperature (Neidhardt & VanBogelen 1987). Several of these HSPs, especially GroEL and DnaK, are also induced in response to infection by bacteriophage, especially bacteriophage lambda (Drahos & Hendrix 1982; Kochan & Murialdo 1982). These proteins have been shown to be necessary in the morphogenesis of lambda production (Friedman et al. 1984) and in replication of the bacteriophage DNA (Zylicz et al. 1989). Mutations in dnaK and groEL render the cells resistant to lambda infection (Friedman et al. 1984; Neidhardt & VanBogelen 1987). Other bacteriophage such as the filamentous phage, fl, and OX174 have also been shown to utilize these host encoded proteins to aid in the life cycle of the bacteriophage (Brissette et al. 1990; Young et al. 1989). Recently, a sigma-32 independent, filamentous phage and
stress induced operon has been described in E. coli (Brissette et al. 1991). In Bacillus subtilis, the GroEL and DnaK homologues have been identified and are components of the bacillus heat shock response (Arnosti et al. 1986; Miller et al. 1991). Carrascosa et al. (1982) described a B. subtilis GroEL homologue which was found to copurify with a bacteriophage 029 protein involved in phage capsid formation. This finding suggests that B. subtilis bacteriophage may also utilize host encoded proteins, in particular some HSPs, for replication and assembly. We wanted to determine if bacteriophage infection in B. subtilis, as shown in E. coli, results in the induction of heat shock proteins. In the present study, we define the induction of the GroEL homologue of B. subtilis following infection with a clear plaquing mutant of the temperate bacteriophage O105. Bacillus subtilis strain YB886 (metB5, trpC2, xin-1, spl3~) was grown at 37~ to a Klett Unit reading of 55 (mid-exponential) (monitored on a
340
(a)
(b)
(c)
97---66--"
Fig. 1. Two-dimensional gel electrophoresis of B. subtilis YB886. (a) 37~ control. (b) 48~ heat shock (10 min). (c) Cells grown at 37~C and infected with 0105clz for 10 min. Spots 8 and 9 are the DnaK and GroEL homologues respectively. The values on the left side are Mrs x 10 3 of standard proteins. Equal amounts of TCA precipitable counts were loaded on each gel.
Klett-Summerson Colorimeter) in Spizizen's minimal medium (Spizizen 1958) containing 40 tzg/ml (final concentration) tryptophane and methionine. At this time, cells were harvested and suspended in minimal medium without methionine. The cells were allowed to equilibrate at 37~C for 5 min prior to either a shift to 48~ or the addition of bacteriophage. Samples (1 ml) of cells were incubated at 37~ (control), 48 ~C, or 37~ with O105clz at a multiplicity of infection of 1 (as determined by plaquing efficiency). Samples for two-dimensional polyacrylamide gel electrophoresis were labeled after 10min with 80~Ci of 35S_methionine (Dupont-New England Nuclear, Wilmington DE) (specific activity, ll00Ci/mmole) for 5min. The cells were then lysed and two-dimensional electrophoresis was performed as described by Miller et al. (1991). Samples for Western blot analysis were incubated for 10, 20, 30, and 40 min, harvested, and then subjected to one-dimensional polyacrylamide gel electrophoresis as described (Streips & Polio 1985). Western blots were developed using a Immunoblot Assay Kit (Bio-Rad, Rockville Centre, NY) with goat anti-rabbit alkaline phosphatase conjugates. To detect whether the GroEL or DnaK homologues of B. subtilis were induced in response to bacteriophage infection, the two dimensional gel electrophoresis system was utilized. Figure la illustrates the relevant portion of the protein pattern
present in B. subtilis strain YB886 during steady state growth at 37~C. Polypeptides designated as 8 and 9 have been previously identified as the DnaK (HSP8) and GroEL (HSP9) homologues respectively (Miller et al. 1991). The induction of these proteins during heat shock for 10min at 48~ is shown in Fig. lb. When cells were infected with O105clz at a MOI of 1, HSP9 was also induced (Fig. lc) when compared to control (uninfected) cells at 37~ (Fig. la). The level of induction remains to be determined, but, in bacteriophage lambda infections of E. coli, GroEL was induced only two fold (Kochan & Murialdo 1982). Thus, the amount of induction might be expected to be low for B. subtilis as well. The induction of HSP 8, the DnaK homologue, was minimal following infection with O105clz (compare Figs la and lc). However, the induction of HSP 8 at 48~C (Fig. lb) in this set of experiments was also low when compared to that in Fig. la. In order to confirm the induction of the GroEL homologue protein, Western blot analysis was done. Figure 2 shows that in this assay the GroEL homologue was induced after 10, 20, and 30 min at 48~ After 20min, the GroEL homologue was induced in O105clz infected cells when compared with the 20min 37~ control. This increase may indicate a requirement for the host encoded protein at this point in the lytic cycle of the bacteriophage. The induction of the GroEL homologue by
341
37~ 10'
20'
46~ 30'
40'
10'
20'
30' 40' 110'
1 105clz
20' 30'
40'
97kDa 66
Fig. 2. Western blot analysis of B. subtilis YB886 proteins using anti-E, coli GroEL antisera. Samples were grown and prepared as described in the text. Equal amounts of unlabled total protein (166 ~g) were loaded in each lane, The values on the left side are M,s x 10-3 of standard proteins.
