Vol. 170, No. 6JOURNAL OF BACTERIOLOGY, June 1988, p. 2816-28260021-9193/88/062816-11$02.00/0Copyright C) 1988, American Society for Microbiology

DNA Supercoiling and the Anaerobic and Growth Phase Regulationof tonB Gene Expression

CHARLES J. DORMAN,* GORDON C. BARR, NIAMH NI BHRIAIN, AND CHRISTOPHER F. HIGGINS

Molecular Genetics Laboratory, Department of Biochemistry, University ofDundee, Dundee DDI 4HN, Scotland

Received 28 December 1987/Accepted 20 March 1988

We show that several interacting environmental factors influence the topology of intracellular DNA. Negativesupercoiling ofDNA in vivo is increased by anaerobic growth and is also influenced by growth phase. The tonBpromoter of Escherichia coli and Salmonella typhimurium was found to be highly sensitive to changes in DNAsupercoiling. Expression was increased by novobiocin, an inhibitor of DNA gyrase, and was decreased byfactors which increase DNA superhelicity. Expression of the plasmid-encoded tonB gene was enhanced by yBinsertions in cis in a distance- and orientation-independent fashion. Both the res site and the TnpR protein of,y8, which is known to function as a type I topoisomerase, were required for this activation. tonB expressionincreased during the growth cycle and was reduced by anaerobiosis. There was excellent correlation betweentonB expression from a plasmid and the level of supercoiling of that plasmid under a wide range of conditions.The chromosomal tonB gene was regulated in a manner identical to that of the plasmid-encoded gene. Thus,the physiological regulation of tonB expression in response to anaerobiosis and growth phase appears to bemediated by environmentally induced changes in DNA superhelicity.

Chromosomal DNA isolated from bacteria is negativelysupercoiled. In vivo supercoiling is determined, at least inpart, by a balance between the relaxing activity of topoisom-erase I and the ATP-dependent supercoiling activity ofDNAgyrase (16, 43, 44, 51, 71). The degree of supercoiling caninfluence a variety of processes, including promoter functionand the initiation of transcription (reviewed in references 17,18, and 72). The possibility that a specific environmentalstimulus might alter DNA supercoiling in vivo and conse-quently contribute to the regulation of transcription of asubset of genes is an attractive idea. In this paper we presentevidence that environmentally induced changes in DNAsupercoiling are an important factor in the regulation of tonBgene expression in response to both anaerobiosis and growthphase.The tonB gene is located near trp at 27 min on the

Escherichia coli genetic map and at 34 min in Salmonellatyphimurium. TonB plays a key role in cellular physiology,being required for many energy-dependent outer membraneprocesses. Mutants lacking TonB are deficient in all high-affinity iron transport systems and vitamin B12 uptake andare resistant to many bacteriophages and colicins (reviewedin references 34, 46, and 48). Although the precise role ofTonB is not known, it seems likely that the protein isrequired to couple energy to outer membrane processes (21,48, 58).

Expression of tonB is repressed anaerobically but isinduced during aerobic growth when iron is oxidized to aninsoluble form and can become limiting (25). The mecha-nisms by which gene expression is regulated in response toanaerobiosis are still poorly understood. When facultativeanaerobes such as E. coli and S. typhimurium switch fromaerobic to anaerobic growth, about 50 new proteins aresynthesized, while the synthesis of other proteins is re-pressed (64, 65). For all anaerobically regulated genes whichhave been examined, regulation is at the level of transcrip-tion (1, 4, 29, 30, 37, 69, 75). The expression of severalanaerobically induced genes requires the Fnr protein (also

* Corresponding author.

called NirA or OxrA) (39, 47, 69). Fnr is a positive regulatoryprotein, strongly resembling the cyclic AMP repressor pro-tein (61, 66). However, the mechanism by which anaerobi-osis is sensed and activates the Fnr protein remains obscure.The expression of other anaerobically regulated genes isindependent of the Fnr gene product (1, 29, 30). Several ofthese genes require a functional oxrC gene product foranaerobic induction (30). oxrC encodes phosphoglucoseisomerase, implying that flux through the glycolytic path-way, or a glycolytic end product, may play a role in theregulation of this class of genes. Again, however, the natureof this signal remains unclear. The picture is further compli-cated by the fact that many anaerobically regulated genes areadditionally influenced by other factors, for example, for-mate or nitrate (13, 67). Thus, it is now becoming apparentthat no single, global anaerobic regulatory pathway oper-ates, but that several interacting networks are togetherresponsible for the observed pattern of anaerobic inductionand repression of gene expression.

It has recently been suggested that DNA supercoiling mayplay a role in the anaerobic response (74). In this paper weprovide evidence for such a role. We show that the anaero-bically regulated tonB promoter is very sensitive to changesin DNA supercoiling. Furthermore, DNA supercoiling invivo is shown to change in response to anaerobic growth andto growth phase. Good correlation was obtained, under awide variety of conditions, between the levels of DNAsupercoiling and tonB expression. It appears that environ-mentally induced changes in DNA supercoiling are respon-sible, at least in part, for the oxygen and growth phaseregulation of tonB. The possibility that these environmen-tally induced changes in DNA supercoiling play a moregeneral role in the anaerobic regulation of gene expression isdiscussed.

MATERIALS AND METHODSBacterial strains and media. The bacterial strains used in

this study are listed in Table 1. Cells were routinely grown ineither LB (56) or the defined minimal medium MMA, sup-plemented with 0.4% glucose as the carbon source (45).

2816

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

DNA SUPERCOILING AND GENE EXPRESSION 2817

TABLE 1. Bacterial strains

Strain Relevant genotype Source or reference

E. coli K-12CSH48 his (+80)thi F' (colV+ colB+ trp) 45GB102 thi pro lac galE A(trp-tonB-opp)467 Strr 28GB291 MC4100 1'(tonB-lacZ) (Xplac Mu 53) This workMBM7060 araC(Am) araD l(argF-lac)U169 trp(Am) malB(Am) rpsL relA thi XplO48 5MC4100 araD139 rpsL150 relAI deoCi thi ptsF25 rbsR flbB5301 A(argF-lac)U169 10PAP1370 pLB8 (colM+ colimm) A. P. PugsleyPB13 F+ cysE tfr-8 recA srl::TnlO M. Jones-Mortimer

S. typhimuriumJF1003 oxiCJ048::Mu dl-8 1JF1141 aniIlO70::Mu dl-8 1JF1292 aniE(phs)1074::Mu dl-8 1JF1325 aniC1052::Mu dl-8 1JF1415 aniHJ069::Mu dl-8 1

Aerobic cultures were grown as a small volume in baffledflasks with vigorous shaking at 37°C. Under these condi-tions, typical anaerobically induced genes remained re-pressed throughout growth. Anaerobic growth was achievedby completely filling sealed bottles with medium and growingwithout shaking or by using a GasPak system (BBL Micro-biology Laboratories). Antibiotics were used at the followingconcentrations; ampicillin, 50 ,ug/ml; chloramphenicol, 20,ug/ml; kanamycin, 25 jig/ml; and tetracycline, 20 jig/ml.Solid media contained 1.5% agar. When required, aminoacid supplements were added to MMA at 300 ,uM. Mac-Conkey-lactose and lactose-tetrazolium plates have beendescribed (45). Colicin BV plates were prepared by streakingthe colicin-producing strain CSH48 across an LB plate andincubating at 37°C for 48 h. During this time, colicinsdiffused from the growing cells into the surrounding agar.Strains to be tested for sensitivity were then streakedperpendicularly to the colicin producer. Colicin-resistantstrains were completely resistant to the colicins, whilesensitive strains showed a 1-cm zone of killing. Resistance orsensitivity to phage 4)80 was determined by cross-streakingon LB plates against a 4)80 lysate (45). Colicin M wasprepared from strain PAP1370 by treating a log-phase culturewith mitomycin C (52). The cells were sonicated, the debriswas removed by centrifugation, the colicin was sterilized byfiltration, and 0.1 ml was added directly to agar plates.

