Vol. 176, No. 12

Growth Phase Variation of Integration Host FactorLevel in Escherichia coli

MARY D. DITTO,'t DENISE ROBERTS,2t AND ROBERT A. WEISBERG`*Section on Microbial Genetics, Laboratory of Molecular Genetics, National Institute of Child Health and

Human Development, Bethesda, Maryland 20892,1 and Cold Sfring Harbor Laboratory,Cold Spring Harbor, New York 11724

Received 12 January 1994/Accepted 6 April 1994

We have measured the intracellular abundance of integration host factor (IHF), a site-specific, het-erodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12.Western immunoblot analysis showed that cultures that had been growing exponentially for severalgenerations contained 0.5 to 1.0 ng of IHF subunits per ,ug of total protein and that this increased to 5 to 6ng/,ug in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentiallygrowing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-typeIHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of theother when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactionsbetween subunits. A mutation that inactivates IHF function but not subunit interaction increased IHFabundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. Weestimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, asurprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites.Nevertheless, small reductions in 11I1F abundance had significant effects on several IHF-dependent functions,suggesting that the wild-type exponential phase level is not in large excess of the minimum required foroccupancy of physiologically important IHF-binding sites.

Integration host factor (IHF) of Escherichia coli is a smallsequence-specific DNA-binding protein. It is a member of thehistone-like family of prokaryotic DNA-binding proteins,which includes the abundant and widely distributed HU pro-tein (12). IHF was first identified and isolated because of itsessential role in X integrative recombination (78). It hassubsequently been shown to play a role in phage DNApackaging, plasmid maintenance, and bacterial gene expres-sion but is not required for cell viability (15). IHF is aheterodimeric protein encoded by two genes, himA and hip (orhimD), located at 38 and 25 min, respectively, on the E. colichromosome (35, 45). The subunits encoded by these genes,IHFot and IHFP, respectively, form a heterodimer that bindsand bends DNA at specific sites (57, 59, 67). IHFa and IHF,are similar to each other, but both are required for IHFfunction in vivo. The related HU protein is a homodimer (or aheterodimer of nearly identical subunits in some bacteria) thatbinds to DNA with little or no sequence specificity (6, 10).Comparison of many IHF-binding sites reveals considerablesequence conservation, and statistical analysis of these se-

quences suggests that the E. coli chromosome contains 80 to100 binding sites (19). Indeed, there is experimental evidencethat IHF interacts with many sites on the bacterial chromo-some and, by so doing, directly affects the expression of manygenes (5, 10a, 14).

In view of the many biological functions that are influencedby IHF, we decided to measure its cellular abundance undervarious conditions of growth and in different genetic back-

* Corresponding author. Mailing address: Bldg. 6B, Room 413,NIH, Bethesda, MD 20892. Fax: (301) 496-0243. Electronic mailaddress: Wia@cu.nih.gov.

t Present address: FDA/CBER, HFM541, Building 29A, Room3B08, Bethesda, MD 20892.

t Present address: Oncogene Science, Uniondale, NY 11553-3649.

grounds. Comparison of the biochemical activity of a crudeextract with that of pure protein in an integrative recombina-tion assay suggests that IHF makes up about 0.1% of total E.coli protein by weight (51). It has also been reported that IHFsynthesis is repressed by IHF and by LexA and that more IHFactivity can be recovered from stationary-phase cultures thanfrom exponential-phase cultures (7, 47, 48). In this article wereport that the abundance of IHF is comparable to that ofribosomal proteins and HU in exponential-phase cultures andincreases 5- to 10-fold in stationary-phase cultures. The level inexponential-phase cultures, although high, is not in largeexcess of the minimum required for biological function.

MATERMILS AND METHODS

Strains and plasmids. Strains and plasmids used in this workare listed in Table 1. Plasmid pDRC164 is a pBR322 derivativewhich contains both the himA and hip coding regions under thecontrol of Pmac31, a hybrid promoter constructed from the lacand malP promoters (75). The Ti terminator from the rmBoperon was included upstream of the mac promoter to preventread-through transcription (65). The IHF genes were sub-cloned from pLhimA-1 and from pKT23 hip23 (52). The himAgene was cloned as a 348-bp BstNI-HaeIII fragment, whichincludes sequences up to 26 bp upstream of the start codon and22 bp downstream of the stop codon. The hip gene was clonedas a 393-bp Fnu4HI fragment, which includes sequences 36 bpupstream of the start codon and 78 bp downstream of the stopcodon.Media and growth conditions. Media and growth conditions

for bacteria and phage were as described previously (49). Cellsfor 3-galactosidase assays were grown at 37°C in MA minimalmedium supplemented with vitamin B1, thymine, arginine (40,ug/ml), tryptophan (80 ,ug/ml), and 0.2% of the appropriate

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TABLE 1. Bacterial strains, plasmids, and phages used in this work

Strain, plasmid, or phage Relevant genotype Source or reference

Bacterial strainsCF3034 N99 zib-563::TnlO M. CashelCF3069 CF3034 Arel4257::Kan AspoT207::cat M. CashelDRC345 A(lacU169) (A RS88 [P,.-lacZ]) This workDRC355 DRC345 himAASma A3(hip::cat) From DRC345 by P1 transductionHN1491 AhimA::cat From K37 (24)K37 45K936 K37 himAG61R (formerly himA42) 45K1298 K37 zdi::TnlO 44K2691 K37 himAASma 23K2704 K37 A3(hip::cat) 24K3374 K37 himAASma A3(hip::cat) 24LE392 64MC1000 8MD84 K2691/pHNa This workMD93 K3374/pHNot This workMD96 K3374/pHN,Ba This workMD97 K3374/pCKR101 This workMD138 K2704/pHN,B This workRJ1617 MC1000 fis-767::Kan 31SG20250 22SG22025 SG20250 rcsA::Kan S. GottesmanSG22094 SG20250 rcsA::Kan clpP::Cam Alon S. GottesmanXPh43 (Mucts) Mu cts62 26YK1340 trpC9941 hupAJ6::Kan hupBll::Cam 76

