IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock

Biochimie (1996) 78, ! 17-122 © Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris

117

lbpA and IbpB, the new heat-shock prote[ns bind to endogenous Escherichia coli proteins aggregated i trace|lullarly by heat shock E Laskowskaa, A Wawrzyn6wb, A Taylora* aDepartment of Biockemistry and bDepartment of Molecular Biology, Univers.:~ of Gda~sk, 80-822 Gda~sk, K.tadki 24, Poland

(Received 25 October 1995; accepted 8 January 1996)

Summary - - IbpA/B, 16 kDa heat-shock proteins were recently described as recognizing heterologous protein inclusion bodies in Escherichia coli cells; the corresponding genes formed an operon regulated by the rpoH gene product, c 32 protein (Burland et al (1993) Genomics 16, 551; Allen et al (1992) J Bacteriol 174, 6938; Chuang et al (1993) Gene 134, 1; Chuang and Blattner (1993) J Bacteriol 175, 5242). We have found that IbpA/Bs also recognize endogenous bacterial proteins aggregated intracellularly by heat shock. IbpA/B proteins were isolated and purified from the aggregates (the S fraction), identified by amino acid microsequencing and used as immunogen for anti-IbpA/B serum preparation. Western blotting with the serum showed that in cells growing at 30°C IbpA/B were located in the bacter'al outer membrane and appeared in the S fraction after heat shock. Then the cellular level of the IbpA/B proteins increased about 20-fold as estimated by densitometry of the Western blots. In the E coli rpoH strain the level of IbpA/B was higher than in wild type before the heat shock and rose to still higher levels after it. This result pointed to a regulation of ibpA/B operon by another factor, besides that of t~32. heat shock / IbpA/B localization / outer men, brahe / I~pA/B level / rpoH

Introduction

Aggregation of cellular proteins upon heat shock could be observed in vivo in Escherichia coli cells as shown by Kucharczyk et al [1]. The aggregated proteins formed a fraction, denoted S, which cosedimented with the membrane fraction in a two-step sucrose density gradient but could be separated in refined gradients. The S fraction appeared in growing Escherichia coil wild type (wt) strains, 15 min after the temperature change from 30°C to 45°C and disappeared rapidly (in 10 min) after transfer to 37°C. The S fraction in rpoH mutants (unable to induce the stress response) was larger than in wt strains and its removal was blocked [1]; it was also retarded by mutations affecting heat-shock inducible proteases. About 30%

*Correspondence and reprints Abbreviations: 2D, two-dimensional; aa, amino acid(s); AU, arbitrary unit(s); CP, cytoplasmic and periplasmic protein fraction; Ibp, i_nclusion _body-associated 12rotein; IM, inner membrane; OM, outer membrane; PAGE, polyacrylamide-gel electrophoresis; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid.

of the proteins of the S fraction were convertible into TCA-soluble material by HtrA protease in vitro (E Laskowska, D Kuczyfiska-Wi~nik, J Sk6rkoGionek, A Taylor (1996) submitted). The S fraction contained numerous proteins visible on gel electropherograms. Heat-shock proteins DnaK and DnaJ bound to these proteins as was shown by ~ tern blotting with corresponding antibodies. The other heat shock proteins, GroEL/ES, GrpE were not associated with the fraction [1]. Supposed'y the removal of the aggregates involved renaturation and proteolysis. To learn more about the process we tried to isolate and purify a protein from the S fraction which would serve as a model for further research on intracellular, thermal denaturation and cell protection from its consequences. The 16 kDa protein, forming one of the strongest bands in the electrophoretic separation pattern of the S fraction, from wt as well as from rpoH mutant, was chosen. The 16 kDa band was found to contain two proteins of similar Mrs separable on 2D gels. Characterization of the proteins revealed their identity with IbpA (HslT) and IbpB (HslS) the newly discovered heat-shock proteins (described as inclusion body associated 12roteins; ibp), recognizing heterologous proteins in E coil cells [2-4]. Our original pur-