Genetic and DNA techniques. Phage P1 vir was used forroutine transduction of genetic markers (62). GB102 wasselected as a spontaneous streptomycin-resistant derivativeof the trp-tonB deletion strain CH483 (28). E. coli strainswere transformed with DNA by the CaCl2 procedure (41). Ingeneral, tonB strains transformed at least 10 times lessefficiently than their parental tonB+ strains; the reason forthis is not known. Restriction endonucleases and DNAligase were from Amersham plc. and were used as specifiedby the manufacturer. Plasmid DNA was purified fromcleared lysates by cesium chloride density gradient centrif-ugation (41) or by the rapid screening technique of Colemanand Foster (14).

Construction of lacZ fusions to the chromosomal tonB gene.Chromosomal tonB-lacZ transcription fusions were con-structed by using the vector Aplac Mu53 (9). A pool ofrandom Xplac Mu53 insertions in the MC4100 chromosomewas made, and cells with insertions in the tonB gene wereselected as being simultaneously resistant to colicins B andV and to phage 4)80 vir (45). This double selection reducedthe possibility of selecting insertions in the genes encodingouter membrane receptors for the individual agents. All

presumptive insertions in tonB were screened on Mac-Conkey-lactose plates to identify those in the correct orien-tation (i.e., expressing lacZ). The fusions were then trans-duced into a nonmutagenized background, selecting forKanr, and rescreened for resistance to colicins B and V andphage 4)80 vir. These transductants grew as small colonies onLB agar and were pink due to secretion of the iron chelatorenterochelin, a characteristic of tonB mutants which becomeiron limited. The presence of a single Aplac Mu insertion at27 min on the chromosome was confirmed by demonstrating95% transductional linkage between the Kanr marker of theMu derivative and various trp mutations. One such fusionstrain, GB291, was used for all further analysis. The fusionjunction in this strain was shown to be within the C-terminalhalf of the tonB structural gene by Southern blotting (datanot shown).Enzyme assays. P-Galactosidase activity was assayed in

cells grown under appropriate conditions as described byMiller (45) with sodium dodecyl sulfate (SDS)-chloroform-permeabilized cells. Units of activity are expressed as de-scribed by Miller (45). Each datum point is an average of atleast three separate determinations, measured on at leasttwo independent cultures. Standard deviations were alwaysless than 8%.

Immunoblotting. Cells (1 ml) grown to an OD6. of 0.5were sedimented by centrifugation and suspended in 200 IlIof Laemmli sample buffer (38), and 25-pul portions wereloaded onto a 10% polyacrylamide-SDS gel (2). After elec-trophoresis, proteins were transferred to nitrocellulose andprobed with anti-TonB antibodies as described (70). Afterwashing, the bound anti-TonB antibodies were detected byalkaline phosphatase staining (33).Measurement of plasmid DNA supercoiling. Plasmid super-

coiling was assessed by separation of topoisomers on chlo-roquine gels. Plasmid DNA was isolated by the method ofColeman and Foster (14) and electrophoresed on a 1%agarose gel containing 25 ,ug of chloroquine per ml. Thesamples were electrophoresed for 20 h at 3 V/cm in TBE (90mM Tris [pH 8.3], 90 mM borate, 10 mM EDTA) containingthe same concentration of chloroquine as the gel itself. At asaturating chloroquine concentrations (25 ,ug/ml), the moresupercoiled topoisomers prior to electrophoresis migratedmost slowly through the gel. Following electrophoresis thegel was washed in distilled water for 4 h to remove chloro-quine prior to staining with ethidium bromide (5 ,ug/ml).

Isolation of y8 insertions. -yb insertions into plasmidpGB144 were isolated essentially as described previously(24). pGB144 was transformed into the F+ strain PB13. A

VOL. 170, 1988

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2818 DORMAN ET AL.

50-,lI amount of an exponential culture of this derivative wasspotted onto an LB-streptomycin-ampicillin plate spreadwith the F- recipient strain GB102. All Smr Apr transcon-jugants were GB102 derivatives which had acquired pGB144containing a -y insertion, following resolution of the trans-ferred cointegrate between the F factor and pGB144.

Plasmids. Plasmid pPAK316, encoding the TnpR proteinof transposon Tn3 (27), was obtained from D. Sherratt. Thisplasmid is a pACYC184 derivative with a modified Tn3element inserted in the tetracycline resistance gene. The Tn3modification involves inversion of an 8.3-kilobase (kb) PstIfragment carrying the res site and the tnpR gene. Thisinversion inactivates the tnpA and bla genes; in addition, asmall PstI fragment, wholly contained within the tnpA gene,has been deleted.

Plasmid pGB144 contains the S. typhimurium tonB genewith about 600 base pairs (bp) of upstream sequence on a1.35-kb BglII-BamHI fragment (28), cloned into the multi-copy vector pGS1406 (11) (Fig. 1A). This plasmid was usedas starting material for all other constructions involving thetonB gene. Plasmid-borne tonB-lacZ fusions were con-structed by cloning the 1.35-kb BglII-BamHI tonB-con-taining fragment from pGB144 between the EcoRI andBamHI sites of plasmid pMLB1034 (5) to give plasmidpGB150 (Fig. 1C). tonB was poorly expressed when pGB150was transformed into a tonB deletion strain (see Results).Protein fusions between tonB and lacZ were obtained bytransforming pGB150 into strain MBM7060 and selecting forLac' derivatives. These arise as a result of spontaneousdeletions that fuse the 3' end of the tonB gene to the 5' endof lacZ (5). One such derivative plasmid, pGB210, wasrescued by transformation back into MBM7060 and shownby restriction mapping to have undergone a deletion of about750 nucleotides, which included the BamHI site of pGB150(Fig. 1iC).

RESULTS

Aberrant activity of TonB expressed from a multicopyplasmid. Plasmid pGB144 contains the tonB gene of S.typhimurium cloned into the multicopy vector pGS1406 (28)(Fig. 1A). In this plasmid, tonB is transcribed from itsphysiological promoter. When transformed into the E. colitonB deletion strain GB102, TonB function was only par-tially restored (Table 2). That is, pGB144 conferred slightsensitivity to colicins B and V and to phage +80 but by nomeans restored sensitivity to that of the wild type or to alevel conferred by the tonB gene cloned on a single-copyvector (28). The possibility that a mutation in or near thetonB gene had arisen during cloning was excluded by repeat-ing the procedure and by DNA sequence analysis of thesubcloned tonB insert (data not shown).The observation that tonB gene function is impaired when

cloned on a multicopy plasmid has been made previously(42; K. Postle, personal communication). It was suggested(42) that this might be due to inhibition of TonB activity byan excess of the protein rather than to an effect on tonB geneexpression. H-owever, in the absence of an assay for theTonB protein, these authors were unable to confirm thismodel. We therefore used anti-TonB antibodies to assess thesynthesis of TonB by Western blotting (Fig. 2). The amountof TonB protein synthesized from the chromosomal genewas so- low as to be undetectable. However, in strainscarrying pGB144, TonB was readily detected. As pGB144substantially overproduces (by at least 10-fold) the TonBprotein and yet fails to restore TonB function to a tonB