PlasmidspCKR101 Ptac vector 40pDRC164 Pmac3l-himA-hip This workpDRC168 Pln35A-lacZ This workpHNa Ptac-himA 24pHN, Ptac-hip A. GranstonpHNota Ptac-hip-himA 24pNK627 pACYC184 lacIq This work

Phages0258 HK022 cItsl2 NIH collectionaY1 A cI857 NIH collectionY1122 A cI857 cinl NIH collectionY1196 A c1857 coslS4 NIH collection

a NIH, National Institutes of Health.

carbon source. Plasmids were selected with 50 ,ug of ampicillinper ml.Measurement of protein concentration. The protein concen-

tration in bacterial cultures was measured as described previ-ously (66) or by determining the optical density at 650 nm(OD650) in a Beckman DU-30 spectrophotometer. Exponen-tial- and stationary-phase cultures of our standard strainscontained approximately 285 ,ug of protein per ml per OD650unit. We measured the number of viable cells in exponential-phase cultures by counting colonies on Luria-Bertani (LB)agar plates or by using the experimentally determined conver-sion factor of 1.5 X 109 viable cells per ml per OD650 unit.

Polyclonal antibodies. Antibodies specific to IHFa andIHF,B were raised against unconjugated synthetic peptidescorresponding to the carboxyl-terminal 20 amino acids of eachsubunit (TFRPGQKLKSRVENASPKDE-COOH for IHFacand KYVPHFKPGKELRDRANIYG-COOH for IHF,) (3).Antiserum that reacted with both IHF subunits was raisedagainst purified wild-type IHF as described previously (24) andwas kindly provided by A. Granston and H. Nash. HU antibodywas kindly provided by L. Huang and R. McMacken.Western immunoblots and measurement of IHF concentra-

tion. We harvested cultures by centrifugation and stored thecell pellets at -70°C. To extract proteins, we resuspendedthawed cells in 0.1 to 0.01 of their original culture volume of 20

mM Tris (pH 7.4)-i mM EDTA-20 mM NaCl-10% glycerol(51). A known number of resuspended cells, as determined bymeasurement of OD650 before harvest, was mixed with loadingbuffer (39) and boiled for 5 mmn, and the entire sample wasapplied to the gel. Equal amounts of cell extract were loaded ineach lane of a given gel. Proteins were fractionated onpolyacrylamide gradient gels (17 to 27%, 11.5 by 16 mm;Integrated Separation Systems) run in a standard Laemmlibuffer (39) or on minigels (Miniplus gels, 8 by 8 cm; IntegratedSeparation Systems) run in modified Laemmli buffer thatcontained Tricine in place of glycine. Known concentrations ofpurified IHF (a kind gift of H. Nash) or purified HU (a kindgift of L. Huang and R. McMacken) were included as stan-dards. The purified standards were mixed with cells deleted forboth himA and hip (for IHF) or both hupA and hupB (for HU)before boiling. Proteins were transferred electrophoretically tonitrocellulose membranes (pore size, 0.2 ,im; Schleicher &Schuell) by using 0.5x Laemmli running buffer in 20% meth-anol (72). Membranes were washed and blocked with 5% skimmilk in 10 mM Tris (pH 8)-150mM NaCl-0.2% Triton X-100.We used antiserum at a 1:500 or 1:2,000 dilution in blockingbuffer according to its source. '251-protein A was used to detectthe bound antibody (29). We quantitated the amount ofradioactivity in the bands with an Ambis radioanalytic imageror by autoradiography and densitometry of the film with a

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CIDTEC video camera interfaced with a Macintosh computerrunning the Image 1.4 program.To verify that we loaded equivalent amounts of protein in

each lane and that the transfer was uniform, we ran duplicategels for several experiments and compared the Coomassieblue-stained gels with stained membranes. We stained themembranes with Ponceau S as described previously (61),except that we omitted sulfosalicylic acid. We also verified theuniformity of transfer and detection by measuring the amountof radioactivity in a band (band X) formed by an unknownprotein that migrated more slowly than genuine IHF subunitsbut that reacted with IHF antiserum (see Results). In accept-able gels the amount of radioactivity in this band varied lessthan twofold in different lanes.

IHF-sensitive lacZ fusion. The activity of the wild-type TnlOtransposase promoter, Pln, is increased three- to fivefold byinactivation of IHF (58a, 66a). This promoter has a noncon-sensus IHF-binding site adjacent to its -35 region (30, 50,50a), and the binding site was changed to consensus to increasethe sensitivity of the promoter to IHF (mutation 35A, num-bered from the ISIO end [see Fig. 6]) (30). A derivative of thispromoter that contains five additional base changes upstreamof the IHF site that are not relevant to this study (5G, 6A, 7G,15C, and 18A) was fused to a promoterless lacZ gene andinserted into the E. coli chromosome as follows. PlasmidpNK2152 is a derivative of pUC19 containing lac sequencesfrom codon 9 of lacZ to the DralIl site in lacY, 67 bp beyondthe lacZ stop codon. A BglII-NheI fragment containing the P1.variant (excised from plasmid pNK2575 [data not shown]) wasligated to Xba-BamHI-cut pNK2152 to generate pDRC168.This construct contains 350 bp of IS10, including the modifiedPln, fused to lacZ. A fusion protein containing 80 amino acidsof IS1O transposase fused to the ninth amino acid of P-galac-tosidase is expressed from the modified Pln. The fusion wascrossed onto X RS88 (65) and integrated into the X attachmentsite in the E. coli chromosome to form strain DRC345. IHFwas provided either from the wild-type chromosomally locatedgenes or from plasmid pDRC164, which is a pBR322 derivativecontaining promoterless himA and hip genes fused to thePmac3l promoter (see above). Pmac31 is repressed by Lacrepressor and induced by isopropyl-,-D-thiogalactopyranoside(IPTG) or maltose (75). Lac repressor was provided by plas-mid pNK627. Cultures were grown overnight, diluted, andgrown to an OD650 of approximately 0.5. For induction of thePmac3l promoter, cells were grown in the presence of glyceroland maltose, with or without IPTG (1 mM). Strains DRC355/pDRC164 and DRC355/pDRC164/pNK627 were grown in thepresence of ampicillin and ampicillin plus tetracycline, respec-tively, to select for the resident plasmids. We assayed P-galac-tosidase as described previously (49), except that we perme-abilized the cells by treatment with chloroform and 0.02%sodium dodecyl sulfate.

