Proc. Natl. Acad. Sci. USAVol. 83, pp. 6959-6963, September 1986Genetics

Heat shock response in Escherichia coli influences cell division(htpR mutant/fam mutant/filament formation/lipoprotein)

TETSUAKI TSUCHIDO*, RUTH A. VANBOGELEN, AND FREDERICK C. NEIDHARDTDepartment of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109-0620

Communicated by Masayasu Nomura, June 2, 1986

ABSTRACT Analysis of a mutant in fam, a pleiotropicgene affecting cell division in Escherichia coli, revealed that thisgene is probably identical to the heat shock regulatory genehtpR. The fam-715 mutant and different htpR mutants werefound to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce thenormal cellular heat shock response. These defects were allcorrected in fam and htpR mutants by complementation withplasmids carrying intact htpR+ or by recombination betweenthese mutant alleles and a plasmid carrying only a portion ofhtpR. These results implicate the E. coli heat shock system inthe regulation of cell division and raise the question of a similarrole in other organisms.

Thefam-715 mutant was originally isolated as a cell envelopemurein lipoprotein mutant (1). Lipoprotein is numerically themost abundant protein in Escherichia coli (2). Its aminoterminus is a lipoamino acid containing three fatty acidresidues attached to glycerylcysteine and is anchored in thehydrophobic domain of the outer membrane (3). At itsopposite end, some, but not all, of the lipoprotein is cova-lently attached through its terminal lysine to the meso-diaminopimelic acid residue of the murein peptidoglycan (4).This bound form of the lipoprotein constitutes the majorattachment between the outer membrane and the mureinsacculus.Mutants in the lipoprotein structural gene (lpp), which

synthesize an abnormal product virtually unlinked to themurein peptidoglycan (and originally designated mipA) (5),and a mutant, which totally lacks all lipoprotein (originallycalled Ipo) (6), exhibit leakage of periplasmic proteins duringgrowth, have increased sensitivity to a variety of toxic agentsand to low ionic strength, and exhibit defective formation ofthe division septum resulting from faulty invagination of theouter membrane (7, 8). Mutants (IkyD) in Salmonellatyphimurium that lack bound lipoprotein, perhaps becauselipoprotein is not linked to murein, display the same char-acteristics (7). All these mutants can still grow and divideand, therefore, the role of the lipoprotein has been regardedas facilitative rather than absolutely essential in differentia-tion of the division septum and maintenance of the integrityof the cell envelope (6, 7).The properties of the fam-715 mutant did not fit this

picture. This lesion is an amber mutation in a strain carryinga temperature-sensitive amber suppressor. At restrictivetemperature the cells cannot grow, and yet have only a fourtimes reduction in the amount of bound lipoprotein (1). Theparadox, that this mutant is more severely growth restrictedthan the Ipo mutant totally lacking lipoprotein, was partiallyresolved when the fam-715 lesion was found not to be in thestructural gene for lipoprotein (9). It has since been generallyassumed that the fam-715 mutation is in a pleiotropic regu-latory gene somehow involved in cell division (6, 9). The

decreased lipoprotein synthesis in the mutant could then beregarded not as the cause of the cell division defect, but eitheras an independent effect of this same regulatory gene or as anindirect consequence of the defect in cell division.The fam gene has been mapped to a region of the chro-

mosome that also contains htpR (9, 10), the gene for theregulatory protein of the heat shock regulon (11). A condi-tional mutation in this gene, htpR165, fails to induce 17proteins that are normally stimulated 5- to 15-fold after a shiftfrom 280C to 420C (12, 13). Eight of the 17 proteins in thisregulon have been identified (reviewed in ref. 13) and appearto be involved in the major macromolecular processes of thecell (DNA replication, RNA synthesis, protein synthesis,protein processing and assembly, and protein degradation).Since both ofthese genes,fam and htpR, have been describedas pleiotropic regulatory genes, we decided to determine therelationship between them.