118 pose was missed, since we did not isolate a model protein undergoing denaturation, however, we have gained new evidence of a possible physiological role of the IbpA/B proteins in bacterial cell protection. T h e y might be involved, together with D n a K and D n M , in the quick r e m o v a l of the a g g r e g a t e d proteins. W h e t h e r their activity leads to renaturation or proteolysis and whether they interact with other heat-shock proteins, remains to be answered. It was shown [2-5] that ibpA and ibpB genes formed one operon regulated by the rpoH gene product, 632 protein, a positive regulator of the heatshock response. We have d e m o n s t r a t e d that the lbpA/B proteins were produced in E coli wt and in the rpoH mutant in detectable amounts, prior to heat shock and then localized to the bacterial outer m e m brane (OM). After heat shock they also appeared in the S fraction. Their cellular level increased considerably in E coil wt as well as in the rpoH mutant after heat shock.

Materials and methods Bacterial strains, growth conditions, subcellularfractionation and preparation of whole cell lysates for 0.1% SDS-15% PAGE Escherichia coil B178 wt (W31OlgalE sup+) was obtained from B Lipifiska (Gdafisk, Poland) [6], CG459 (K165)

rpoH(Am) and its parent strain CG458 (SC122) iac(Am) trp(Am) pho(Am) real(Am) supC(Ts) from D A n g (Geneva, Switzerland) [7, 81. The bacteria were grown in 100 ml of LB medium [91 at 30°C with aeration to Am = 0.25, then shifted to 45°C for 15 rain and further grown at 37°C. Samples were removed at time 0 (before the temperature shift to 45°C), after 15 rain at 45°C, and at 25th and 35th min during growth at 37"C for either cell fractionation (100 ml) or for preparation of whole cell lysates (1 ml). Cell harvesting, gentle lysis, fractionation by ultracentrifugation in sucrose density gradients and control of separation exactness was performed as described earlier [ 1]. Briefly, samples were chilled quickly to 4°C and the cells were sedimented and subjected to spheroplastization in 1 M sucrose solution, using egg white lysozyme in Tris-HCl buffer (pH 8) supplemented with EDTA, according to Witholt et al [10]. The spheroplasts were subjected to sonication in a Vibra cell 72408 (Bioblock Scientific, France) sonifier. Unbroken cells were removed by centfifugation and the supernatant was used for the membrane fractionation, essentially as described by lshidate et al[ 111 and modified by Kucharczyk et al [I ]. The supernatant was layered on a two-step sucrose density gradient (SG0) and centrifuged for 90 min in a Beckman SW41 Ti rotor at 240 000 g. Four 1 ml CP subfractions were collected from the top of the gradient; they contained soluble cytoplasmic and periplasmic proteins. Opalescent fractions of OM and IM, also containing the heat-aggregated proteins were collected together (crude membrane fraction) and submitted to fractionation in a six-step sucrose density gradient (SGI) (for details see [ 1]). After centrifugation in a Beckman SW41 Ti rotor at 240 000 g for 16 h, 30 subfractions were collected from the bottom (total volume 12 ml). Aliquots were taken from the SGI subfractions for determination

of protein concentration, NADH-oxidase activity and refractive index for calculation of sucrose density. The subfractions corresponding to four discernible fractions wele pooled and denoted S (density, 1.26 g ml-l), OM (1.22 g ml-l), A (1.18 g ml -l) and IM (1.14 g ml-l). The detailed analysis of the fractions was presented in [1]; it was shown by lipid labelling with [laC]-glycerol that the S fraction did not contain the following: lipid (contrary to OM and IM), NADH-oxidase activity (the marker of IM), the OmpA protein (the OM marker, detected by Western blotting with the corresponding antibody) nor lipopolysaccharide (LPS), as assayed by the Limulus test [12]. Therefore, it was concluded that the S fraction contained only protein, though it cosedimented with the membranes in SG0. The whole cell iysates were prepared for estimation of induced synthesis of 16 kDa protein(s). A600 was the measure of the cell number (1 A6oo = 8 x 108 cells/ml). The bacteria were quickly sedimented, resuspended in 40 Bi of lysis buffer [131, heated (100°C, 5 rain) and used for 0.1% SDS-15% PAGE. An equivalent of 108 bacterial cells was used for each well.

