Direct CIII–HflB interaction is responsible for the inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli r 32 by kCIII Sabyasachi Halder 1 , Subhamoy Banerjee 1,2 and Pradeep Parrack 1 1 Department of Biochemistry, Bose Institute, Kolkata, India 2 Madhav Institute of Technology & Science, Gwalior, India Development of the temperate coliphage k depends on a set of phage-specified regulatory proteins that inter- act with host target proteins [1–4]. Among several effects on host protein synthesis, k provokes the over- production of some bacterial proteins induced in the heat shock response. This effect depends on early gene expression encoded by the leftward (p L ) transcription unit of k [5,6]. CIII is a k-specific regulatory protein from the p L operon with a potential for host inter- action. It is a small 54-residue protein that favors the lysogenic response to infection by stabilizing kCII, the transcriptional factor that favors lysogeny and is responsible for primary control of the lambda develop- mental decision for lysis or lysogeny [4,7–9]. In the absence of CIII, CII is rapidly degraded by the ATP- dependent host metalloprotease HflB (FtsH). The molecular mechanism for CIII-mediated inhibition of the proteolysis of CII by HflB involves CIII–HflB interaction [10,11]. CIII itself is a substrate of HflB [12]. It has also been reported that CIIIC, the central helical domain of CIII, is resistant to HflB proteolysis, and is a more effective inhibitor than the full-length protein [11]. Apart from promoting lysogeny by lambda, CIII protects Escherichia coli r 32 , the heat shock-specific sigma factor, which is also a substrate of HflB [13,14]. This effect generates a heat-shock response in the cell [15,16]. Maximal production of CIII prolongs the heat-induced synthesis of E. coli heat shock proteins even at low temperature [17]. The half-life of r 32 ( 2 min) is increased approximately fourfold upon overproduction of CIII, resulting in an overproduction of heat shock proteins and rapid inhibition of cell growth [15]. The molecular mechanism of the CIII- mediated inhibition of proteolysis of r 32 by HflB is unknown. We tried to address this issue by studying the effects of CIII and CIIIC on the proteolysis of Keywords antiproteolytic activity; heat shock; lysogeny; kCII; r 32 Correspondence P. Parrack, Department of Biochemistry, Bose Institute, P-1 ⁄ 12, C.I.T. Scheme VIIM, Kolkata 700 054, India Fax: +91 33 2355 3886 Tel: +91 33 2569 3227 E-mail: pradeep@bic.boseinst.ernet.in (Received 28 April 2008, revised 14 July 2008, accepted 28 July 2008) doi:10.1111/j.1742-4658.2008.06610.x The CIII protein of bacteriophage lambda exhibits antiproteolytic activity against the ubiquitous metalloprotease HflB (FtsH) of Escherichia coli, thereby stabilizing the kCII protein and promoting lysogenic development of the phage. CIII also protects E. coli r 32 , another substrate of HflB. We have recently shown that the protection of CII from HflB by CIII involves direct CIII–HflB binding, without any interaction between CII and CIII [Halder S, Datta AB & Parrack P (2007) J Bacteriol 189, 8130–8138]. Such a mode of action for kCIII would be independent of the HflB substrate. In this study, we tested the ability of CIII to protect r 32 from HflB digestion. The inhibition of HflB-mediated proteolysis of r 32 by CIII is very similar to that of kCII, characterized by an enhanced protection by the core CIII peptide CIIIC (amino acids 14–41 of kCIII) and a lack of interaction between r 32 and CIII. Abbreviation GST, glutathione S-transferase. FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4767 r 32 by HflB in vitro. Our results show that both CIII and CIIIC inhibit the HflB-mediated proteolysis of r 32 in vitro, and that CIIIC is a more effective inhibitor. We also found that there is no interaction between CIII and r 32 . From these results, we suggest that the inhibition of r 32 by CIII is due to a direct CIII–HflB interaction. Results In vitro proteolysis of r 32 by HflB requires ATP The proteolysis of r 32 by HflB is very rapid and requires the DnaK–DnaJ–GrpE chaperone machine in vivo [18–20]. However, this proteolysis is much slower in vitro, because of a lack of chaperone machin- ery. The in vitro proteolysis requires ATP [14]. We examined the proteolysis of purified r 32 -C-his (1.2 lm) by glutathione S -transferase (GST)–HflB (0.8 lm) in the absence or presence of ATP (5 mm). Digestion after specified time intervals was assayed by 11% SDS ⁄ PAGE (Fig. 1). It is clear that the HflB-mediated degradation of r 32 could not proceed without ATP. In this respect, r 32 resembles kCII. Inhibition of HflB-mediated proteolysis of r 32 by CIII and by CIIIC The effect of CIII and CIIIC on the HflB-mediated proteolysis of r 32 was checked by treating r 32 -C-his (1.2 lm) with GST–HflB (0.8 lm) in the presence of CIII (100 lm) for varying time intervals (Fig. 2A). It was found that r 32 was partially protected in the pres- ence of CIII, and  65% of r 32 remained undigested after 80 min, compared with  40% for the control (r 32 alone, Fig. 2C). The inhibitory action of CIIIC on the proteolysis of r 32 by HflB was also assayed in the presence of CIIIC (60 lm) instead of CIII (Fig. 2B). It is clear that in this Fig. 1. ATP-dependence of proteolysis of r 32 by HflB. (Upper) Band corresponding to r 32 on 11% SDS ⁄ PAGE, in the presence or absence of 5 m M ATP. Numbers on the top of each lane indicate the time of digestion in min. (Lower) Densitometric scan showing the amount of r 32 -C-his (1.2 lM) remaining after proteolysis with GST–HflB (0.8 l M). Each data point represents the mean value from three identical experiments. A B C Fig. 2. Inhibition of HflB-mediated proteolysis of r 32 by CIII or CIIIC. The bands corresponding to r 32 -C-his (1.2 lM) remaining after proteolysis with GST–HflB (0.8 l M) in the absence or presence of (A) His 6 –CIII (100 lM) or (B) CIIIC (60 lM) are shown. Numbers on the top of each lane indicate the time of digestion in min. Samples were run on (A) a 15% SDS ⁄ PAGE, or (B) a 17.5% SDS ⁄ PAGE. (C) A densitometric scan of the above bands showing the amount of r 32 remaining after proteolysis, for r 32 alone ( )or in the presence of CIII ( ) or CIIIC (d). Each data point represents the mean value from three identical experiments. Protection of r 32 from HflB by kCIII S. Halder et al. 4768 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS case the proteolysis, with  85% of r 32 remaining undigested after 80 min (Fig. 2C), was inhibited more effectively than with intact CIII. The above experiments on the inhibition of the pro- teolysis of r 32 by HflB were also carried out in the presence of varying amounts (up to 200 lm) of CIII or CIIIC (Fig. 3). In this case, proteolysis was terminated after 40 min. Stronger inhibition by CIIIC is also evi- dent from these experiments, with  95% of r 32 remaining undigested in the presence of 40 lm CIIIC (Fig. 3, lower). r 32 interacts with HflB but does not interact with CIII The interaction between r 32 and HflB was tested in an in vitro GST pull-down assay. GST-tagged HflB was bound to glutathione-Sepharose beads followed by the addition of r 32 . The proteins were analyzed on an 11% SDS ⁄ PAGE and visualized by western blotting with anti-his as the primary antibody. The same exper- iment was repeated with GST protein as a negative control. It was observed that r 32 co-eluted with GST– HflB but not with GST protein (Fig. 4A), implying that r 32 interacts specifically with GST–HflB. The interaction between r 32 and CIII was assayed in an in vitro Ni 2+ -nitrilotriacetic acid pull-down assay. The Ni 2+ -nitrilotriacetic acid bound His 6 –CIII was mixed with r 32 (with the 6· histidine tag removed) and incubated at 4 °C for 4 h. The Ni 2+ -nitrilotriace- tic acid beads were washed with 1· NaCl ⁄ P i and eluted by boiling with 1· sample buffer. Proteins were ana- lyzed on 17.5% SDS ⁄ PAGE (Fig. 4B). It was observed that r 32 did not co-elute with CIII, implying that r 32 does not interact with CIII. Discussion Phage protein CIII works as an antiprotease against E. coli HflB and protects kCII by directly binding to the protease [10,11], without any detectable interaction with the substrate kCII [11]. Thus, the protection of Fig. 3. Inhibition of HflB-mediated proteolysis of r 32 by different concentrations of CIII or CIIIC. The bands corresponding to r 32 -C- his (1.2 l M) remaining after proteolysis with GST–HflB (0.8 lM) for 40 min in the presence of His 6 –CIII or CIIIC (up to 200 lM) are