Bacteriophage burst size. Cells to be infected were grownovernight, diluted 100-fold, and grown for several hours in LBsupplemented with 10mM MgSO4 and 0.2% maltose. Culturescontaining different amounts of IHF (see below) were centri-fuged, resuspended in 10 mM MgSO4, and infected at multi-plicities of less than 0.1 phage per cell with X c1857 cinl, AcoslS4 c1857, HK022 cItsl2, A cI857, or Mu cts62. Phage wasmixed with cells and allowed to adsorb for 15 min at 30°C. A1-ml aliquot of cells was incubated in parallel for 15 min at30°C and then set aside for Western analysis. After 15 min, 5ml of prewarmed growth medium (42°C for HK022, Mu, and AcoslS4 and 32°C for A cinl) with the same composition as thestarting medium was added to 0.1 ml of infected cells. Aliquotswere removed immediately after dilution and at intervals

A.

IHF

X- U

a-'Ii

B* Antibody:(t IHF 1

- 14.3

- 6.5

1 2FIG. 1. Identification of IHF subunits. (A) The Western blot was

treated with antibody to IHF. Lanes: 1, purified IHF (150 ng) wasmixed with strain K3374 (himAASma A3[hip::cat]) before extraction; 2,extract of strain K3374. The molecular masses of standards (inkilodaltons) are indicated on the right. a and a indicate the two IHFsubunits, and X indicates the position of an unknown cellular proteinthat reacts with our antiserum (see text). (B) A blot of an extract ofstrain MD96, which produces both subunits of IHF, was reacted withanti-IHFa, anti-IHF, and anti-IHFP antiserum, as indicated. The laneswere formed with an Immunetics Miniblotter25. The top band in themiddle lane corresponds to the band labeled X in panel A.

afterwards, treated with chloroform, and centrifuged, and thesupernatants were assayed for phage. Unadsorbed phage wasestimated from the aliquot removed immediately after dilu-tion. Burst size is the increase in phage titer after 120 min (forlambda) or 180 min (for HK022 and Mu) divided by the titerof infected cells. To increase IHF or IHFa abundance, cellslacking chromosomal IHF genes and carrying plasmid pHN,Baor pHNa (24) were grown in LB medium supplemented withampicillin, 10 mM MgSO4, 0.2% maltose, and 1 mM IPTG.

Labeling of cells and two-dimensional electrophoresis. Sat-urated cultures grown in minimal M9-glucose medium werediluted 100-fold in the same medium, grown to an OD650 of 0.6to 0.7, and labeled by addition of 50 ,uCi of [35S]methionine to1 ml of growing cells for 5 min. Cells were processed and gelswere run as described previously (32).

Overexpression of IHF. We overproduced IHF subunits byusing fusions of promoterless himA and/or hip genes to the Ptacpromoter in plasmids pHNa, pHN1, or pHNIa (24, 40). Thesource of IHF1, pHN,3, was derived from the same parentplasmid as pHN13x (24) by a complete cut with HindIII. Toinduce overproduction, we added 1 mM IPTG to cultures of aplasmid-carrying host deleted for the himA and hip genes(K3374). We observed some expression from these plasmidseven without addition of IPTG, and pHN,Ba produces nearlywild-type levels of IHF even in the presence of the Lacjqrepressor (data not shown).

RESULTS

Identification and measurement of IHF subunits by West-ern blots. We measured IHF levels in whole-cell extracts byusing Western blot analysis with polyclonal antiserum (seeMaterials and Methods). The antiserum detects several pro-teins, and we identified IHFa and IHF13 by their mobilities,their absence in extracts of a mutant deleted for both the himAand hip genes, and their reappearance when we added purifiedIHF to mutant extracts (Fig. 1A and 2). The a and 1 subunitswere distinguished by the higher mobility of the latter (51, 52),by the use of subunit-specific antisera (Fig. 1B), and by analysis

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E. COLI INTEGRATION HOST FACTOR 3741

himA A A A + + *hipIHF

A A + A + *

........~~~~.I.._m

-K00

5+

-x

- 14.3

-a

1 2 3 4 5 6FIG. 2. Identification of IHF subunits with mutants. Extracts of expo-

nentially growing cultures were applied to the gels. The relevant genotypeis indicated in lines 1 and 2, and the presence or absence of 75 ng ofpurified IHF is indicated in line 3 above the Western blot. The bandsformed by the subunits, the X protein, and a molecular mass marker (inkilodaltons) are indicated to the right. Lanes: 1 and 2, strain K3374; 3,strain K2691; 4, strain K2704; 5, strain K37; 6, no cell extract (asterisk).

of cells deleted for either himA or hip (Fig. 2). The ratio ofIHFao to IHFP was the same in extracts as in purified protein.Figures 1A and 2 also show a cross-reacting band, labeled X,that migrates more slowly than IHFot and whose level did notsystematically vary under any of the conditions we used. In theexperiments described below, we applied equal amounts of

. 40

0~0) 3=LIcm2 -0c b

1 A

0-200

6

400 600 800Minutes

X 10

*~~~-.0

LO_ 1 x,-o0

l .11000 1200

FIG. 4. Increase in IHF abundance as cells enter stationary phase.An overnight culture of strain K37 in LB medium was diluted 1:2,000in the same medium and incubated at 37°C. The first sample was takenafter 4 h (see text). The blot was scanned with an AMBIS radioanalyticimager to determine the amount of IHF, and the results are plotted asa function of time of incubation. Symbols: O, OD650 of the culture; 0,amount of IHF.