Protein purification 16 kDa protein was isolated from the S fraction of B 178 wt, at the time of its maximal development (15th min at 45°C). i) For antigen preparation, for rabbit immunization, proteins of the S fraction were separated by 0.1% SDS-15% PAGE [13] (60 V/ era, 4 h, at room temperature), electrotransferred to PVDF membrane (Milipore Co, Bedford, USA) stained with PonceauS (Sigma, St Louis, USA) cut out and eluted [14]. ii) For microsequencing the 16 kDa proteins were isolated from twodimensional gel. Isoelectrofocusing in the first dimension was performed according to O'Farrell [151 (1.6% pH 6-8 and 0.4% pH 3.5-10 carrier ampholyte mixture; Pharmacia, Uppsala, Sweden) and in the second dimension as described above. Electrotransfer to PVDF was carried out by the method described by Wilson and Yuan [16].

Preparation of polyclonal antibodies, and immunodetection of 16 kDa (lbpA/B)proteins The use of eluted protein as immunogen, rabbit immunization and immune serum testing, was carried out by standard techniques [14]. 200 lag of eluted 16 kDa protein was used for a two-fold injection. Thus obtained, anti-IbpA/B-serum reacted with the immunogen (0.5 lag) in a dilution of 1:200. Undiluted aliquots were stored at -70°C. For IbpA/B immunodetection the anti-lbpA/B antibody, followed by anti-rabbit IgG peroxidase conjugate (Sigma, St Louis, USA) as second antibody, wi~h 4-chioro-l-naphtol and H202 (Serva, Heidelberg, Germany) as substrates were used.

Analytical techniques Protein microsequencing was performed in a 473A Applied Systems proteins sequencer (A Wawrzyn6w). A sample for the microsequencing was prepared as described in [16], Blotted spots on PVDF were introduced directly into the protein sequencer. Densitometry was carried out by scanning electropherograms by the UVP (Ultra-Violet Products) EASY densitometry system (Cambridge, UK). The Laemmli method [131 was used for 0.1% SDS-15% PAGE, and O'Farrell [15] for 2D electrophoresis. Silver staining was according to [17]. Total protein was determined by the Bradford method [I 8].

119

Results and discussion

Microsequencing of 16 kDa proteins and their identification as IbpA and IbpB proteins For microsequencing, the 16 kDa proteins were isolated from the S fraction obtained from B178 after heat shock (fig !A). The following N-terminal, 14-aa sequences of the proteins were determined:

i) M R N F D L S P L Y R S A I . . . ii) M R N F D L S P L M R Q W I . . . The computer search of the Swiss Prot database by FASTA program revealed that the N-terminal sequences were identical to those of IbpA and IbpB proteins, respectively. The genes of ibpAB [2] or hslTS [3, 4] operon were mapped at 82.5 rain on the E coli chromosome. The proteins IbpA and IbpB showed 52% identity of the aa sequence and therefore cross-reacted with antibodies [2].