shown in the upper panels. Numbers on the top of each lane indi- cate the concentration of His 6 -CIII or CIIIC (in lM). Lane C indicates the control lane showing undigested r 32 . The samples were run on a 15% (upper) or 17.5% (middle) SDS ⁄ PAGE. (Lower) Densitomet- ric scan of the above bands depicting the amount of r 32 remaining after proteolysis in the presence of CIII ( ) or CIIIC (d). Each data point represents the mean value from three identical experiments. A B Fig. 4. In vitro binding of HflB-r 32 and CIII-r 32 . (A) Interaction between GST–HflB and r 32 -C-his was tested by GST pull-down fol- lowed by 11% SDS ⁄ PAGE, and immunoblotting with anti-His Ig. Lane 1, r 32 -C-his (control); lane 2, fraction pulled down with GST– HflB; lane 3, fraction pulled down with GST. (B) Absence of interac- tion between His 6 –CIII and r 32 (without the histidine tag) as obtained from Ni-nitrilotriacetic acid pull-down, followed by 17.5% SDS ⁄ PAGE and Coomassie Brilliant Blue staining. Lane 1, His 6 -CIII alone; lane 2, r 32 alone; lane 3, fraction pulled down. S. Halder et al. Protection of r 32 from HflB by kCIII FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4769 CII by CIII works at the protease level, rather than at the substrate level. Nevertheless, competition between CII and CIII for interaction with HflB also influences the proteolysis of CII [11], because CIII itself is a sub- strate of HflB [12]. CIIIC, the central region of CIII, is not a substrate of HflB, and acts as a better inhibitor for digestion of CII by HflB, than CIII [11]. Is this mode of antiproteolytic action of CIII a general mode of CIII activity, or does it apply only for CII? We examined the mode of the inhibitory action of CIII on proteolysis of the heat shock sigma factor r 32 , another substrate of HflB. Like proteolysis of CII, r 32 proteol- ysis requires ATP. Both CIII and CIIIC inhibit this proteolysis, with CIIIC exhibiting stronger inhibition both as a function of time (Fig. 2) or as a function of concentration (Fig. 3). Under the conditions of our experiment, near-total inhibition by CIIIC could be observed, whereas in the presence of CIII, only partial inhibition was achieved. As in the case of CII [10,11], CIII appears to work as an inhibitor for the proteoly- sis of r 32 through direct interaction with the protease, characterized by a lack of interaction between r 32 and CIII (Fig. 4). As for CII, competition between r 32 and CIII for binding to HflB would also decide the extent and efficiency of protection of r 32 by CIII, because both are HflB substrates. The relative binding affinity for r 32 –HflB interaction and CIII–HflB interaction would play an important role in the inhibition of proteolysis of r 32 by HflB. Interestingly, both CII and r 32 are cytosolic sub- strates for HflB. In addition, HflB also acts on several membrane-associated substrates, for which the mecha- nism of proteolysis is probably somewhat different [21]. Whether k CIII would act as an inhibitor for such substrates (e.g. SecY, YccA, Foa) remains to be seen. However, the biological connection between kCIII and such substrates is poor, and it is unlikely that CIII would have any role in the proteolysis of substrates like SecY or YccA by HflB. We think that the primary role of CIII is associated with k lysogeny. In this respect, the antiproteolytic activity of CIII needs to be short-lived, made possible by the fact that CIII is also an HflB substrate. CIII, however, acts via direct inter- action with HflB, which may be enabled by the bind- ing of its central helical region to the substrate-entry cleft of HflB [22], as pointed out previously [11]. This probably makes CIII a general inhibitor for the cyto- solic substrates of HflB, accounting for its protection of r 32 . The intriguing question that follows is why would a lambda protein stabilize the E. coli heat shock sigma factor? r 32 is an unstable protein [23,24] with a rapid turnover during normal growth and a transient stabilization during heat shock, followed by rapid deg- radation [18,24]. The level of r 32 in E. coli cells is tightly controlled, through the interactions of the DnaK–DnaJ–GrpE machinery and by HflB-mediated degradation. Interestingly, these latter proteins that promote the degradation