A.ngIHF 15 30 60

B. 1200-

1000

U)

C 800-

>141uE 6000

:11400

200

90 12

0 20 40 60 80 100

ng IHF

FIG. 3. IHF calibration curve. Known amounts cwere added to strain K3374 before processing for VW(A) The amounts of purified IHF are indicated abovblot was probed with anti-IHF antibody. (B) Plotbetween the intensity of both subunits and the amounThe intensities of the IHFot and IHF, bands were

densitometry.

total protein to each gel lane and used the level of the X bandas well as a general protein stain to confirm uniformity ofloading, transfer, and detection across the gel (see Materials

0° and Methods). The results we present rely on Western blots inwhich the lane-to-lane fluctuation in the amount of the X bandwas less than twofold, and we therefore believe that lane-to-lane comparisons of the amount of IHF are accurate to withina factor of 2 in a given gel. To calibrate our assay, we mixeddifferent known amounts of purified IHF with extracts of ahim-A hip double deletion mutant and measured the intensitiesof the bands formed by IHFot and IHFP. The relation betweenIHF loaded and IHF detected was essentially linear over a10-fold range (Fig. 3). To estimate the amount of intracellularIHF, we included at least two lanes with different amounts ofpurified IHF standard mixed with an extract of a doubledeletion mutant in each gel.IHF is abundant and accumulates in the stationary phase.

To measure the abundance of IHF and its dependence ongrowth phase, we diluted an overnight broth culture of strainK37 2,000-fold and incubated it in LB medium for about 4 h,which allowed a 260-fold increase in mass or approximatelyeight doublings, before beginning sampling (Fig. 4). The initialsamples, taken while the cells were growing exponentially,contained 0.5 to 1.0 ng of IHF dimers per p.g of total proteinor about 8,500 to 17,000 molecules per viable cell, if all of thesubunits are dimerized. The abundance began to increase

120140 about the time cell growth began to slow and reached amaximum of almost 6 ng of IHF per ,ug of protein after 18 h ofincubation. This level did not change upon further incubation)f purified IHF for 48 h (data not shown). We note that the amounts of several

ie the blot. The peptides that cross-react with our IHF antibody did not varyof the relation with growth phase in the same manner as did IHF (only theI of IHF added. band formed by protein X is shown in Fig. 1A, 2, and SA). Wedetermined by conclude that IHF is an abundant protein in steady-state

exponentially growing cells, increases in amount when the cells

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A.

OD65

Tir-ne.09 0.2 0.5 0.8 4.3

0 40' 90'1 1 0' 1800

.S- .o- iow ."- - -x_ * * 'm.4 - 1?

1-4

B.

5

4

0. 3

-r

CT

0 100 20C 300 200G 600M flutes

0'o2000

FIG. 5. Decrease in IHF abundance as cells enter exponentialphase. An overnight culture of strain K37 in LB medium was diluted1:50 in the same medium and incubated at 37°C. The first sample was

taken immediately after dilution. (A) Autoradiogram of a Westernblot treated with anti-IHF antibody. (B) The blot was scanned with an

AMBIS radioanalytic imager to determine the amount of IHF, and theresults are plotted as a function of time of incubation. Symbols: O,OD650 of the culture; 0, amount of IHF.

enter the stationary phase, and continues to increase inamount for some time thereafter.To measure the rate of decrease of IHF subunit abundance

upon initiation of exponential growth, we diluted a similarovernight culture 50-fold and began sampling immediately. Wefound that the abundance decreased from about 4.5 to about1.5 ng/,ug as the cell density increased from 0.09 to 0.5 OD650unit (Fig. 5). Upon further incubation, IHF abundance grad-ually increased to its late-stationary-phase level. The decreasein IHF abundance can be roughly accounted for by assumingthat little or no new IHF is synthesized immediately afterdilution of the stationary-phase cells, that the preexisting IHFis diluted as the cell mass increases, and that new IHF synthesisresumes at a characteristic exponential-phase rate as theabundance falls.We used the same method to estimate the abundance of HU

protein in these extracts. HU is related to IHF in sequence andis believed to be very abundant. We found 2.1 ng of HU dimersper pug of total protein or 32,000 molecules per cell in cells thathad been diluted 1:50 from the overnight culture and grown toa density of 0.67 OD650 unit in LB medium (gel not shown).This is in reasonable agreement with a previous estimate ofHU abundance of 2 to 3 ng/,ug of total protein or 30,000 to50,000 molecules per cell (12, 60).

Miller and Nash (48) were unable to detect IHF by two-dimensional gel electrophoresis of pulse-labeled proteins ex-

tracted from wild-type cells, suggesting that its rate of synthesisis low. We have done similar experiments with extracts of

exponential-phase cultures of strains K37, K3374, and MD96labeled for 5 min of growth in medium containing [35S]methi-onine. We identified a spot that corresponds to the IHFEsubunit as judged by its absence in extracts of a hip deletionmutant and its intensification in extracts of an IHF-overpro-ducing strain (gel not shown). We were unable to identifyunambiguously a spot corresponding to IHFox. The amount oflabel per methionine residue in the IHFP spot, determined byscanning the dried gel, was about one-third that of ribosomalprotein S10, which was identified by the published coordinates(56). This result suggests that the rate of synthesis of IHF,B isone-third that of S10, since we have no reason to think thateither protein turns over rapidly (see below). The low intensityof the S10 spot in the previous two-dimensional gel analysis(48) suggests that the IHF3 spot was not seen because it wasbelow the limit of detection.IHF subunits are stable when protein synthesis is blocked.