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Fig 1. Electrophoretic separation i,attem of heat-aggregated proteins (S fraction) and immunodetection of the IbpA/B proteins in subcellular fractions of E coli. A. 20 l.tg of protein (S fraction) was separated by 0.1% SDS-15% PAGE and silver stained [17]. 16 kDa proteins (lbpA/B) are marked by arrow heads. B. The bacterial cultures were grown in LB [9] at 30°C to A600 = 0.25 (0-time), then shifted to 45°C for 15 min and further grown at 37°C. Time at which samples for fractionation were removed from the bacterial culture is marked on the top. Subcellular fractions: OM, IM, S and CP were separared by sucrose density gradient centrifugation and the exactness of the separation was checked by determination of sucrose density of the fractions (S fraction, 1.26 g ml-i; OM, 1.22 g ml-l; IM, 1.14 g ml-l) and NADH-oxidase activity (absent from S and OM, present in IM) [1] (see also page 4). Each fraction was subjected to 0.1% SDS-15% PAGE, and Western blotting with anti-IbpA/B serum in a 1:200 dilution followed by goat-anti-rabbit IgG-HRP (Sigma, St Louis) as the second antibody and 4-chloro-lnaphtol and H202 as substrates. The purified IbpA/B protein was used as control (B, on the left side).

120

CeH fractionation and localization of IbpA/B proteins in subcellular fractions The wt cell fractionation according to Kucharczyk et al [ 1] in the first, two-step sucrose density gradient resulted in separation of a fraction CP from an insoluble, crude membrane fraction, containing cosedimenting aggregated proteins. The insoluble fraction was further fractionated in the six-step sucrose density gradient. Cells fractionation from 0-time culture yielded the fractions OM and IM. In the sample removed after 15 min at 45°C a new fraction denoted S appeared. Its buoyant density (1.26 g/ml-~) was higher than that of OM (1.22 g/ml-n). It was proved that it does not contain any membrane fragments but consists of numerous aggregated proteins. Their state of aggregation prevented entry into 8% nondenaturing gel thus leaving the path empty [ 1]. Figure 1 presents the electrophoretic separation pattern of the S fraction proteins from B 178 w~ in denaturing gel (A) and the immunoblotting of proteins of subcellular fractions: S, OM, IM, and CP with antiIbpA/B antibodies (B). The 16 kDa proteins, purified by elution from one-dimensional gel served as the marker. At 0-time (30°C) the S fraction was absent (the corresponding subfractions were devoid of protein) IbpA/B proteins were present exclusively in OM. After heat shock, at time of the maximal development of the S fraction (the 15th min at 45°C) IbpA/B proteins were found in both OM and S fractions, a faint spot appeared also in CP. At the 25th and 35th rain (37°C) the S fraction was already absent (no protein detectable in the corresponding subfractions), but IbpA/B proteins were well visible in OM and as faint bands in CP. This showed the ability of lbpA/B proteins to associate with the heat-aggregated cellular proteins. The lbpA/B protein band from different cellular fractions shows a slight difference in its mo~ility in gel electrophoresis (fig 1), We suppose that i~ is an artefact arising from the presence of membrane components in OM fraction, which could interact with lbpA/B proteins affecting their mobility. Our attention was turned to differences in the intensity of the protein bands in the time course of the experiment,

Estimation of lbpAIB protein levels in Escherichia coli wt (CG458) and rpoH mutant (CG459) Increase of the IbpA/B protein levels before and after heat shock was estimated in samples of whole cell lysates removed from the CG458 (wt) and CG459 (rpoH-) cultures: i) grown at 30°C, immediately before heat shock (time 0); ii) 15 min after transfer to

45°C; and iii) at 25th and 35th rain of culture growth at 37°C (fig 2). The level of IbpA/B was hardly detectable in the lysate of the wt strain (though, measurable as 3 AU) at 0-time. However, IbpA/B proteins were clearly visible at this time in OM (fig 1). The rise of the protein level followed the kinetics of the heat shock response in the wt strain: it reached a maximum after the 25th min of the experiment (57 AU) and dropped in the next 10 min (39 AU). But it was striking that in rpoH, the IbpA/B protein level was remarkably higher than in wt at 0-time (32 AU), and rose further up to 100 AU in the 35th min of the experiment. Three repetitions gave comparable results. Though the densitometry of Western blots cannot be considered as a quantitative analytical techpique, it allows for comparative estimation. The increase of IbpA/B proteins in the rpoH strain did not seem to fit the expression of an operon regulated solely by 632. The presence of the heat-shock promoter recognizable by a32 upstream of the ibpA/B operon was documented [2-5], therefore one might expect that more complicated regulation also involved other factor(s). Genes of heat-shock regulon may have two or more differently regulated promoters [19]. It was tempting to speculate on possible 6 E involvement, because of a notion that it may be res-