O105clz in Fig. 2 appears less than Fig. lc. This is likely due to differences in detecting induction by 35S-methionine incorporation (Fig. lc) versus total GroEL (Fig. 2). These data provide further evidence for a possible role of HSPs in the morphogenesis of bacteriophage in B. subtilis. A t present, no HSPs are known to be required absolutely for the development of any B. subtilis bacteriophage. The isolation of HSP mutants should provide more definitive data about the requirements for HSPs in bacteriophage morphogenesis in B. subtilis.
Acknowledgements We would like to thank C. Georgopoulos (University of Utah) for the GroEL antiserum and R.E. Yasbin (University of Maryland, Baltimore County) for the bacteriophage O105clz. This work was supported by a National Science Foundation EPSCOR grant to U.N.S.
References Arnosti DN, Singer VL & Chamberlin MJ (1986) Characterization of heat shock in Bacillus subtilis. J. Bacteriol. 168: 1243-1249 Brissette JL, Russel M, Weiner L & Model P (1990) Phage shock protein, a stress protein of Escherichia coli. Proc. Natl. Acad. Sci. USA 87:862-866 Brissette JL, Weiner L, Ripmaster TL & Model P (1991) Characterization and sequence of the Escherichia coli stress-induced psp operon. J. Mol. Biol. 220:35-48 Carrascosa JL, Garcia JA & Salas M (1982) A protein similar to Escherichia coil groEL is present in Bacillus subtilis. J. Mol. Biol. 158:731-737 Drahos DJ & Hendrix RW (1982) Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins. J. Bacteriol. 149:1050-1063 Friedman DI, Olson ER, Georgopoulos C, Tilly K, Herskowitz I & Banuett F (1984) Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol. Rev. 48:299-325 Kochan J & Murialdo H (1982) Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection. J. Bacteriol. 149:1166-1170 Miller BS, Kennedy TE & Streips UN (1991) Molecular characterization of specific heat shock proteins in Bacillus subtilis. Curt. Micro. 22:231-236 Neidhardt FC & VanBogelen RA (1987) Heat shock response.
342 In: Neidhardt FC, Ingraham JL, Magasanik B, Low KB, Schaechter M & Umbarger HE (Eds) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol 2 (pp 1334--1345).Washington, DC: American Society for Microbiology Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA 44:1072-1078
Streips UN & Polio FW (1985) Heat shock proteins in bacilli. J. Bacteriol. 162:434--437 Young KD, Anderson ILl & Hafner RJ (1989) Lysis of Escherichia coli by the bacteriophage OX174 E protein: Inhibitionof lysis by heat shock proteins. J. Bacteriol. 171:4334-4341 Zylicz M, Ang D, Liberek K & Georgopoulos C (1989) Initiation of lambda DNA replication with purified host-and bacteriophage-encoded proteins: the role of the DnaK, DnaJ, and GrpE heat shock proteins. EMBO J. 8:1601-1608
Short communication
Bacteriophage O105clz induces the GroEL-homologue protein in Bacillus subtilis Robin R. Staples ~, Brian S. Miller & Uldis N. Streips*
Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, Kentucky 40292, USA; (;present address: Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA; * requests for offprints) Received 9 August 1991; accepted 18 November 1991
Key words: Bacillus subtilis, GroEL, heat shock, phage induction Abstract
Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue of Bacillus subtilis was shown to be induced upon infection with 0105clz, a clear plaque mutant of the temperate bacteriophage O105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homoiogue when cells were infected with 0105clz.