FIG. 1. Structure of tonB plasmids. (A) pGB144 is an 11.2-kbderivative of pGS1406 (11) containing the S. typhimurium tonBgene. The origin of replication (rep), the P-lactamase gene (bla), thetruncated E. coli lac operon (1acZYA'), and the tonB gene areindicated. The construction of pGB320, pGB321, pGB324, andpGB325 is described in the text. pGB157 to pGB162 are derivativesof pGB144 into which -yS was inserted. The locations and orienta-tions of these insertions are indicated. (B) Structure of thepGB144::-yb derivative pGB158. Plasmids pGB310 and pGB311 wereconstructed from pGB158 by deleting specific sequences. The DNAretained in these constructs and the restriction endonuclease sitesused are indicated. (C) Structures of the tonB+ plasmid pGB150 andits Lac' derivative, pGB210. pGB210 arose as a spontaneous Lac'deletion from pGB150, the extent of which is indicated. B, BamHI;C, ClaI; H, Hindlll; R, EcoRI; S, SmaI.

deletion strain, this is strong evidence for the model (42) thatexcess TonB protein inhibits its own function. The mecha-nism by which this is achieved is obscure but may be related

A.

pG324/pGB321

p68331

pGB62

J. BACTERIOL.

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

DNA SUPERCOILING AND GENE EXPRESSION 2819

TABLE 2. Complementation of TonB function by theplasmid-encoded tonB gene

Plasmida Description Zone of killing(mm)b

pGB144 Multicopy tonB+ 3X opp8c Single-copy tonB+ 10pGS1406 Vector alone (tonB) 0pGB156 pGB144tonB::-y 0pGB157 to pGB162 pGB144::-y8 10pGB310 pGB144 tnpR res 3pGB311 pGB144 tnpR+ res+ 10pGB320 pGB144 tnpR+ res+ 10pGB321 pGB144 tnpR+ res+ 10pGB324 pGB144 X 3pGB325 pGB144 tnpR res+ 3pGB144 + pPAK316 (tnpR+) 3pGB325 (res+) + pPAK316 Lethal

(tnpR+)a See Fig. 1 and text for details of plasmid constructions.b Zone of killing by colicins B and V was measured as described in

Materials and Methods.Described in reference 28.

to the fact that, when overproduced, the TonB protein formsaggregates within the cell (unpublished results).tonB expression is activated by -y& insertions in cis. During

analysis of the tonB plasmid pGB144, we observed that theability to restore TonB function to a tonB deletion strain wasenhanced by insertion of the -yb transposon into the plasmid.Insertions of -yb into pGB144 were selected at random, andthe location and orientation of the insertions were deter-mined by restriction mapping (24, 53) (Fig. 1A). Insertions of-yb into the rep or bla genes were not obtained, presumablybecause they are lethal. Insertions elsewhere in the plasmidgave two distinct phenotypes (Table 2). As expected, inser-tions in the tonB structural gene eliminated the low-levelTonB activity of pGB144 (e.g., pGB156; Table 2). However,all other yb insertions into pGB144 (pGB157 to pGB162)increased TonB activity such that the derivative plasmidsfully restored colicin sehsitivity to the tonB deletion strainGB102. Plasmids carrying these ryb insertions were found to

OJ a3 aN man ci. E

C,j miC' mCMCC) CN

UJ c ) L3

AXB>

FIG. 2. Synthesis of TonB from pGB144 and its -yb insertionderivatives. Cell extracts were separated by electrophoresis, trans-ferred to nitrocellulose, and probed with anti-TonB antibodies.GB102 (AtonB) is the host strain. A, TonB protein; X, alkalinephosphatase, detected by the staining technique; B, breakdownproduct of TonB.

direct synthesis of at least fivefold more TonB than pGB144(Fig. 2). Thus, -yb insertions cause an increase in expressionof the plasmid-encoded tonB gene. This was shown to be aresult of increased transcription from the tonB promoter byNorthern (RNA) blotting (data not shown). Activation oftonB expression by yb was independent of the orientation ofthe transposon and its distance from the tonB gene. Further-more, tonB expression from these plasmids was regulatednormally by anaerobiosis and iron (see below). Thus, acti-vation of tonB transcription cannot be due to readthroughfrom an outward-reading -yb promoter. The fact that activa-tion of tonB expression by yb was both distance and orien-tation independent is reminiscent of eucaryotic enhancersand can best be explained by changes in promoter topology(see below).

Activation of tonB expression requires the tnpR gene and ressite of -y8. One -yb insertion derivative pGB144 (plasmidpGB158) was selected for further study. Removal of the yend of the transposon, by digesting with EcoRI and religating(Fig. 1B), had no effect on tonB expression; the resultantplasmid, pGB311, remained fully TonB+ (Table 2). In con-trast, removal of additional -yb sequences, including theresolvase gene tnpR and the res site, by digestion of pGB158with BamHl and religation produced a plasmid (pGB310,Fig. 1B) which was phenotypically TonB-. This suggeststhat activation of tonB expression by ryB is associated withthe function of the resolvase.To test this hypothesis, a 2.4-kb HindIII fragment from -y8,

encoding the TnpR protein and the res sequence, was ligatedin both orientations into the unique HindIII site of pGB144,giving plasmids pGB320 and pGB321 (Fig. 1A). BothpGB320 and pGB321 were fully TonB+ (Table 2). To ex-clude the possibility that tonB activation was simply due toan increase in plasmid size, the 2.3-kb HindlIl fragment ofphage lambda was also cloned into the HindIll site ofpGB144 to give pGB324; this fragment had no effect on tonBexpression. Although when TnpR and res were providedtogether, tonB expression was activated, the res site alonewas insufficient; when the res site alone was cloned intopGB144, as a 450-bp SmaI-ClaI fragment, the resultantplasmid (pGB325) failed to express tonB to any greaterextent than pGB144 (Fig. 1A and Table 2). Similarly, theTnpR protein alone was not sufficient to activate tonBtranscription. Thus, when the TnpR+ plasmid pPAK316 wascotransformed with pGB144 (which does not contain the ressite) into GB102, tonB was not activated. In conclusion,neither TnpR nor res alone is sufficient to activate the tonBpromoter.To demonstrate unambiguously that both res and TnpR

were required for activation of tonB expression, we at-tempted to transform GB102 simultaneously with pGB325(containing the res site but not the tnpR gene) and the tnpR+plasmid pPAK316. Although GB102 could be transformedsimultaneously with pGB325 (res+) and the vectorpACYC184, pPAK316 (a tnpR+ derivative of pACYC184)could not be introduced together with pGB325. One rare AprCmr transformant that was obtained was completely TonB-,and when pGB325 was isolated from this strain and retrans-formed into GB102, it failed to show even the basal level ofTonB function normally seen for pGB325 (Table 2). Thus,when forced to coexist with a TnpR+ plasmid, pGB325accumulated mutations which totally inactivated the tonBgene. The reason for this lethality is unclear and prevents aconclusive demonstration that TnpR can function in trans toactivate tonB expression. Nevertheless, it is clear thatactivation of tonB expression required both the TnpR pro-

VOL. 170, 1988

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2820 DORMAN ET AL.