To determine the stability of IHF within cells, we compared itsabundance before and after incubation of cells in the presenceof chloramphenicol, which blocks protein synthesis. In oneexperiment, two different dilutions of an overnight culture ofstrain K37 were grown in LB medium to cell densities of 0.4and 1.4 OD650J units, at which point 170 ,ug of chloramphenicolper ml was added to both. We found that the abundance ofIHF subunits remained constant at 1.7 and 2.5 ng/,ug, respec-tively, for 3.5 h after chloramphenicol addition in both cultures(gel not shown). In a second experiment, an overnight cultureof SG20250 was diluted directly into LB medium containingchloramphenicol. We found that IHF subunit abundance wasconstant for 3 h after dilution but declined to about 50% of itsinitial value after 6 h of incubation (gel not shown). Theseresults show that IHF is stable in cells incubated for 3 h ormore in medium containing chloramphenicol and suggest thatit is stable in growing cells.

Effects of changes in genetic background and growth condi-tions on IHF subunit level. We found that two widely used E.coli K-12 strains, MC1000 and LE392, are similar to strain K37in the level and growth-phase-dependent pattern of accumu-lation of IHF subunits (data not shown). This is also true forstrain DRC345, which we used for experiments describedbelow. Accumulation of IHF during stationary phase alsooccurred in mutants unable to synthesize the DNA-bindingproteins HU and Fis (strains YK1340 and RJ1617, respective-ly), in a relA spoT double deletion mutant (strain CF3069)which makes neither guanosine tetraphosphate nor guanosinepentaphosphate and which has multiple amino acid auxotro-phies (79), and in strains unable to synthesize the proteasesencoded by the clpP and Ion genes (21) (strains SG22094 andSG22025) (data not shown). The amount of IHF per cell andits accumulation during stationary phase is not altered bygrowing strain MC1000 at 32 or 42°C instead of 37°C or bygrowth in minimal-glucose salts medium instead of LB broth.We have noticed that a TnJO insertion located about 300 bpdownstream of himA (strain K1298) (44) reduces the abun-dance of both subunits in overnight cultures to 0.3 to 0.5 of thelevel found in the parental K37 strain. The reason for thisreduction is unknown. We found that this TnJO insertionmagnified the effect of certain himA and hip mutations on theabundance of the unaffected subunit (data not shown).

Effect of IHF mutations and overproducing plasmids onIHF subunit level. Previous attempts to overproduce IHFshowed that the level of each of the overproduced IHFsubunits is increased by the presence of the other, presumablybecause of the stabilizing effect of the IHFo-IHFI interaction(52). Results of more recent work are consistent with thisassumption (24). We determined the effects of mutations that

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TABLE 2. IHF subunit levels in various strainsa

Level of:Strain Relevant genotype

IHFa IHFIB

K37 himA+ hip+ 1.0 1.0K1298 himA+ hip' zdi::TnlO 0.3-0.5c 0.5K2691 himAASma 0 0.5-1.0CHN1491 AhimA::cat 0 0.2K2704 A3(hip::cat) 0.3-0.5c 0K3374 himAASma A3(hip::cat) 0 0K936 himAG61R 4 4MD84d himAASma/pHNot 17 1.0MD93d himAASma A3(hip::cat)IpHNot 6e-16 0MD96 himAASma A3(hip::cat)IpHNPoa 1 2MD96d himAASma A3(hip::cat)/pHNPa 10 10MD138d A3(hip::cat)IpHNP 1.3 3.8

a Overnight cultures (except as noted) of each strain were grown in LBmedium at 37°C and analyzed for IHF subunit levels as described in Materialsand Methods.

b All levels are normalized to those found in strain K37.cDifferent values were obtained in different experiments.d These cultures were grown in the presence of 1 mM IPTG.eThis value is from an exponentially growing culture.

inactivate IHF on the levels of the two subunits in stationary-phase cells. Deletion of either himA or hip eliminates the bandformed by the directly altered subunit, as expected, andreduces the amount of the unaltered subunit to an extent thatdepends on the particular deletion (Table 2). Both thehimA::cat and A3hip::cat substitution mutations are expectedto prevent synthesis of even truncated polypeptides, yet theeffect of the himA substitution on IHF,B is somewhat moresevere than the effect of the hip substitution on IHFa. This isconsistent with other results (see below). Therefore, IHFa inthe absence of its partner may be marginally more stable orsynthesized at a somewhat higher rate (or both) than IHFI inthe absence of its partner. The reduction of IHF,B level causedby himAASma, which should truncate the IHFa polypeptide atresidue 65 (23), is quite modest. A plausible explanation of thesmall effect was suggested by Granston and Nash (24), whodetected IHFa-ASma-IHFP heterodimers. Therefore thetruncated IHFa peptide interacts with and presumably par-tially stabilizes IHFP. We fail to see the band formed by thetruncated polypeptide, perhaps because it is too small or haslost critical epitopes. The mutually stabilizing effect of the twosubunits is not apparent in the experiment reported in Fig. 2,in which we saw only a small reduction, if any, in IHF, and amodest increase in IHFao when the partner subunit was absent.These cells, unlike those used in the experiments reported inTable 2, were harvested in the exponential phase, and wesurmise that the undimerized subunits are less stable in thestationary phase.

Previous work suggests that IHF represses the transcriptionof IHFa (42, 47). We confirmed that the G61R mutation inhimA (= himA42, the mutant used in the previous studies)substantially increased the level of both subunits (Table 2). Weattribute this increase to the formation of stable IHFot-G61R-IHF3 dimers that are unable to repress subunit synthesis. Infact, Granston and Nash (24) have detected such dimers.Presumably the deletion mutants described above also fail torepress IHF synthesis, but the increased synthesis does notcompensate for the decreased stability of the wild-type subunit.We also investigated the effect of overexpression of one orboth IHF genes on the abundance of the two subunits. It isknown that fusion of the powerful phage APL promoter to theIHF genes results in overproduction of the subunits and death

of the cells (13, 52). For these studies we directed IHF genetranscription with the less powerful Ptac promoter, anticipatingthat the cells might survive overproduction. Introduction of aplasmid carrying a Ptac-hip-himA fusion (pHN3ao) into ahimAASma hipA3::cat double mutant resulted in a 10-foldincrease in both IHFoL and IHFI after overnight growth inbroth supplemented with inducer (Table 2). In the absence ofinducer, these cells grew somewhat more rapidly, achieved aslightly higher final OD650' and showed little or no overpro-duction. Clearly a 10-fold excess of IHF is well tolerated by thecells. Fusion of Ptac to himA (in plasmid pHNot) stronglyincreased the level of IHFoa, and this single-subunit overpro-duction did not require the presence of active IHF, (Table 2).The mobility of overproduced IHFot was identical to that ofIHFot from crude extracts of wild-type cells or from purifiedIHF, and we saw no specific degradation products (gel notshown). A similar fusion of Ptac to hip (in plasmid pHN,)resulted in much less striking overproduction of IHF,B, perhapsbecause of the greater instability of this subunit in the absenceof stoichiometric amounts of its partner (Table 2).