Introduction
In Escherichia coli there are at least 17 sigma-32 dependent heat shock proteins (HSPs) induced, when cells are subjected to a sudden increase in temperature (Neidhardt & VanBogelen 1987). Several of these HSPs, especially GroEL and DnaK, are also induced in response to infection by bacteriophage, especially bacteriophage lambda (Drahos & Hendrix 1982; Kochan & Murialdo 1982). These proteins have been shown to be necessary in the morphogenesis of lambda production (Friedman et al. 1984) and in replication of the bacteriophage DNA (Zylicz et al. 1989). Mutations in dnaK and groEL render the cells resistant to lambda infection (Friedman et al. 1984; Neidhardt & VanBogelen 1987). Other bacteriophage such as the filamentous phage, fl, and OX174 have also been shown to utilize these host encoded proteins to aid in the life cycle of the bacteriophage (Brissette et al. 1990; Young et al. 1989). Recently, a sigma-32 independent, filamentous phage and
stress induced operon has been described in E. coli (Brissette et al. 1991). In Bacillus subtilis, the GroEL and DnaK homologues have been identified and are components of the bacillus heat shock response (Arnosti et al. 1986; Miller et al. 1991). Carrascosa et al. (1982) described a B. subtilis GroEL homologue which was found to copurify with a bacteriophage 029 protein involved in phage capsid formation. This finding suggests that B. subtilis bacteriophage may also utilize host encoded proteins, in particular some HSPs, for replication and assembly. We wanted to determine if bacteriophage infection in B. subtilis, as shown in E. coli, results in the induction of heat shock proteins. In the present study, we define the induction of the GroEL homologue of B. subtilis following infection with a clear plaquing mutant of the temperate bacteriophage O105. Bacillus subtilis strain YB886 (metB5, trpC2, xin-1, spl3~) was grown at 37~ to a Klett Unit reading of 55 (mid-exponential) (monitored on a
340
(a)
(b)
(c)
97---66--"
Fig. 1. Two-dimensional gel electrophoresis of B. subtilis YB886. (a) 37~ control. (b) 48~ heat shock (10 min). (c) Cells grown at 37~C and infected with 0105clz for 10 min. Spots 8 and 9 are the DnaK and GroEL homologues respectively. The values on the left side are Mrs x 10 3 of standard proteins. Equal amounts of TCA precipitable counts were loaded on each gel.
Klett-Summerson Colorimeter) in Spizizen's minimal medium (Spizizen 1958) containing 40 tzg/ml (final concentration) tryptophane and methionine. At this time, cells were harvested and suspended in minimal medium without methionine. The cells were allowed to equilibrate at 37~C for 5 min prior to either a shift to 48~ or the addition of bacteriophage. Samples (1 ml) of cells were incubated at 37~ (control), 48 ~C, or 37~ with O105clz at a multiplicity of infection of 1 (as determined by plaquing efficiency). Samples for two-dimensional polyacrylamide gel electrophoresis were labeled after 10min with 80~Ci of 35S_methionine (Dupont-New England Nuclear, Wilmington DE) (specific activity, ll00Ci/mmole) for 5min. The cells were then lysed and two-dimensional electrophoresis was performed as described by Miller et al. (1991). Samples for Western blot analysis were incubated for 10, 20, 30, and 40 min, harvested, and then subjected to one-dimensional polyacrylamide gel electrophoresis as described (Streips & Polio 1985). Western blots were developed using a Immunoblot Assay Kit (Bio-Rad, Rockville Centre, NY) with goat anti-rabbit alkaline phosphatase conjugates. To detect whether the GroEL or DnaK homologues of B. subtilis were induced in response to bacteriophage infection, the two dimensional gel electrophoresis system was utilized. Figure la illustrates the relevant portion of the protein pattern