A B

FIG. 3. Effect of novobiocin on the distribution of pGB210topoisomers. An overnight culture of cells containing plasmidpGB210 was diluted 200-fold in MMA and grown aerobically in theabsence (lane A) or presence (lane B) of 20 ,ug of novobiocin per ml.Once the ODWO reached 0.2, plasmid DNA was isolated andtopoisomers were separated on a 0.5% agarose gel containingchloroquine (25 ,ug/ml). At this concentration of chloroquine, thetopoisomers that were more relaxed prior to electrophoresis mi-grated more rapidly through the gel.

tein and the res site to which it binds. As TnpR, wheninteracting with the res sequence, is known to function as a

type I topoisomerase (36) and cause structural perturbationsin the DNA (26), the simplest explanation for the above datais that altered topology activates transcription from the tonBpromoter, particularly in view of the data presented below.

tonB expression is sensitive to novobiocin. The activation oftonB by yb can best be interpreted in terms of topologicaldependence of the tong promoter. Unfortunately, plasmidscarrying -yB insertions are too large to measure linkingnumber readily. We therefore used a smaller tonB-lacZplasmid, pGB210, which enabled us to determine the level ofsupercoiling for the very same plasmid from which tonB wasbeing expressed. This plasmid expresses a hybrid TonB-LacZprotein (Fig. 1C), and P-galactosidase synthesis reflectstranscription from the tonB promoter. To obtain directevidence that the tonB promoter is DNA supercoiling sensi-tive, we examined the effects of novobiocin. Novobiocin is aspecific inhibitor of DNA gyrase and normally results inrelaxation of intracellular DNA (23). An overnight culture ofcells containing pGB210 encoding a tonB-lacZ fusion pro-tein, was diluted 200-fold and grown aerobically in MMA inthe presence or absence of novobiocin (20 ,g/ml). Underthese conditions, this concentration of novobiocin was sub-lethal and had little effect on growth rate (data not shown).At an OD 0" of 0.2, P-galactosidase activity was assayed andfound to be increased by growth in the presence of novobi-ocin (3,594 U versus 1,407 U). Samples of cells from thesame cultures were harvested, pGB210 plasmid DNA was

extracted, and topoisomers were separated on a chloroquinegel (Fig. 3). As expected, DNA from novobiocin-treatedcells was more relaxed than DNA from untreated cells.Thus, relaxation of the plasmid DNA led to an increase in

tonB expression. Further evidence for novobiocin sensitivityof the tonB promoter is presented below.Growth phase-dependent tonB expression correlates directly

with the level ofDNA supercoiling. Cells of GB102 containing

pGB210 were grown aerobically in MMA, and at varioustime intervals samples were taken for 3-galactosidase assayand to prepare plasmid DNA. tonB expression was growthphase dependent, increasing ninefold throughout the growthcycle (Fig. 4A). Growth phase regulation of tonB expressionhas not been reported previously, and such regulation issomewhat unusual (15). When plasmid pGB210 DNA wasexamined, the DNA was found to become increasinglyrelaxed as growth proceeded (Fig. 4B). The linking numberof other unrelated plasmids, pACYC184 and pLK4 (12, 55),varied similarly in response to growth phase (data notshown). There was excellent correlation between the in vivolevels of supercoiling of pGB210 and tonB expression fromthis plasmid; the more relaxed the plasmid, the more effi-ciently tonB was transcribed. This is consistent with theresults obtained for novobiocin treatment above.Chromosomal and plasmid-encoded tonB are regulated sim-

ilarly. To assess the effects of anaerobiosis and iron onplasmid-encoded tonB expression, the P-galactosidase activ-ity of cells carrying pGB210 was assayed under a variety ofgrowth conditions (Table 3). In anaerobically grown cells,tonB expression was repressed by excess iron. However,excess iron failed to repress tonB when cells were grownaerobically. Thus, neither iron nor anaerobiosis alone issufficient to repress tonB expression.Because data obtained for the plasmid-encoded tonB gene

might not reflect the normal chromosomal regulation, ex-pression of tonB was also analyzed with chromosomaltonB-lacZ fusions. Strain GB291 carries a Xplac Mu inser-tion in the E. coli tonB gene, directing synthesis of atonB-lacZ transcriptional fusion (see Materials and Meth-ods). GB291 was grown under appropriate conditions, and,-galactosidase activity was assayed (Table 3). As for theplasmid-encoded gene, transcription from the tonB promoterwas repressed by excess iron under anaerobic conditions butnot aerobically. In the absence of iron, anaerobic growth didnot repress tonB expression and normal aerobic expressionwas observed. These data are similar to those obtained byHantke (25) and show that regulation of tonB gene expres-sion on plasmid pGB210 reflects regulation in its normalchromosomal context. It should be noted that excess irondid not repress the plasmid-encoded tonB gene as com-pletely as it did the chromosomal gene. This is presumablydue to titration of the Fur protein, which mediates ironrepression of tonB (3, 60).

It is important to exclude the possibility that the aerobicinduction of tonB expression is distinct from iron-mediatedregulation and is not simply due to iron limitation caused bythe oxidation of iron to an insoluble form. Two lines ofpublished evidence argue against this. First, other geneswhich are repressed by the same iron-binding regulatoryprotein (Fur) as tonB are not derepressed during aerobicgrowth (22, 25). Second, in fur mutants, which lack ironregulation, tonB is still derepressed by aerobic growth (25).Finally, because the chromosomal fusion strain GB291 lacksa functional TonB protein, it was important to exclude thepossibility that the absence of TonB itself resulted in ironlimitation. GB291 was therefore transformed with theTonB+ plasmid pGB158. However, even in the presence ofa functional TonB protein, iron failed to repress tonB-lacZexpression during aerobic growth (data not shown). Thus,oxygen regulation of tonB expression is clearly distinct fromthe Fur-mediated iron regulation.

Anaerobic growth affects plasmid DNA supercoiling. Weshowed above that expression of the tonB gene correlateswith changes in DNA supercoiling, increasing as the tem-

J. BACTERIOL.

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

DNA SUPERCOILING AND GENE EXPRESSION 2821

A 6000

5000 FUco

0

m'e*)

U,

B')>> 'O 4-)

Z Z <z t-

4000 F

3000 1

2000 1

1000 1-

00

O.D.

2

600FIG. 4. Growth phase regulation of tonB expression correlates directly with changes in plasmid supercoiling. Cells containing the

tonB-lacZ fusion plasmid pGB210 were grown in MMA under fully aerobic conditions. At appropriate intervals during growth (monitored bymeasuring the OD6w), samples were harvested and used for ,B-galactosidase assays and for determination of plasmid topoisomer distribution.(A) 13-Galactosidase activity plotted as a function of growth phase (OD6.). (B) Plasmid topoisomer distribution as a function of growth phase(OD6w). Plasmid DNA was separated on a 0.5% agarose gel containing chloroquine (25 ,ug/ml). At this concentration of chloroquine, thetopoisomers that were more relaxed before electrophoresis migrated more rapidly through the gel. Samples were taken for ,3-galactosidaseand plasmid topoisomer preparations at the OD6. values shown above the gel.

plate is relaxed. If this supercoiling sensitivity plays a role inthe regulation of tonB expression, it is important to demon-strate that supercoiling levels in vivo change in response tothose environmental factors which influence tonB expres-sion. Since iron alone is insufficient to repress tonB, itseemed possible that an anaerobically induced topologicalchange might also be required for repression. PlasmidpACYC184 (12) DNA was isolated from strain GB291 grownaerobically and anaerobically. Plasmid DNA isolated fromcells grown anaerobically was more negatively supercoiledthan DNA isolated from cells grown aerobically, with anincrease of up to 10 supercoils (Fig. 5). The same was foundfor plasmid DNA isolated from the tonB+ strain MC4100,showing that TonB itself plays no role in the anaerobicchanges in supercoiling, and also for an entirely unrelatedplasmid pLK4 (55; data not shown). When anaerobicallygrown cells were treated with the gyrase inhibitor novobio-