Biological effects of decreasing IHF abundance. The abun-dance of IHF subunits seems high relative to that of othersite-specific DNA-binding proteins (4) and to the estimatednumber of IHF-binding sites (19). To find how much IHF isrequired for biological function, we placed himA and hipexpression under the control of promoter Pmac3l (plasmidpDRC164) (75). In cells in which a plasmid carrying this fusionwas the only source of IHF, the IHF abundance variedbetween 0.3 and 3.8 ng/,ug in different cultures (see below).Isogenic cells in which the only source of IHF was thechromosomal himA and hip genes had IHF levels that rangedfrom 1.6 to 4.2 ng/4ig when grown under similar conditions. Wemeasured the effects of such variation on several biologicalfunctions.

(i) Promoter response. We determined the effect of reducingIHF abundance on the activity of an IHF-repressible pro-moter, a derivative of P,. of IS10, fused to a lacZ reporter gene(see Materials and Methods). IHF is believed to repress thispromoter by binding near its -35 region, and the derivative weused has an IHF-binding site with an improved match toconsensus. We attempted to vary the IHF level by usingdifferent carbon sources and a plasmid containing the gene forlac repressor (see Materials and Methods). The level of1-galactosidase activity was 80 to 120 U in cells with no IHFand 1 to 3 U in cells with wild-type IHF levels (Fig. 6, solid andopen squares). Although there is considerable scatter in thedata (see below), it is clear that ,-galactosidase activity beganto increase significantly when IHF subunit abundance fellbelow approximately 1 ng/,ug (Fig. 6, circles and triangles).This is our estimate of IHF abundance in steady-state expo-nential-phase himA+ hip' cultures, which argues strongly thatthe wild-type IHF level is not greatly in excess of that neededfor occupancy of the consensus IHF-binding site in the Plnderivative we used. The scatter in the data may be attributableto fluctuation among IHF gels and possibly to the variety ofexperimental conditions we used. Because of this variabilityand because we unexpectedly failed to see a convincing effectof lac repressor and growth in different carbon sources on IHFabundance, we cannot draw a more precise conclusion fromthese data.

(ii) Phage growth. Several bacteriophages and bacterio-phage mutants are dependent on IHF for growth. We mea-sured phage yields of cells containing various levels of IHFafter infection with phages Mu, A cinl, or HK022, all of whichrequire IHF for plaque formation (46, 76a, 78). The yields ofall three phages decreased with decreasing IHF abundance

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GTGAGAGATcGcGTGATMT1TGGTMMATCMTMGTGGTGGATAC ...plN-ecz.HK022, and A coslS4, in strain MD93, which lacks IHFP but

-G-T-.--G-T-T- I-T- ...I has high levels of IHFox because it contains an overproducingIHF Consensus -35 plasmid (Table 2). We found that the yields of these phages in

-.....I...........n MD93 were indistinguishable from those in strain MD97, theIHF-Proteced RegionIHF-deficient control, and much lower than those in MD96,the wild-type control (Table 4). The efficiency of plating ofthese phages on the same set of hosts is consistent with the

E1 measurements of yields (data not shown). We conclude that4 - IHFot, which we estimate was present at about 10 times its

°5 normal abundance (Table 2), cannot satisfy the IHF require-02 ment of these phages in vivo. Since IHFa specific activity in

3 .3 vitro in the absence of IHF3 is at least 1/10 that of het-14 erodimeric IHF (77), its inactivity in vivo cannot be explained

simply by reduced abundance but must be due, at least in part,03

* DRC 34(QHF+) to reduced specific activity. Perhaps the overproduced subunit2 3 0 DR55/pDRC164 is altered or sequestered in vivo in some way that does not

A DRC355/pDRC1S4. pNK627 change its mobility in our gels. We note that Bear et al. (2)o3 DRC355 (IHF-)

have argued that the IHF requirement of X cosl54, which7 5d01 needs IHF to package its DNA, can be partially filled by IHFa.

60 Our data do not conflict with theirs, because we used different

028 Al mutations and conditions, but we clearly do not support theirCb3 9 2 3 1 conclusion. It is possible that the hip mutants they used make

0 I, . , . low levels of IHF,B. We have not asked if overproduction of0 20 40 60 so 100 120 IHFP suppresses a himA deletion, because our experiments

P GALACTOSIDASE UNITS suggest that this subunit is not well overproduced (Table 2;data not shown) and because it is less active than IHFot in vitro