of r 32 are themselves pro- duced as a result of a stress response [13,25], being transcribed from heat shock promoters involving r 32 . They may be part of mechanisms that allow the bacte- rium to respond quickly to changing nutritional and environmental conditions. When a temperate virus like lambda takes up the lysogenic pathway in response to stressed conditions of the host, the phage functions must closely follow host conditions so that a correct developmental decision can be taken. During the estab- lishment of lysogeny, stabilisation of r 32 by kCIII may lead to elevated production of the bacterial protease lon that is transcribed from heat shock promoters [26] and degrades the phage protein N [27], indirectly helping the lysogenic response. It is known that k lysogeny is reduced in lon mutants [27,28]. Alternatively, inhibition of HflB by kCIII would lead to increased levels of r 32 , causing elevated production of the heat shock proteins DnaJ, DnaK and GrpE which promote the replication of k [29,30]. Such an event would serve to keep the lysogenized cell prepared for stress-induced induction while degradation by HflB is compromised. Various pathways, sometimes even antagonistic [31], are known to regulate the concentration of r 32 in E. coli. Lambda could be taking advantage of this regulatory network and act at multiple levels of host–virus interactions. Experimental procedures Materials Various fine chemicals, reagents and enzymes were obtained from Sigma-Aldrich (New Delhi, India), USB (Cleveland, OH, USA), Merck Limited (Mumbai, India) and Sisco Research Laboratory (Mumbai, India). Resins, primers and columns were used as described in Halder et al. [11]. Purification of proteins and peptides Purification of r 32 was carried out according to Chattopad- hyay and Roy [32] and Sambrook et al. [33]. The NUT-21 strain containing pUHE 211-1 was grown at 30 °Cin1L of 2XYT medium [34] with 100 lgÆmL –1 ampicillin and 50 lgÆmL –1 kanamycin. At A 600  1, isopropyl thio-b-d- galactopyranoside was added to a final concentration of 0.5 mm. Cells were grown for a further 20 min and poured into cold tubes. All subsequent steps were performed at 4 °C. After centrifugation at 1900 g for 10 min, the cell Protection of r 32 from HflB by kCIII S. Halder et al. 4770 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS pellet was resuspended in 18 mL of ice-cold buffer L (50 mm phosphate buffer, pH 7.9, 300 mm KCl, 50 mm iso- leucine, 50 mm phenylalanine) with 20 lgÆmL )1 of phenyl methanesulfonyl fluoride and disrupted by sonication. The cell lysate was centrifuged for 45 min at 14 500 g. The supernatant was loaded onto a 3-mL Ni 2+ -nitrilotriacetic acid-agarose column pre-equilibrated with buffer L at a rate 0.4 mLÆmin –1 . The column was subsequently washed with 40 mL of buffer L followed by 10 mL of buffer L plus 15 mm imidazole. Nickel-bound proteins were eluted with 30 mL of 15–150 mm imidazole gradient in buffer L. Pure fractions of r 32 proteins were dialyzed against two changes of 1 L of 50 mm phosphate buffer, pH 7.9, containing 300 mm KCl and 50% glycerol. HflB was overexpressed as a GST fusion protein from plasmid pAD101 containing the hflB gene cloned in vec- tor pGEX4T and was purified with a glutathione-Sepha- rose column (GSTrap FF; Amersham Biosciences, Sweden). His 6 –CIII was obtained by overexpressing recombinant plasmid pAB905 containing the cIII gene and purified with Ni-nitrilotriacetic acid column as described previously [11]. A 28-residue peptide, CIIIC, was chemically synthesized and purified by reverse-phase C 18 column (hypersil) by water–acetonitrile gradient as stated previously [11]. Measurements of inhibition of proteolysis by HflB The activity of CIII (or CIIIC) was measured by its ability to inhibit HflB-mediated proteolysis of r 32 [14]. Reaction mixtures (50 lL) were prepared by taking 60 pmol of r 32 in buffer P (50 mm Tris ⁄ acetate, pH 7.2, 100 mm NaCl, 5mm MgCl 2 ,25lm Zn-acetate, 1.4 mm b-mercaptoetha- nol) containing 5 mm ATP. CIII or CIIIC (up to 200 lm) was also added. Proteolysis was carried out by adding HflB (0.8 lm) followed by incubation at 37 °C for specified time intervals. Reactions were stopped by the addition of 5 mm EDTA and SDS ⁄ PAGE loading buffer, followed by heating