TABLE 3. Regulation of tonB-lacZ expression from thechromosome and from the multicopy plasmid pGB210a

Growth conditions P-Galactosidase activity (U)

02 Iron GB291 pGB210(chromosomal) (plasmid)

- - 169 4,760- + 37 522+ - 172 4,504+ + 159 3,417

a Cells were grown to early exponential phase (O.D.6m = 0.3) in MMA,aerobically or anaerobically as indicated. For aerobic conditions, the culturewas shaken vigorously in a large vessel; under these conditions, characteristicanaerobically induced genes remain repressed. For anaerobic conditions,growth was in a filled and sealed vessel without shaking. Iron excess (+) wasachieved by adding 100 ,uM FeCl3; iron-limiting growth (-) was achieved byadding 200 p.M dipyridyl to the medium.

cin, the effects of anaerobiosis were reversed and supercoil-ing was restored to a level approximating that of aerobicallygrown cells (Fig. 5). Novobiocin inhibits the GyrB subunit ofDNA gyrase (23), and thus the increase in negative super-coiling of DNA in anaerobically grown cells may depend onDNA gyrase activity.Oxygen regulation of tonB expression is mediated by

changes in supercoiling. We showed above that, in highlyaerated cells, tonB expression increases with growth phaseand correlates directly with growth phase-induced relaxation

A B C

FIG. 5. Anaerobic growth increases DNA supercoiling. The re-porter plasmid pACYC184 was isolated from overnight cultures ofGB291 grown in MMA aerobically (lane A), anaerobically (lane B),and anaerobically in the presence of novobiocin (20 ,ug/ml) (lane C).Topoisomers were separated on a 1% agarose gel containing chlo-roquine (25 ,ug/ml). Under these conditions, the topoisomers thatwere more relaxed prior to electrophoresis migrated faster throughthe gel.

VOL. 170, 1988

1

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2822 DORMAN ET AL.

400

4000

0

Im, 3000

0"A 2000

1000

06

I a I ~

0 1 2 3 4 5 0 1 2OD. 600 O.D. 600

FIG. 6. Expression of the plasmid- and chromosomally encoded tonB genes is similarly supercoiling dependent. (A) Cells harboring thetonB-lacZ fusion plasmid pGB210 were grown semiaerobically (see text) in LB with (*) or without (E) the gyrase inhibitor novobiocin (20jig/ml). 1-Galactosidase activity was assayed at the indicated ODwo values. Expression of ,3-galactosidase increases with growth phase untilconditions begin to become anaerobic, at which point expression begins to decline. (B) The chromosomal tonB-lacZ fusion strain GB291 wasgrown semiaerobically in LB with (*) or without (El) novobiocin (20 ,ug/ml). At the indicated OD6. values, samples were taken and assayedfor P-galactosidase activity.

of the DNA template. If anaerobically induced increases inDNA supercoiling play a role in the anaerobic repression oftonB expression, anaerobic growth would be expected toreverse the growth phase induction of expression. This wasshown to be the case. tonB expression from pGB210 was

assayed at various points throughout growth in a half-filled,capped Universal bottle. Under these semianaerobic condi-tions, when growth exceeded on OD6. of about 0.3, theculture began to become anaerobic and expression of tonBdecreased (Fig. 6A). Anaerobiosis was shown to be respon-sible for the change in tonB expression, as various typicalanaerobic genes were induced at the same stage of growth(data not shown); in addition (Fig. 4), if extensive effortswere made to maintain aerobicity, these anaerobic geneswere not induced and tonB remained derepressed. We alsoshowed that the effects observed above were not due to ironlimitation, as the induction profiles were unaltered by theaddition of excess iron to the LB medium (data not shown).Finally, novobiocin partially reversed the anaerobically in-duced increase in negative superhelicity of plasmid DNA(Fig. 5). We therefore examined the effects of novobiocin onthe anaerobic repression of tonB and found that the drugprevented the decrease in tonB expression normally ob-served as the cells became anaerobic; in the presence ofnovobiocin, expression was maintained at a level essentiallythe same as for cells maintained under fully aerobic condi-tions. These data strongly support the model that anaerobicrepression of tonB expression is mediated by anaerobicallyinduced increases in DNA supercoiling.The above experiments were carried out on the plasmid-

encoded tonB gene. We have shown that anaerobic regula-tion of tonB expression from plasmid pGB210 is very similarto that of the chromosomally encoded gene (see above). Wetherefore repeated the above experiment on strain GB291,which harbors a chromosomal tonB-lacZ fusion (Fig. 6B).Again, tonB expression increased with growth phase until anOD6. of about 0.3 was reached, when expression began todecrease. As for the plasmid-encoded gene, the effect ofanaerobiosis was reversed by novobiocin. Thus, the aerobic-anaerobic regulation of chromosomal tonB gene expressionalso appears to be mediated by changes in DNA supercoil-ing.Not aUl anaerobically induced genes are supercoiling depen-

dent. To assess how general the sensitivity of anaerobically

regulated genes to supercoiling might be, transcriptionallacZ fusions to a variety of oxygen-regulated genes wereexamined. These were fusions to the oxiC, aniC, aniE, aniH,and aniI loci, which have been mapped to very differentlocations on the S. typhimurium chromosome (Table 4) (1).The fusions displayed a variety of regulatory features. LiketonB, the oxiC fusion was anaerobically repressed. The fourani fusions are induced by anaerobic growth, but to verydifferent extents (1). Anaerobic cultures of each of thesefusion strains were grown in the presence and absence ofnovobiocin, and P-galactosidase activity was assayed (Table4). The various fusions displayed very different responses tonovobiocin. Three of the fusions (oxiC, aniC, and aniE) werecompletely unaffected by novobiocin treatment, expressionof aniI was repressed, and expression of aniH was in-creased. Although care must be taken when interpreting theresults of inhibitor experiments, these results at least suggestthat not all oxygen-regulated promoters respond in a similarmanner to changes in DNA supercoiling.

DISCUSSION

It is well established that the expression of a number ofgenes is influenced by the level ofDNA supercoiling, both invitro and in vivo (17, 18, 27a, 32, 57, 72). Thus, the idea thatenvironmentally induced changes in superhelicity play a rolein the regulation of gene expression is attractive. In thispaper we show that anaerobiosis and growth phase influencethe level of DNA supercoiling in vivo. Furthermore, these

TABLE 4. Effect of novobiocin on the expression of variousanaerobically regulated genesa

,3-Galactosidase activity (U)Strain Gene Approx. maplocation (min) With Without

novobiocin novobiocin

JF1003 oxiC::Mu dl-8 28 450 474JF1141 anil::Mu dl-8 35 130 45JF1292 aniE::Mu dl-8 42 205 251JF1325 aniC::Mu dl-8 93 47 41JF1415 aniH::Mu dl-8 81 347 996

a Cells were grown to mid-exponential phase anaerobically in LB, with orwithout 20 ,ug of novobiocin per ml, and ,B-galactosidase activity was assayedas described in Materials and Methods.