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Fig 2. lmmunoblotting of IbpA/B proteins and estimation of their level in CG458 (wt) and CG459 (rpoH). The bacterial cultures were grown and heat treated as described in the legend to figure 1. The whole-cell lysates were prepared from 1 ml samples of the cultures removed at the times indicated on the top. Electrophoresis and immunoblotting was carded out as described in the legend to figure 1. The densitometrically estimated IbpA/B protein level in each sample is expressed in arbitrary units at the figure's bottom.

121 ponsible for processes occurring in th~ extracytoplasmic space [20-23] to which IbpA/B and the OMproteins belong. The slight induction of ibpAB operon in the rpoH mutant was visible on a Western blot shown by Allen et al in figure 8B [2], though it was not considered in the discussion of their results. The IbpAB protein level was considerably lower in the rpoH mutant than in wt strain in their experiment, contrary to our results. The discrepancy is explainable by the different conditions of the experiments. We used LB medium and the bacterial cultures were submitted to heat shock at a density corresponding to A600 = 0.25, while M9 was used in [2] and heat shock was applied at the culture density equal to 125 Klett U (As.~0= 1.1 ). Moreover, the temperature regime was different since after heat shock growth was continued at 42°C in [2]. Nevertheless, it follows from both types of experiments that the level of IbpAB increased in the rpoH mutant cells with the course of time after heat shock. The presence of IbpAB, the recently discovered heat-shock proteins in the S fraction, suggest that they may be involved in the removal or renaturation of the heat-aggregated proteins. However, elucidation of the role of IbpAB, needs further efforts. At present it is not even known whether they may have proteolytic or chaperone activities, or both, as was found recently for CIpX [ 24] and CIpA [25].

Conclusions 1) Two 16 kDa proteins constituting one of the strongest bands in the protein separation pattern of the S fraction (intracellularly heat-aggregated, endogenous proteins), are identical with IbpA/B heatshock proteins as found by aa microsequencing and the search of the Swiss Prot data base by FASTA program. 2) lbpA/B proteins were localized to cell OM. After heat shock they were also found associated with proteins of the S fraction of E coli. This seems to paint to their physiological role. Since the aggregated proteins were shown to disappear rapidly from wt cells, one may suppose the IbpA/B participate in renaturation or proteolysis of the aggregated, endogenous proteins. 3) In wt cells of E coli the lbpA/B protein level rose after heat shock. 4) Contrary to expectations, after heat-shock, the level of the IbpA/B proteins also rose in the rpoH mutant. The heat-shock promoter, recognizable by ¢~32 was identified upstream of the ibpAB operon by [2-5] and its inducibility was delr:onstrated [2-4]. Our results indicate that some unidentified factors also participate in the ibpA/B operon regulation.

Acknowledgments This work was supported by grants from the Polish Committee for Scientific Research (KBN, No 4 4386 91 02) and the Foundation for Polish Science (BIMOL 20/93). For the bacterial strains we thank B Lipifiska (Gdafisk, Poland) and D Ang (Geneva, Switzerland).