IG. 6. Response of an IHF-repressible promoter to different (77).1 v s.r - -. s - s - - *- *.* .s .. - Ilevels of IHF. The sequence of the IHF-binding site in the P1,-IacZ

transcriptional fusion used to monitor promoter response is shown atthe top, where base 1 is the end of ISIO (see text). The regionfootprinted by IHF (Morisato and Kleckner [SOa], as cited in reference30) the IHF consensus sequence, and the -35 region of P,. areindicated by dashed lines. The graph shows P-galactosidase activity asa function of IHF abundance. The specific activity of ,B-galactosidaseand the abundance of IHF were determined in cells that contain achromosomal copy of the P,n-lacZ fusion and had grown for four to sixdoublings after a 100-fold dilution of the overnight cultures (seeMaterials and Methods). The source of IHF in these cells was eitherthe chromosomal IHF genes (strain DRC345) or plasmid IHF genesunder the control of the Pmac3l promoter (plasmid pDRC164 [seetext]). Plasmid pNK627 contains the lacIq gene. The symbols indicatethe strains, and the numbers next to the symbols indicate the growthmedia. All media except medium 8 are minimal (MA) supplementedwith the indicated carbon source(s) to 0.2%. Strains DRC355/pDRC164 and DRC355/pDRC164/pNK627 were grown in the pres-ence of ampicillin and ampicillin plus tetracycline, respectively, toselect for the resident plasmids. Media: 1, glycerol; 2, glycerol, maltose,and 1 mM IPTG; 3, glucose; 4, glucose, maltose, and 5 mM IPTG; 5,glycerol and maltose; 6, glycerol and 1 mM IPTG; 7, glycerol and 5 mMIPTG; 8, LB medium with 10 mM MgSO4, maltose, and 1 mM IPTG;9, maltose and 1 mM IPTG.

(Table 3). The IHF requirements for growth of Mu and X cinlappear to be satisfied by IHF binding to a single identified sitein each phage (20, 25, 38, 73, 74), but the basis of the HK022IHF requirement is unknown.

Effect of increasing IHFu abundance. Recent work showsthat either IHFa. or IHFI, when present in sufficient quantity,binds specifically to IHF sites and satisfies the in vitro require-ment for IHF heterodimer in A site-specific recombination (77;see also reference 82). Previous work with mutants shows thatin vivo, both subunits are required for integrative recombina-tion as well as for other IHF-dependent processes (35, 45) (fora possible exception, see reference 2). The requirement forboth subunits in vivo might reflect the reduced level of onesubunit in the absence of the other. To test this hypothesis, wemeasured the yields of three IHF-requiring phages, A cinl,

DISCUSSION

We report that exponentially growing bacterial culturescontain about 0.5 to 1 ng of IHF per p.g of total protein or

8,500 to 17,000 molecules per cell. This is equivalent to 7 to 14,uM (if the volume of a cell is 2 ,um3 under our conditions [11]).This estimate is not significantly different from an independentmeasurement based on immunoprecipitation of labeled pro-tein (12a). The abundance of IHF increased to about 6 ng/,ugin late-stationary-phase cultures and then decreased whenstationary-phase cells were diluted into fresh medium andallowed to resume growth. The increase began when cellgrowth began to slow and continued for about 6 h. Thedecrease began when the cell mass of the diluted stationary-phase cells started to increase and continued for severaldoublings. These changes in IHF abundance could reflectalterations in relative rates of transcription, translation, or

protein degradation. Of these possibilities, protein degradationis the least likely, since the IHF level remained constant forseveral hours in the presence of chloramphenicol. We have noinformation about the rate of translation of IHF mRNA.Unpublished work of M. Aviv, H. Giladi, A. Oppenheim, andG. Glaser (1) supports the hypothesis that the relative tran-scription rate of IHF genes increases in the stationary phase.We note that Bushman et al. (7) previously reported thatIHF-dependent DNA-binding activity in cell extracts increasedthree- to fourfold when the cells entered the stationary phase,although this result may depend on the method of extraction(69).

Since IHF appears stable, the decrease in its abundanceupon resumption of exponential growth is best accounted forby assuming that its synthesis temporarily stops or slows. It istempting to speculate that new synthesis stops because the highIHF concentration prevents transcription of IHF genes uponresumption of exponential growth and that synthesis resumeswhen cell growth lowers the IHF concentration below the pointneeded to repress transcription. Indeed, himA transcriptiondoes appear to be repressed by IHF (42, 47), and the abun-

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TABLE 3. Phage burst sizes and IHF levelsa

Strainb Xcinl burst size' IHF leveld HK022 clts burst sizec IHF leveld Mu clts burst sizec IHF leveld

DRC345 33 (±21%; n = 3) 2.8 (+4%; n = 2) 5,761 (+39%; n = 4) 3.4e 29 (±25%; n = 3) 3.4eDRC355/pDRC164 1 (±6%; n = 4) 1 (+40%; n = 5) 520 (±52%; n = 4) 1.4 (±30%; n = 4) <1 1.5 (±39%; n = 3)DRC355 <1 0 <1 0 <1 0

a The burst sizes of the indicated phage strains and the IHF levels in the cultures immediately after mock infection were measured as described.b DRC345 is himA+ hip+, DRC355 is himAASma A3hip::cat, and pDRC164 is a plasmid that carries the IHF genes under the control of the Pmac31 promoter (see

text).c The numbers are burst sizes corrected for unadsorbed input phage (see Materials and Methods), and the numbers in parentheses are the standard errors of the

means (expressed as a percent of the mean) followed by the number of experiments (n).d IHF levels are expressed as nanograms of IHF per gram of total protein (see Materials and Methods). For numbers in parentheses, see footnote c.e Only one experimental determination was made.

dance of both IHFox and IHF, was increased by thehimnAG61R mutation, which leads to the formation of inactiveheterodimers (Table 2) (24). According to this model, IHFautorepression is bypassed or becomes less effective in station-ary-phase cells.Our experiments do not directly reveal if the IHF subunits

we detected were in the heterodimeric form before extraction,but the following observations suggest that a substantial frac-tion were. First, our estimate of IHF abundance in exponen-tially growing cells agrees with an estimate derived from thedata of Nash and Robertson (51), who assayed the IHF activityof purified protein and crude extracts. Second, the relativeamounts of the two subunits in extracts of wild-type cells arethe same as in purified protein. Third, deletion of one IHFgene reduces the level of the unaffected subunit, suggestingthat formation of heterodimers stabilizes the subunits in vivo(24, 52; see above). If most IHF subunits within the cell are inthe active, heterodimeric form, the number of heterodimersper cell vastly exceeds an estimate of the number of specificIHF-binding sites (19) and also exceeds the abundance of mostother E. coli proteins that recognize specific DNA sites (4).Indeed, the number of IHF heterodimers per cell is compara-ble to that of ribosomes and HU protein. Despite this, theamount of IHF in exponentially growing cells is not far abovethe minimum required for full occupancy of at least some