J. BACTERIOL.

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

DNA SUPERCOILING AND GENE EXPRESSION 2823

environmentally induced changes in superhelicity appear tobe important for the normal regulation of at least oneoxygen-regulated gene, the tonB gene, of both E. coli and S.typhimurium.The tonB gene is required for a number of outer membrane

processes, including the uptake of iron chelates and vitaminB12. Expression of tonB is regulated by iron availability,mediated by the Fur protein, which acts as a repressor in thepresence of iron (3, 25, 60). However, even in the presenceof excess iron, tonB expression is induced aerobically inde-pendently of iron availability and the Fur protein. Theaerobic induction of tonB expression is probably of somephysiological importance as iron is oxidized aerobically,loses solubility, and becomes less available for uptake intothe cell.The tonB promoter is highly sensitive to changes in DNA

topology. Expression, both from a multicopy plasmid andfrom the chromosome, is increased by the DNA gyraseinhibitor novobiocin. Under anaerobic growth conditionswhich increase negative supercoiling (see below), tonBexpression is repressed, and there is also excellent correla-tion between the growth phase-dependent changes in DNAsupercoiling and expression of tonB. We have shown else-where that osmZ mutations and increased medium osmolar-ity, both of which increase plasmid and chromosomal super-coiling (27a), decrease tonB expression. Further evidencethat the tonB promoter is topologically sensitive comes fromstudies on the cloned gene. Plasmid-encoded tonB expres-sion was increased by the transposable element -yb. ybactivated tonB only when present in cis, and its activity wasboth distance and orientation independent. This is reminis-cent of eucaryotic enhancers and can best be explained by atopological effect on tonB promoter activity. A more detailedanalysis of the -yb sequences required to activate tonBconfirmed this interpretation. Only the TnpR protein and itsbinding site, res, appear to be required. The TnpR protein isknown to function as a type I topoisomerase and can alterDNA structure when it interacts at the res site (36). It shouldbe pointed out that, to exhibit topoisomerase activity, twores sites are required on the target plasmid. Presumably theplasmid dimerizes temporarily to provide two res sites whichare resolved by the TnpR protein, altering the linking num-ber. An alternative possibility is that binding of the TnpRprotein to the res site might, in itself, alter DNA topology(26), which, when recognized by DNA gyrase, would resultin a compensatory adjustment in linking number. There areat least two precedents for activation or inhibition of pro-moter function by a nearby transposable element, both ofwhich are best interpreted on the basis of topological effects.Expression of the cloned ebg gene of E. coli is reduced by -ybinsertions in an orientation- and distance-independent man-ner (68). These data are analogous to our own, apart from thefact that ebg is inhibited rather than stimulated by -yb.Similarly, IS insertions near the chromosomal bgl operonactivate the normally cryptic bgl promoter (54). It is believedthat this is a result of an insertion sequence-induced changein local DNA topology (54); the bgl promoter is known to besensitive to altered DNA topology (16, 51) and can also beactivated by mutations in a trans-acting gene, osmZ, whichalter chromosomal supercoiling in vivo (27a).

If the topological sensitivity of the tonB promoter plays arole in the physiological regulation of tonB expression, it isimportant to show that DNA topology is influenced bynormal environmental stimuli. It now appears that DNAtopology in growing cells is in a dynamic state and isinfluenced by a variety of external factors. We show here

that the linking number of plasmid DNA decreases withgrowth phase but increases as cells become anaerobic. Thereversal of this effect by novobiocin suggests that DNAgyrase activity is required for the anaerobically inducedincrease in DNA supercoiling. In addition, we have previ-ously shown that plasmid linking number is affected bygrowth medium osmolarity (27a), and a recent report impliesa role for nutrient shifts (6). Thus, there is complex controlof in vivo topology. This finding has considerable implica-tions for the fine-tuning of gene expression and the experi-mental conditions under which studies should properly beundertaken. As these results would predict, many osmoti-cally sensitive genes are also affected by anaerobiosis andanaerobically regulated genes are affected by osmotic stress(unpublished data). These studies involved monitoring plas-mid supercoiling and not the chromosome directly. How-ever, the fact that the chromosomal tonB gene responds tonovobiocin, anaerobiosis, and growth phase in a mannervery similar to that of the plasmid-encoded gene implies thatsimilar changes in superhelicity are occurring on the chro-mosome. It has been suggested that chromosomal DNAfrom anaerobically grown cells might be more highly super-coiled than that from aerobic cells (74; K. Drlica, personalcommunication). We do not yet know how anaerobiosisaffects the linking number of DNA in vivo. The fact thatthere is an effect on plasmids of different sequence and onthe chromosome implies a rather general specificity. Anae-robiosis may directly alter the activity of topoisomerase I orDNA gyrase. Alternatively, there may be additional, as yetunidentified, topoisomerases. A further possibility is thatanaerobiosis affects the interaction between general DNA-binding proteins, such as the histonelike HU proteins, andDNA. It is known that the binding of such proteins caninfluence DNA topology (7, 19).The changes in DNA supercoiling in response to growth

conditions seem to be important for the growth phase andanaerobic regulation of tonB expression. There is an excel-lent correlation between the levels of plasmid supercoilingand tonB expression from the same plasmid over a widerange of growth phases and anaerobiosis. The fact thatnovobiocin reverses the effects of anaerobiosis on supercoil-ing and similarly overcomes the anaerobic repression oftonB provides strong support for this model. Furthermore,since growth phase, anaerobiosis, and novobiocin affectexpression of the chromosomal tonB gene in a mannersimilar to that of the plasmid-encoded gene, changes insupercoiling must also be responsible for the anaerobicregulation of tonB in its normal chromosomal context.The observation that anaerobic growth causes DNA to

become increasingly supercoiled raises the question of howspecificity is achieved. First, of course, many promoters arenot significantly sensitive to changes in supercoiling (17).Those few promoters which are exquisitely sensitive toDNA supercoiling presumably include those which showlarge induction ratios after a shift between aerobiosis andanaerobiosis. In addition, many genes not normally consid-ered anaerobically induced do exhibit minor variations inexpression after a shift to anaerobiosis (unpublished results).This may well be a reflection, with little physiologicalsignificance, of anaerobically induced changes in chromo-somal supercoiling. A further factor which may be importantis genetic geography. The chromosomal locations of genesmay affect their responses to altered supercoiling. There isevidence that the bacterial chromosome is divided intotopologically distinct domains (63, 73), and these may bedifferently affected by anaerobiosis. Since genes which are

VOL. 170, 1988

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2824 DORMAN ET AL.

anaerobically responsive are scattered around the chromo-some and are frequently close to genes which are unaffectedby anaerobiosis, this is not a complete explanation. Addi-tional factors, such as the histonelike DNA-binding proteins(e.g., HU, Fis, and IHF) or even events such as transcrip-tion, can have local effects on topology and gene expression(7, 40, 50). Thus, local genetic context may well play a rolein determining sensitivity to global topological changes.The possibility that an increase in DNA supercoiling

associated with anaerobic growth forms a primary compo-

nent of the anaerobic switch is an attractive one. Such a keyrole was suggested on the basis that some DNA gyrase

mutants are unable to grow anaerobically and topoisomeraseI mutants are unable to grow aerobically (74). We have beenunable to confirm these results. Well-defined topA deletions(55) are fully viable aerobically. Presumably, in the anaero-bic constitutive mutants described by Yamamoto andDroffner (74), additional mutations had accumulated. Fur-thermore, their data were obtained with nonisogenic deriv-atives and in genetic backgrounds (LT2 and LT7) which are

known to differ in their anaerobic enzymes (59). It seems

more probable that, while anaerobically induced changes intopology play a key role in the regulation of some genes,

they are unlikely to be solely responsible for the anaerobicswitch. Despite suggestions that anaerobically inducedgenes might form a global regulon, it is becoming increas-ingly clear that several distinct (but perhaps overlapping)regulatory networks are involved. Certain anaerobicallyregulated promoters are supercoiling sensitive, includingtonB and the nifH promoter of a cyanobacterium (35), and,at least for tonB, anaerobic changes in DNA supercoilingseem to be the primary means of regulation. For suchpromoters, additional factors are also often superimposed,such as regulation of tonB by iron and the fur gene product.Indeed, the binding of such specific regulatory proteinsmight also be influenced by topology. Nevertheless, many

anaerobically regulated genes are completely insensitive tonovobiocin and therefore are unlikely to be topologicallyregulated. Furthermore, a large subset of anaerobicallyinduced genes are dependent on the positive regulatoryprotein Fnr. Supercoiling changes would appear to be irrel-evant for such promoters, although the possibility that Fnrcan only interact productively when the DNA is in thecorrect topological state should be considered. Certain Fnr-binding sites are surrounded by sequences which may wellbe sensitive to changes in topology or form bends (31). Thus,changes in DNA supercoiling seem to be likely to provideone of several levels of control which must be considered inconcert with other regulatory processes in attempting tounderstand the mechanisms by which the anaerobic re-

sponse is regulated.