in a boiling water-bath for 5 min. The amount of r 32 that remained after proteolysis was analyzed after SDS ⁄ PAGE of the samples followed by Coomassie Brilliant Blue stain- ing and quantitation in a gel documentation system (Bio- Rad Gel Doc 1000). Removal of the histidine tag To get a native r 32 protein without the C-terminal 6· histi- dine tag, r 32 ( 100 lg) was mixed with thrombin (1 unit) and incubated overnight at 22 °C. The protein mixture was then passed through a 1 mL benzamidine column (Amer- sham) for removal of thrombin. Finally, the native protein was separated from the mixture by treatment with Ni 2+ – nitrilotriacetic acid beads. The flow-through contained only native proteins, devoid of the histidine tag. In vitro binding assay The interaction between HflB and r 32 was studied by in vitro GST pull-down assay. A 50 lL aliquot of bound GST–HflB (25 lg) was mixed with 30 lgofr 32 in buffer P. The final volume was made up to 500 lL and incubated overnight on a rotating machine at 4 °C. Before binding, 5% of the input solution was kept aside separately and was used as a control for comparison. The proteins were analyzed in an 11% SDS ⁄ PAGE and by western blotting using anti-his Ig. As a negative control, the same experiment was also performed with GST protein replacing GST–HflB. The interaction of histidine-tagged proteins with other proteins was examined by in vitro Ni 2+ -nitrilotriacetic acid pull-down assay. Purified His 6 –CIII was immobilized at 4 °C for 1 h on Ni 2+ -nitrilotriacetic acid beads in buffer A containing 10 mm imidazole and washed with buffer P. Fifty microliters of His 6 –CIII bound bead ( 25 lg) was mixed with 30 lg r 32 . The final volume was made up to 500 lL and incubated on a rotating machine at 4 °C for 4 h. The beads were then washed three times with buffer P and resuspended in SDS gel loading buffer for elution of proteins from the beads. The eluted proteins were separated by 17.5% SDS ⁄ PAGE and visualized using Coomassie Brilliant Blue staining. Acknowledgements The authors would like to thank Professor Siddhartha Roy, Indian Institute of Chemical Biology, Kolkata, for the gift of the E. coli strain NUT-21 containing pUHE211-1 that was used to purify the r 32 protein. S.H. was supported by fellowships from CSIR, India, and by Bose Institute. Subhamoy Banerjee is a summer student at the Bose Institute, from Madhav Institute of Technology & Science, Gwalior, India. References 1 Echols H (1980) Bacteriophage k development. In The Molecular Genetics of Development (Leighton TJ & Lo o mis WF, eds), pp. 1–16. Academic Press, New York, NY. 2 Herskowitz I & Hagen D (1980) The lysis–lysogeny decision of phage k: explicit programming and respon- siveness. Annu Rev Genet 14, 399–445. 3 Friedman DI, Olson ER, Georgopoulos C, Tilly K, Herskowitz I & Banuett F (1984) Interactions of bacte- riophages and host macromolecules in the growth of bacteriophage k. Microbiol Rev 48, 299–325. 4 Echols H (1986) Bacteriophage k development: tempo- ral switches and the choice of lysis or lysogeny. Trends Genet 2, 26–30. 5 Drahos DJ & Hendrix RW (1982) Effect of bacterio- phage lambda infection on synthesis of groE protein S. Halder et al. Protection of r 32 from HflB by kCIII FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4771 and other Escherichia coli proteins. J Bacteriol 149, 1050–1063. 6 Kochan J & Murialdo H (1982) Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection. J Bacteriol 149, 1166–1170. 7 Hoyt MA, Knight DM, Das A, Miller HI & Echols H (1982) Control of phage lambda development by stabil- ity and synthesis of cII protein: role of the viral cIII and host hflA, himA and himD genes. Cell 31, 565–573. 8 Rattray A, Altuvia S, Mahajna G, Oppenheim AB & Gottesman M (1984) Control of phage lambda cII activ- ity by phage and host functions. J Bacteriol 159, 238–242. 9 Banuett F, Hoyt MA, Mcfarlane L, Echols H & Her- skowitz I (1986) HflB, a new Escherichia coli locus regu- lating lysogeny and the level of bacteriophage lambda CII protein. J Mol Biol 187, 213–224. 10 Kobiler O, Rokney A & Oppenheim AB (2007) Phage lambda CIII: a protease inhibitor regulating the lysis– lysogeny decision. PLoS ONE 2, e363. 