References 1 Kucharczyk K, Laskowska E, Taylor A (1991) Response of Escherichia coli cell membranes to induction of iambda cl857 prophage by heat shock. Mol Microbiol 12, 2935-2946 2 Allen SP, Polazzi JO, Gierse JK, Easton AM (1992) Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli..I Bacteriol 174, 6938-6q47 3 Chuang SE, Burland V, Plunkett G llI, Daniels DL, Blattner FR (1993) Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli. Gene 134. 1-6 4 Chuang SE, Blattner FR (1993) Characterization of twenty-six new heat shock genes of Escherichia coli. J Bacteriol 175, 5242-5252 5 Burland V, Plunkett Ill G, Daniels DL, Blattner FR (1993) DNA sequence and analysis of 136 kilobases of the Escherichia coil genome: organizational symmetry around the origin of replication. Genomics 16, 55 !-556 6 Lipifiska B, Fayet O, Baird L, Georgopouios C (1989) Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J Bacteriol 171, 1574-1584 7 Cooper S, Ruetinger T (1975) A temperature sensitive nonsense mutation affecting the synthesis of a mayor protein of Escherichia coli K 12. Mol Gen Genet 139, 167-176 ~' Neidhardt FC, van Bogelen RA, Lau ET (1983) Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli. J Bacteriol 153.597-603 9 Sambrook J, Fritsch EE Maniatis T (1989) Molecular cloning. A Laboratory matmal, 2nd edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY lO Witholt B. Boekhout M, Brock M, Kingma J. van Heerikhuizen H. de Leij L. (1976) An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem 74, 160-170 I| lshidatc K, Crcegcr ES, Zrike J, Deb S, Glauner B, MacAlister TJ. Rothfield LI (1986) Isolation of differentiated membrane domains from Escherichia coli and Sabnonella ~,phimurium. including a fraction containing attachIlllen| sites between the inner and OIlier nlenlbl'ane~ i|nd IJle iiltlreiil skeletotl ol the cell envelope..! Biol Chem 261,428--443 12 Yin TE, Galanos C, Kinsky S, Bradshaw RA, Wessler S, Luderitz O. Sarmiento M (1972) Picogram sensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid A from Gram-negative bacteria. Biochem Biophys Acta 261, 284-289 13 Laemmli UK (1970) Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 227, 680--685 14 Szewczyk B, Harper DR (1993) Use of blotted proteins as immunogens. In: Protein blotting - a practical approach (Dunbar B, ed) IRL Press, Oxford. 189-205 15 O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007--402 I 16 Wilson KJ, Yuan PM (1989) Protein and peptide purification. In: Protein sequencing, a practical approach (Findlay JBC. Geisow MJ. eds) Oxford Univ Press, New York, 1--41 17 Hempelmann E, Kaminsky R (1986) Long term stability of colours after silver staining. Ehwtrophoresis 7, 481 18 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254

122 19 Georgopoulos C, Liberek K, Zylicz M, Ang D (1994) Properties of the heat shock proteins of E~cherichia coli and the au,oregulation of the heat shock response. In: The biology of heat shock proteins and molecular chaperones (Morimoto RI, Tissieres A, Georgopoulos C, eds) Cold Spring Harbor Laboratory Press, New York, 209-249 20 Wiilfing C, PlUckthun A, (1994) Microreview: Protein folding in the periplasm of Escherichia coli. Mol Microbiol 12, ¢~85-692 21 Mecca,s J, Rouviere PE, Erickson JW, Donohue TJ, Gross CA (1993) The activity of o E, an Escherichia coil heat-inducible a-factor, is modulated by expression of outer membrane proteins. Genes Dev 7, 2618-2628

22 Rouviere PE, De las Penas A, Mecsas J, Lu CZ, Rudd KE, Gross CA (1995) rpoE the gene encoding the second heat-shock sigma factor, c E, in Escherichia coli. EMBO J 14, 1032-1042 23 Raina S, Missiakas D, Georgopoulos C (1995) The rpoE gene encoding the lie (¢~24)heat shock sigma factor of Escherichia coli. EMBO J 14, 1043- 1055 24 Wickner S, Gottesm~n S, Skowyra D, Hoskins J, Mckenney K, Maurizi MR (1994) A molecular chaperone, CIpA, functk, :s like DnaK and DnaJ. Proc Natl Acad Sci USA 91, 12218-12222 25 Levchenko I, Luo L, Baker TA (1995) Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev 9, 2399-240