ACKNOWLEDGMENTS

We are grateful to D. H. Boxer, D. W. Brighty, K. Drlica, M.Gallagher, S. Pearce, and J. A. Cole for useful discussions and thankJ. Foster, K. Postle, A. P. Pugsley, C. Schnaitman, and D. Sherrattfor bacterial strains or plasmids.C.F.H. is a Lister Institute Research Fellow.

LITERATURE CITED1. Aliabadi, Z., F. Warren, S. Mya, and J. W. Foster. 1986.

Oxygen-regulated stimulons of Salmonella typhimurium identi-fied by Mu d(Ap lac) operon fusions. J. Bacteriol. 165:780-786.

2. Ames, G. F.-L. 1974. Resolution of bacterial proteins by poly-acrylamide gel electrophoresis on slabs. J. Biol. Chem.249:634 644.

3. Bagg, A., and J. B. Neilands. 1987. Ferric uptake regulationprotein acts as a repressor, employing iron(II) as a cofactor tobind to the operator of an iron transport operon in Escherichiacoli. Biochemistry 26:5471-5477.

4. Barrett, E. L., H. S. Kwan, and J. Macy. 1984. Anaerobiosis,formate, nitrate, and pyrA are involved in the regulation offormate hydrogenlyase in Salmonella typhimurium. J. Bacte-riol. 158:972-977.

5. Berman, M. L., and D. E. Jackson. 1984. Selection of lac genefusions in vivo: ompR-lacZ fusions that define a functionaldomain of the ompR gene product. J. Bacteriol. 159:750-756.

6. Blake, V. L., and J. D. GralIa. 1987. Changes in linking numberof supercoiled DNA accompany growth transitions in Esche-richia coli. J. Bacteriol. 169:4499-4506.

7. Bliska, J. B., and N. R. Cozzarelli. 1987. Use of site-specificrecombination as a probe of DNA structure and metabolism invivo. J. Mol. Biol. 194:205-218.

8. Borowiec, J. A., and J. D. Gralla. 1987. All three elements of thelac pS promoter mediate its transcriptional response to DNAsupercoiling. J. Mol. Biol. 195:89-97.

9. Bremer, E., T. J. Silhavy, and G. M. Weinstock. 1985. Trans-posable Aplac Mu bacteriophages for creating lacZ operonfusions and kanamycin resistance insertions in Escherichia coli.J. Bacteriol. 162:1092-1099.

10. Casadaban, M. J. 1976. Transposition and fusion of the lacgenes to selected promoters in Escherichia coli using bacterio-phage lambda and Mu. J. Mol. Biol. 104:541-555.

11. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro genefusions that join an enzymatically active ,B-galactosidase seg-ment to amino-terminal fragments of exogenous proteins. J.Bacteriol. 143:971-980.

12. Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloning vehiclesderived from the P1SA cryptic miniplasmid. J. Bacteriol. 134:1141-1156.

13. Cole, J. A. 1988. Assimilatory and dissimilatory reduction ofnitrate to ammonia. Soc. Gen. Microbiol. Symp. 42:281-329.

14. Coleman, D. C., and T. J. Foster. 1981. Analysis of the reduc-tion in expression of tetracycline resistance determined by thetransposon TnJO in the multicopy state. Mol. Gen. Genet. 182:171-177.

15. Connell, N., Z. Han, F. Moreno, and R. Kolter. 1987. AnEscherichia coli promoter induced by the cessation of growth.Mol. Microbiol. 1:195-202.

16. DiNardo, S., K. A. Voelkel, R. Sternglanz, A. E. Reynolds, andA. Wright. 1982. Escherichia coli DNA topoisomerase I mu-tants have compensatory mutations in DNA gyrase genes. Cell31:43-51.

17. Drlica, K. 1984. Biology of bacterial deoxyribonucleic acidtopoisomerases. Microbiol. Rev. 48:273-289.

18. Drlica, K. 1987. The nucleoid, p. 91-103. In F. C. Neidhardt(ed.), Escherichia coli and Salmonella typhimurium: cellularand molecular biology. American Society for Microbiology,Washington, D.C.

19. Drlica, K., and J. Rouviere-Yaniv. 1987. Histonelike proteins ofbacteria. Microbiol. Rev. 51:301-319.

20. Elison, M. J., M. J. Fenton, P. S. Ho, and A. Rich. 1987.Long-range interactions of multiple DNA structural transitionswithin a common topological domain. EMBO J. 6:1513-1522.

21. Evans, J. S., B. A. Levine, I. P. Trayer, C. J. Dorman, and C. F.Higgins. 1986. Sequence-imposed structural constraints in theTonB protein of E. coli. FEBS Lett. 208:211-216.

22. Fleming, T. P., M. S. Nahlik, and M. A. McIntosh. 1983.Regulation of enterobactin iron transport in Escherichia coli:characterization of ent::Mu d(Apr lac) operon fusions. J. Bac-teriol. 156:1171-1177.

23. Gellert, M., M. H. O'Dea, T. Itoh, and J. Tomizawa. 1976.Novobiocin and coumermycin inhibit DNA supercoiling cata-lyzed by DNA gyrase. Proc. Natl. Acad. Sci. USA 73:4474-4478.

24. Guyer, M. S. 1978. The -yS sequence is an insertion sequence. J.Mol. Biol. 126:347-365.

25. Hantke, K. 1981. Regulation of ferric iron transport in Esche-

J. BACTERIOL.

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

DNA SUPERCOILING AND GENE EXPRESSION 2825

richia coli K12; isolation of a constitutive mutant. Mol. Gen.Genet. 182:288-292.

26. Hatfull, G. F., S. M. Noble, and N. D. F. Grindley. 1987. The -ybresolvase induces an unusual DNA structure at the recombina-tional crossover point. Cell 49:103-110.

27. Heffron, F., B. J. McCarthy, H. Ohtsubo, and E. Ohtsubo. 1979.DNA Sequence analysis of the transposon Tn3: three genes andthree sites involved in the transposition of Tn3. Cell 18:1153-1163.

27a.Higgins, C. F., C. J. Dorman, D. A. Stirling, L. Waddell, I. R.Booth, G. May, and E. Bremer. 1988. A physiological role forDNA supercoiling in the osmotic regulation of gene expressionin S. typhimurium and E. coli. Cell 52:569-584.

28. Hiles, I. D., L. M. Powell, and C. F. Higgins. Peptide transportin Salmonella typhimurium: molecular cloning and characteri-zation of the oligopeptide permease genes. Mol. Gen. Genet.206:101-109.