MATERIALS AND METHODSBacterial Strains and Their Construction. The E. coli htpR

mutant strain, K165, and its parent, SC122, have beendescribed (11, 14). E. coli strain ST715, carrying thefam-715mutation, has been described (1, 9); it was kindly provided byJames T. Park (Tufts University School of Medicine). Con-struction of recA56 derivatives of strains was done byconjugation using strain JC10240, which is an Hfr straincontaining recA56 srJ300::TnJO. Introduction of other plas-mids into various strains was done by transformation usingconventional methods (15), and drug-resistant transformantswere selected.Media and Culture Conditions. Liquid cultures were grown

aerobically at specified temperatures in rotary water bathshakers in defined MOPS medium with 0.4% glucose andsupplemented with 19 amino acids (minus methionine), fivevitamins, and four bases (16).

Labeling and Resolution of Cellular Proteins on Two-Dimensional Gels. Cultures were grown at 280C to an OD420of 0.3 and then shifted to 44°C. A 2-ml sample was pulsed for5 min with [35S]methionine (1098 Ci/mmol; 50 ,uCi/ml; 1 Ci= 37 GBq) starting 3 min after the temperature shift, and thenchased with 0.167 ml of 0.2 M methionine. Extracts ofcultures were prepared as described (17), and proteins wereresolved on O'Farrell two-dimensional polyacrylamide gels(18).

Microscopic Examination of Cells. Cultures were grown at28°C to an OD420 of 1.0. The cultures were then shifted to42°C or 44°C. Cultures were diluted at various times tomaintain the optical density between 1.0 and 2.0. Sampleswere taken prior to the temperature shift and at 30-minintervals after the shift for observation in a Leitz phase-contrast microscope.Measurement of Cell Number. Cultures were grown aero-

bically in supplemented MOPS medium at 28°C to an OD420

*Present address: Department of Fermentation Technology, OsakaUniversity, Osaka, Japan.

6959

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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6960 Genetics: Tsuchido et al.

of 0.3 and then shifted to 42°C or 44°C. Cell number wasdetermined in a model ZB Coulter Counter after diluting the

Proc. Natl. Acad. Sci. USA 83 (1986)

samples appropriately in a NaCl (8.5 g/liter)/formaldehyde [5ml of 40% (vol/vol) per liter] solution.

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FIG. 1. Synthesis of individual proteins in various E. coli strains after a shift from 28°C to 44C. The autoradiograms show the incorporationof [35S]methionine during a 5-min period either before the shift or from 3 to 8 min after the shift to 44°C. (A) Strain M7 (fam+), 28°C. (B) Same,44°C. (C) Strain ST715 (fam-715), 44°C. (D) Strain ST715 (fam-715) with plasmid pFN97 (htpR+) at 44°C. The pattern of synthesis in both ofthe mutant series of 28°C resembled that of strain M7 (A). The boxes surround heat shock proteins (13), which are numbered in B as follows:1, B25.3; 2, B56.5 (groEL); 3, B66.0 (dnaK); 4, C14.7; 5, C15.4 (groES); 6, C62.5; 7, D33.4; 8, D48.5; 9, D60.5 (lysU); 10, F10.1; 11, F21.5;12, F84.1; 13, G13.5; 14, G21.0; 15, H94.0 (lon). The protein product of recA is circled.

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Proc. Natl. Acad. Sci. USA 83 (1986) 6961

RESULTS

The fam-715 Mutant Is Defective in the Heat Shock Re-sponse. Strain ST715 (fam-715) was grown at 280C and shiftedto 440C. Growth continued at the higher temperature, but atonly one-fifth the rate exhibited by the wild-type parent strainM7. Samples were labeled for brief periods before and afterthe shift and processed by two-dimensional gel electropho-resis to reveal the pattern of synthesis of individual proteins.The results (Fig. 1) showed that heat shock proteins in thefam mutant, with the exception of the IysU gene product,were not induced.The fam-715 Mutation Is Complemented by htpR' and

Yields Wild-Type Recombinants by Crosses with Part of thehtpR' Sequence. Hybrid plasmids containing various seg-ments of DNA in the htpR region were transformed intostrain ST715 (fam-715) and strain K165 (htpR165) and theirrecA derivatives. Transformants were isolated by selectionfor ampicillin resistance. The chromosomal segments carriedby the various plasmids are shown in Fig. 2.