11 Halder S, Datta AB & Parrack P (2007) Probing the antiprotease activity of kCIII, an inhibitor of the Esc- herichia coli metalloprotease HflB (FtsH). J Bacteriol 189, 8130–8138. 12 Herman C, Thevenet D, D’Ari R. & Bouloc P (1997) The HflB protease of Escherichia coli degrades its inhib- itor lambda CIII. J Bacteriol 179, 358–363. 13 Herman C, Thevenet D, D’Ari R & Bouloc P. (1995) Degradation of r 32 , the heat shock regulator in Escheri- chia coli, is governed by HflB. Proc Natl Acad Sci USA 92, 3516–3520. 14 Tomoyasu T, Gamer J, Bukau B, Kanemory M, Mori H, Rutman AJ, Oppenheim AB, Yura T, Yamanaka K, Niki H et al. (1995) Escherichia coli FtsH is a mem- brane bound, ATP-dependent protease which degrades the heat-shock transcription factor r 32 . EMBO J 14, 2551–2560. 15 Bahl H, Echols H, Straus DB, Court D, Crowl R & Georgopoulos CP (1987) Induction of the heat shock response of E. coli through stabilization of r 32 by the phage kCIII protein. Genes Dev 1, 57–64. 16 Kornitzer D, Altuvia S & Oppenheim AB (1991) The activity of CIII regulator of lambdoid bacteriophages resides within a 24-amino-acid protein domain. Proc Natl Acad Sci USA 88, 5217–5221. 17 Ang D, Chandrasekhar GN, Zylicz M & Georgopoulos CP (1986) Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature. J Bacteriol 167, 25–29. 18 Straus D, Walter W & Gross CA (1990) DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of r 32 . Genes Dev 4, 2202–2209. 19 Tatsuta T, Joo DM, Calendar Y, Akiyama Y & Ogura T (2000) Evidence for an active role of the DnaK chap- erone system in the degradation of r 32 . FEBS Lett 478, 271–275. 20 Tomoyasu T, Ogura T, Tatsuta T & Bukau B (1998) Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in E. coli. Mol Microbiol 30, 567–581. 21 Ito K & Akiyama A (2005) Cellular functions, mecha- nisms of action, and regulation of FtsH protease. Annu Rev Microbiol 59, 211–231. 22 Bieniossek C, Schalch T, Bumann M, Meister M, Meier R & Baumann U (2006) The molecular architecture of the metalloprotease FtsH. Proc Natl Acad Sci USA 103, 3066–3071. 23 Grossman AD, Straus DB, Walter WA & Gross CA (1987) Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev 1, 179–184. 24 Tilly K, Spence J & Georgopoulos C (1989) Modulation of stability of the Escherichia coli heat shock regulatory factor r 32 . J Bacteriol 171, 1585–1589. 25 Yura T, Nagai H & Mori H (1993) Regulation of the heat-shock response in bacteria. Annu Rev Microbiol 47, 321–350. 26 Gottesman S (1984) Bacterial regulation: global regula- tory networks. Annu Rev Genet 18, 415–441. 27 Maurizi MR (1987) Degradation in vitro of bacterio- phage lambda N protein by Lon protease from Escheri- chia coli. J Biol Chem 262, 2696–2703. 28 Walker JR, Usser CL & Allen JS (1973) Bacterial cell division regulation: lysogenization of conditional cell division lon ) mutants of Escherichia coli by Bacterio- phage Lambda. J Bacteriol 111, 1326–1332. 29 Liberek K, Georgopoulos C & Zylicz M (1988) The role of the Escherichia coli DnaK and DnaJ heat shock pro- teins in the initiation of bacteriophage k DNA replica- tion. Proc Natl Acad Sci USA 85, 6632–6636. 30 Johnson C, Chandrasekhar GN & Georgopoulos C (1989) Escherichia coli DnaK and GrpE heat shock pro- teins interact both in vivo and in vitro. J Bacteriol 171, 1590–1596. 31 Morita MT, Kanemori M, Yanagi H & Yura T (2000) Dynamic interplay between antagonistic pathways con- trolling the r 32 level in Escherichia coli. Proc Natl Acad Sci USA 97, 5860–5865. 32 Joo DM, Ng N & Calendar R (1997) A r 32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affin- ity. Proc Natl Acad Sci USA 94, 4907–4912. 33 Chattopadhyay R & Roy S (2002) DnaK–sigma 32 interaction is temperature dependent: implication for the mechanism of heat shock response. J Biol Chem 277, 33641–33647. 34 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning, A Laboratory Manual, Vol. 3 (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Protection of r 32 from HflB by kCIII S. Halder et al. 4772 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS . Direct CIII HflB interaction is responsible for the inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli r 32 by kCIII Sabyasachi. The molecular mechanism for CIII-mediated inhibition of the proteolysis of CII by HflB involves CIII HflB interaction [10,11]. CIII itself is a substrate of HflB [12].