present in B. subtilis strain YB886 during steady state growth at 37~C. Polypeptides designated as 8 and 9 have been previously identified as the DnaK (HSP8) and GroEL (HSP9) homologues respectively (Miller et al. 1991). The induction of these proteins during heat shock for 10min at 48~ is shown in Fig. lb. When cells were infected with O105clz at a MOI of 1, HSP9 was also induced (Fig. lc) when compared to control (uninfected) cells at 37~ (Fig. la). The level of induction remains to be determined, but, in bacteriophage lambda infections of E. coli, GroEL was induced only two fold (Kochan & Murialdo 1982). Thus, the amount of induction might be expected to be low for B. subtilis as well. The induction of HSP 8, the DnaK homologue, was minimal following infection with O105clz (compare Figs la and lc). However, the induction of HSP 8 at 48~C (Fig. lb) in this set of experiments was also low when compared to that in Fig. la. In order to confirm the induction of the GroEL homologue protein, Western blot analysis was done. Figure 2 shows that in this assay the GroEL homologue was induced after 10, 20, and 30 min at 48~ After 20min, the GroEL homologue was induced in O105clz infected cells when compared with the 20min 37~ control. This increase may indicate a requirement for the host encoded protein at this point in the lytic cycle of the bacteriophage. The induction of the GroEL homologue by
341
37~ 10'
20'
46~ 30'
40'
10'
20'
30' 40' 110'
1 105clz
20' 30'
40'
97kDa 66
Fig. 2. Western blot analysis of B. subtilis YB886 proteins using anti-E, coli GroEL antisera. Samples were grown and prepared as described in the text. Equal amounts of unlabled total protein (166 ~g) were loaded in each lane, The values on the left side are M,s x 10-3 of standard proteins.
O105clz in Fig. 2 appears less than Fig. lc. This is likely due to differences in detecting induction by 35S-methionine incorporation (Fig. lc) versus total GroEL (Fig. 2). These data provide further evidence for a possible role of HSPs in the morphogenesis of bacteriophage in B. subtilis. A t present, no HSPs are known to be required absolutely for the development of any B. subtilis bacteriophage. The isolation of HSP mutants should provide more definitive data about the requirements for HSPs in bacteriophage morphogenesis in B. subtilis.
Acknowledgements We would like to thank C. Georgopoulos (University of Utah) for the GroEL antiserum and R.E. Yasbin (University of Maryland, Baltimore County) for the bacteriophage O105clz. This work was supported by a National Science Foundation EPSCOR grant to U.N.S.
References Arnosti DN, Singer VL & Chamberlin MJ (1986) Characterization of heat shock in Bacillus subtilis. J. Bacteriol. 168: 1243-1249 Brissette JL, Russel M, Weiner L & Model P (1990) Phage shock protein, a stress protein of Escherichia coli. Proc. Natl. Acad. Sci. USA 87:862-866 Brissette JL, Weiner L, Ripmaster TL & Model P (1991) Characterization and sequence of the Escherichia coli stress-induced psp operon. J. Mol. Biol. 220:35-48 Carrascosa JL, Garcia JA & Salas M (1982) A protein similar to Escherichia coil groEL is present in Bacillus subtilis. J. Mol. Biol. 158:731-737 Drahos DJ & Hendrix RW (1982) Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins. J. Bacteriol. 149:1050-1063 Friedman DI, Olson ER, Georgopoulos C, Tilly K, Herskowitz I & Banuett F (1984) Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol. Rev. 48:299-325 Kochan J & Murialdo H (1982) Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection. J. Bacteriol. 149:1166-1170 Miller BS, Kennedy TE & Streips UN (1991) Molecular characterization of specific heat shock proteins in Bacillus subtilis. Curt. Micro. 22:231-236 Neidhardt FC & VanBogelen RA (1987) Heat shock response.
342 In: Neidhardt FC, Ingraham JL, Magasanik B, Low KB, Schaechter M & Umbarger HE (Eds) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol 2 (pp 1334--1345).Washington, DC: American Society for Microbiology Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA 44:1072-1078
Streips UN & Polio FW (1985) Heat shock proteins in bacilli. J. Bacteriol. 162:434--437 Young KD, Anderson ILl & Hafner RJ (1989) Lysis of Escherichia coli by the bacteriophage OX174 E protein: Inhibitionof lysis by heat shock proteins. J. Bacteriol. 171:4334-4341 Zylicz M, Ang D, Liberek K & Georgopoulos C (1989) Initiation of lambda DNA replication with purified host-and bacteriophage-encoded proteins: the role of the DnaK, DnaJ, and GrpE heat shock proteins. EMBO J. 8:1601-1608