Transformants containing the plasmid pFN97, which car-ries the wild-type htpR gene, grew at high temperature andexhibited a normal heat shock response in both recA andrecA' recipients. Plasmid pFN92, which carries all but theterminal 15 C-terminal amino acid codons of the htpR geneand which produces a biologically inactive fusion protein(19), did not complement the fam-715 lesion in recA recipi-ents but did yield wild-type recombinants in recA' cells. Thelatter result shows that pFN97 is not merely suppressingfam-715 by overproduction of the htpR gene product, but istruly complementing the mutation. Since the htpR fragmentis the only segment carried by pFN92, the fam-715 lesionwould seem to be within the htpR gene.The htpR Heat Shock Mutant Is Defective in Cell Division.

Cultures of strain K165 and its parent, SC122, and of strainST715 and its parent, M7, were grown at permissive temper-ature (280C) and then shifted to restrictive temperatures (420Cand 440C, respectively). Samples were removed at intervalsfor phase-contrast microscopy and for enumeration using aCoulter counter (Fig. 3). At restrictive temperature eachmutant grew in filaments that in the two mutants were similarin appearance. Both normal parental strains displayed animmediate shift in rate of increase in mass (optical density).

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FIG. 3. Growth and cell division of htpRl65 andfam-715 mutantsafter a temperature shift. Cultures were grown aerobically in glucose-supplemented MOPS medium at 280C to an OD420 of 0.3 and thenshifted to 420C (A and C) or 44TC (B and D). Cell number wasdetermined on a Coulter counter Model Z13. (A and C) A shift from280C to 420C in the following strains: o, SC122 (htpR+); z, K165(htpR165); and A, K165 containing plasmid pFN97 (htpR+). (B andD) A shift from 280C to 440C in the following strains: c, M7 (fam+);7, ST715 (fam-715); and a, ST715 containing pFN97 (htpR+). In allpanels * represents the parental strain at 28TC.

Cell division continued at the pre-shift rate for approximately45 min before accelerating to its final, post-shift rate. Al-though the rate and extent ofgrowth at high temperature weredifferent in the two mutants, both exhibited a markedinhibition of cell division at the time of the shift and neverrecovered from this state despite, at least in the case of thefam mutant, extensive mass increase. The mutant strainscontaining normal htpR on plasmid pFN97 behaved identi-cally to their respective, normal parents. In addition, thehtpR165 allele in a strain (CAG2046) with a "cleaner" geneticbackground (21) exhibited the response described for strainK165.

DISCUSSIONThe heat shock response is a universal cellular response toabrupt, increases in the environmental temperature of anorganism (13, 22, 23). Despite intensive work on the natureof the genes involved in the response and the mechanism oftheir control, no central role for the heat shock response inkeeping with its genetic conservation has emerged. To date,most ideas have centered on DNA damage (24, 25) andprotein denaturation (26-28), two cellular events caused byagents that induce the heat shock response. These ideas havemerit, but need to be more fully developed and tested.Mutants of the bacterium E. coli deficient in the heat shock

response are potentially useful for this problem because theyoffer the possibility of learning what consequences befall acell unable to respond normally to increased temperature.These mutants have been used to identify the regulatorygene, htpR, and to map (11, 29), clone (19), and sequence (20,30) it. This gene is now known to encode an alternative o-likesubunit of RNA polymerase (20, 31). The chromosomalregion around htpR includes genes (ftsE, ftsS, dnaM, and