specific binding sites. Expression of an IHF-repressible pro-moter began to increase and growth of several IHF-dependentbacteriophages began to decrease when we artificially reducedIHF abundance to less than 1.0 ng/p,g of total protein, aboutthe level found in exponentially growing wild-type cells. Esti-mates of the dissociation constants of IHF from some func-tional binding sites are in the range of 1 to 80 nM (16, 37; seealso reference 14), considerably lower than our estimate ofintracellular IHF concentration in exponentially growing cells(7 to 14 ,uM). Therefore, the decrease in site occupancy causedby a modest reduction in the IHF concentration suggests thatmost of the intracellular IHF is not free.The number of known IHF sites (5, 14, 19) is sufficient to

bind only a very small fraction of the total IHF. Are the surplusheterodimers bound to DNA, and what is their function? Onepossibility is that there are a large number of undiscoveredsites that resemble known sites in sequence and bindingaffinity. Although a substantial number of new sites hasrecently been discovered (54), it is highly unlikely that enoughremain to bind more than a small fraction of the surplus IHF.A second and more likely possibility is that much of the surplusIHF is bound to a large number of low-affinity DNA sites andthat this binding has a function(s) that is not yet apparent.Indeed, recent work suggests that quasi-specific binding of IHFto phage X attachment site variants that lack the normalbinding sites promotes X excisive recombination in vitro (62a).

We are unable to identify functions for IHF bound to low-affinity sites in vivo, perhaps because of functional overlap ofabundant DNA-binding proteins (see below).The abundance of IHF increased 5- to 10-fold during the

transition from steady-state exponential growth to late station-ary phase, and we assume that there are equivalent increases inthe intracellular IHF concentration and IHF-to-DNA ratio.Landy and his collaborators, namely, Bushman et al. (7) andThompson et al. (69), have argued that an increase in IHFconcentration favors prophage integration and blocks excisionand that IHF abundance is thus used by the prophage as a wayof coupling the directionality of recombination to the growthphase of the cell. Although this is an attractive idea and is onethat could also apply to other IHF-responsive processes, morerecent in vivo footprinting results from the Landy laboratorysuggest that the decrease in IHF abundance that occurs duringthe transition from stationary to exponential phase does notalter the occupancy of high-affinity binding sites within the Xattachment site (68). This result suggests that such sites do notrelease IHF during this transition and that any release thatoccurs is from low-affinity sites. Additional support for thisconclusion comes from the observation that synthesis of 3-ga-lactosidase from the IHF-sensitive Pln-lacZ fusion describedabove does not vary with growth phase in a himA + hip-' strain(data not shown). We are ignorant of the physiological conse-quences, if any, of release of IHF from low-affinity sites.Perhaps it promotes some of the changes that are associatedwith transition into or out of stationary phase (36, 63).However, the absence of severe growth defects or stationary-phase recovery problems in IHF-deficient strains suggests thatany such role of the growth phase regulation of IHF is eitherredundant or subtle or both.IHF has been grouped with HU, H-NS, and Fis as nucleoid-

associated proteins on the basis of their high abundance, low

TABLE 4. IHFat overproduction and phage growtha

Yield of5:Strain Subunit present

X cosl54 X cinl X HK022

MD96 IHFot and IHFP 62 60 184 500MD93 IHFax only 0.6 4 22 2.5MD97 Neither 0.7 3 14 1.4

a Cells deleted for both chromosomal IHF genes and containing plasmids thatoverproduced both subunits (MD96), IHFat only (MD93), or neither subunit(MD97) were infected with the indicated phage, and the yields of phage perinfected cell were measured (see Materials and Methods). The abundance of theIHF heterodimer of IHFc in cultures of MD96 or MD93 grown under identicalconditions was 10 and 6 ng/,ug, respectively (Table 2). The observed dependenceof wild-type X growth on IHF is consistent with previous observations (37).

b PFU per cell.

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3746 DITTO ET AL.

molecular weight, solubility at low pH, and association withDNA (62). Several groups have noted that some phenotypes ofmultiple mutants that lack IHF and HU or that lack IHF, HU,and H-NS are more severe than the phenotypes of singlemutants (18, 33, 34, 43, 81). H-NS, like IHF, is a small andabundant protein that can bind specifically to DNA (55, 58, 70,80). Two of the nucleoid-associated proteins, in addition toIHF, are growth phase regulated. The abundance of E. coliH-NS protein is reported to increase in the stationary phase (9)(although there are conflicting results for Salmonella typhi-munum [28]). The abundance of E. coli Fis protein also varieswith growth phase, but it does so with a pattern that differsmarkedly from that of IHF: there is a very dramatic increase inthe Fis level when stationary-phase cells are diluted intogrowth medium and a rapid decrease after the onset of growth(la, 53). This fluctuation alters site occupancy in vivo: aFis-binding site within the X attachment site was occupiedduring exponential growth but not after cells had entered thestationary phase (68). HU, as far as we are aware, is not growthphase regulated and, unlike IHF, H-NS, and Fis, shows nocapacity for site-specific DNA binding. HU and IHF areknown to be widely distributed in prokaryotes (16a, 17, 27, 41,71; see also reference 12). Despite these hints of globalimportance, none of the nucleoid-associated proteins seemsabsolutely essential for cell propagation under laboratoryconditions. It remains to be seen if this apparent dispensabilityis a consequence of extensive functional overlap or reflects ourignorance of the competitive pressures faced by these organ-isms outside of the laboratory.

ACKNOWLEDGMENTSWe are grateful to Drew Granston and Pam Jones for their help; to

Gadi Glaser, Anca Segal, and Milton Werner for communicatingunpublished results; and to Don Court and Howard Nash for theircomments on the manuscript.

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