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Proc. Natl. Acad. Sci. USA 83 (1986)

fam) implicated in cell division and in the control of lipopro-tein synthesis and cell lysis (10). It has become important toclarify the relationship between these genes and htpR. Ourstudy of one of these genes,fam, indicates that it is identicalto htpR.Thefam-715 mutation ofE. coli was isolated in a search for

mutants defective in murein lipoprotein synthesis (1). Thisamber mutation is carried in a strain, ST715, containing atemperature-sensitive amber suppressor. At restrictive tem-perature the mutant cells form filaments and produce abnor-mally low quantities of murein lipoprotein (9). Whether thelow lipoprotein level is responsible for the cell division defectis uncertain, because a mutant with a deletion in the struc-tural gene for lipoprotein, Ipp, grows normally (6). On theother hand, different Ipp mutants do exhibit cell divisiondefects (5, 7, 8). It is known thatfam is not the structural genefor lipoprotein, but rather is a gene with a processing orregulatory function that affects one or more proteins neces-sary for cell division (9).From the evidence in this paper we believe that htpR is the

regulatory gene postulated to harbor the fam-715 mutation.The phenotypes offam-715 and htpR-165 are virtually indis-tinguishable. Both the cell division defect and the heat shockdefect are exhibited by each mutant. The only difference inphenotype is that in the fam-715 mutant one can detectinduction at high temperature of one particular heat shockprotein, tRNALYs syntase form II, the product of the lysUgene (Fig. 1). This protein, however, is known to differ fromthe other 16 heat shock proteins in being induced by a varietyof conditions (including growth in the presence of peptidessuch as glycylleucine, and mutational lesions in metK), whichleave the others unchanged (32). We assume that strainST715 at 44TC produces some internal condition that acti-vates lysU by this alternative mechanism. The genetic evi-dence strongly indicates that the fam-715 lesion is withinhtpR, specifically in the fragment carried by plasmid pFN92.Two related questions are raised by our findings. First, is

murein lipoprotein a heat shock protein? If it were, thereduced synthesis observed in the fam-715 cells at hightemperature would seem to be readily explained as a conse-quence of reduced amount of htpR gene product. Two factsargue somewhat against this interpretation. The promoter of

-35 -10lpp (TTCTCA; TGTAAT) (33) resembles more the normal(o-70) promoter consensus than that of the known heat shockpromoters (34). Second, the known heat shock proteinsvisible on gels display continued synthesis after the shift to44°C (see Fig. 1), while lipoprotein synthesis actually declines(1). Neither of these facts rigorously excludes lpp as aheat shock gene, and direct measurement of transcription invivo and in vitro is needed to settle the point.Whether or not lpp is a heat shock gene controlled by htpR,

what the involvement of htpR is in cell division has becomethe paramount question. Our results show that failure tomount a heat shock response is accompanied by faulty celldivision and consequent filament formation after a shift up intemperature. This defect in cell division may be due to thefailure to induce one or more of the heat shock proteins.None have a known function in cell division, but we haveobserved that temperature-sensitive mutants in three differ-ent heat shock proteins (products of dnaK, groEL, andgroES) form filaments at restrictive temperature (unpub-lished experiments).

Alternatively, the cell division defect in htpR mutantsmight be caused by activation of some other damage controlsystem that blocks cell division, such as the SOS response(35). In this regard, inspection of the data in Fig. 1 shows thatthe product of recA, a gene of the SOS regulon, is not inducedby the growth-restricting conditions in htpR mutants. Othercell division regulators, however, could be playing a role.

That temperature has a profound effect on cell division iswell known in both prokaryotes and eukaryotes. Normal E.coli cells exhibit a transient tendency to elongate upon anincrease in temperature (Fig. 3 and refs. 36 and 37). Thisbehavior is in fact quite widespread among bacteria and haseven been reported for psychrophiles and psychrotrophsexposed to temperature near their upper limit (38-40).Despite obvious differences in the details of how bacteria,protozoa, and mammalian cells divide, in all of them tem-perature shifts of one kind or another have been reported toinduce division synchrony (41). Several models of cell divi-sion have been built on this observation (41). It is tempting tospeculate that the major mystery of the heat shock re-sponse-its universal genetic conservation-has its basis inpart in some universal characteristic of cell division.

We are grateful to Vicki Vaughn for construction of plasmidpFN119 and to A. J. Clark, C. A. Gross, and J. T. Park for bacterialstrains. This investigation was supported by Grant GM 17892 fromthe National Institutes of Health (F.C.N.). The Japanese Ministry ofEducation is thanked for providing a fellowship to T.T.

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