29. Jamieson, D. J., and C. F. Higgins. 1984. Anaerobic regulationof a peptide transport gene in Salmonella typhimurium. J.Bacteriol. 160:131-136.

30. Jamieson, D. J., and C. F. Higgins. 1986. Two geneticallydistinct pathways for transcriptional regulation of anaerobicgene expresion in Salmonella typhimurium. J. Bacteriol. 168:389-397.

31. Jayaraman, P. S., T. C. Peakman, S. J. W. Busby, R. V.Quincey, anid J. A. Cole. 1987. Location and sequence of thepromoter of the gene for the NADH-dependent nitrite reductaseof Escherichia coli and its regulation by oxygen, the Fnr proteinand nitrite. J. Mol. Biol. 196:781-788.

32. Jovanovich, S. B., and J. Lebowitz. 1987. Estimation of theeffect of coumermycin A1 on Salmonella typhimurium promot-ers by using random operon fusions. J. Bacteriol. 169:4431-4435.

33. Knecht, D. A., and R. L. Dimond. 1984. Visualization ofantigenic proteins on Western blots. Anal. Biochem. 136:180-184.

34. Konisky, J. 1979. Specific transport systems and receptors forcolicins and phages, p. 319-359. In M. Inouye (ed.), Bacterialouter membranes. John Wiley & Sons, New York.

35. Kranz, R. G., and R. Haselkorn. 1986. Anaerobic regulation ofnitrogen-fixation genes in Rhodopseudomonas capsulata. Proc.Natl. Acad. Sci. USA 83:6805-6809.

36. Krasnow, M. A., and N. R. Cozzarelli. 1983. Site-specificrelaxation and recombination by the Tn3 resolvase: recognitionof the DNA path between oriented res sites. Cell 32:1313-1324.

37. Kuritzkes, D. R., X.-Y. Zhang, and E. C. C. Lin. 1984. Use of4(glp-lac) in studies of respiratory regulation of the Escherichiacoli anaerobic sn-glycerol-3-phosphate dehydrogenase genes(glpAB). J. Bacteriol. 157:591-598.

38. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

39. Lambden, P. R., and J. R. Guest. 1976. Mutants of Escherichiacoli K12 unable to use fumarate as an anaerobic electronacceptor. J. Gen. Microbiol. 97:145-160.

40. Liu, L. F., and J. C. Wang. 1987. Supercoiling of the DNAtemplate during transcription. Proc. Natl. Acad. Sci. USA84:7024-7027.

41. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

42. Mann, B. J., C. D. Holroyd, C. Bradbeer, and R. J. Kadner.1986. Reduced activity of TonB-dependent functions in strainsof Escherichia coli. FEMS Microbiol. Lett. 33:255-260.

43. Menzel, R., and M. Geliert. 1983. Regulation of the genes for E.coli DNA gyrase: homeostatic control of DNA supercoiling.Cell 34:105-113.

44. Menzel, R., and M. Gellert. 1987. Fusions of the Escherichiacoli gyrA and gyrB control regions to the galactokinase gene areinducible by coumermycin treatment. J. Bacteriol. 169:1272-1278.

45. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

46. Neilands, J. B. 1982. Microbial envelope proteins related to iron.Annu. Rev. Microbiol. 36:285-309.

47. Newman, B. M., and J. A. Cole. 1978. The chromosomallocation and pleiotropic effects of mutations of the nirA + gene ofEscherichia coli K12: the essential role of nirA+ in nitritereduction and in other anaerobic redox reactions. J. Gen.Microbiol. 106:1-12.

48. Postle, K., and R. F. Good. 1983. DNA sequence of theEscherichia coli tonB gene. Proc. Natl. Acad. Sci. USA 80:5235-5239.

49. Postle, K., and R. F. Good. 1985. A bidirectional rho-indepen-dent transcription terminator between the E. coli tonB gene andan opposing gene. Cell 41:577-585.

50. Pruss, G. J., and K. Drlica. 1986. Topoisomerase I mutants: thegene of pBR322 that encodes resistance to tetracycline affectsplasmid DNA supercoiling. Proc. Natl. Acad. Sci. USA 83:8952-8956.

51. Pruss, G. J., S. H. Manes, and K. Drlica. 1982. Escherichia coliDNA topoisomerase I mutants: increased supercoiling is cor-rected by mutations near gyrase genes. Cell 31:35-42.

52. Pugsley, A. P., and P. Reeves. 1976. Characterization of group Bcolicin-resistant mutants of Escherichia coli K-12: colicin resis-tance and the role of enterochelin. J. Bacteriol. 127:218-228.

53. Reed, R. R., G. I. Shibuya, and J. A. Steitz. 1982. Nucleotidesequence of -yb resolvase gene and demonstration that its geneproduct acts as a repressor of transcription. Nature (London)300:381-383.

54. Reynolds, A. E., S. Mahadevan, J. Felton, and A. Wright. 1985.Activation of the cryptic bgl operon: insertion sequences, pointmutation and changes in superhelicity affect promoter strength.UCLA Symp. Mol. Cell. Biol. New Ser. 20:265-277.

55. Richardson, S. M. H., C. F. Higgins, and D. M. J. Lilley. 1984.The genetic control of DNA supercoiling in Salmonella typhi-murium. EMBO J. 3:1745-1752.

56. Roth, J. R. 1970. Genetic techniques in studies of bacterialmetabolism. Methods Enzymol. 17A:3-35.

57. Sanzey, B. 1979. Modulation of gene expression by drugsaffecting deoxyribonucleic acid gyrase. J. Bacteriol. 138:40-47.

58. Sauer, M., K. Hantke, and V. Braun. 1987. Ferric-coprogenreceptor FhuE of Escherichia coli: processing and sequencecommon to all TonB-dependent outer membrane receptor pro-teins. J. Bacteriol. 169:2044-2049.

59. Sawers, R. G., D. J. Jamieson, C. F. Higgins, and D. H. Boxer.1986. Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium.J. Bacteriol. 168:398-404.

60. Schaffer, S., K. Hantke, and V. Braun. 1985. Nucleotide se-quence of the iron regulatory gene fur. Mol. Gen. Genet. 200:110-113.

61. Shaw, D. J., D. W. Rice, and J. R. Guest. 1983. Homologybetween CAP and Fnr, a regulator of anaerobic respiration inEscherichia coli. J. Mol. Biol. 166:241-247.

62. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984.Experiments with gene fusions. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

63. Sinden, R. R., and D. E. Pettiohn. 1981. Chromosomes in livingEscherichia coli cells are segregated into domains of supercoil-ing. Proc. Natl. Acad. Sci. USA 78:224-228.

64. Smith, M. W., and F. C. Neidhardt. 1983. Proteins induced byanaerobiosis in Escherichia coli. J. Bacteriol. 154:336-343.

65. Smith, M. W., and F. C. Neidhardt. 1983. Proteins induced byaerobiosis in Escherichia coli. J. Bacteriol. 154:344-350.

66. Spiro, S., and J. R. Guest. 1987. Activation of the lac operon ofEscherichia coli by a mutant FNR protein. Mol. Microbiol.1:53-58.

67. Stewart, V. 1988. Nitrate respiration in relation to facultativemetabolism in enterobacteria. Microbiol. Rev. 52:170-213.

68. Stokes, H. W., and B. G. Hall. 1984. Topological repression ofgene activity by a transposable element. Proc. Natl. Acad. Sci.USA 81:6115-6119.

69. Strauch, K. L., J. B. Lenk, B. L. Gamble, and C. G. Miller.1985. Oxygen regulation in Salmonella typhimurium. J. Bacte-

VOL. 170, 1988

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

2826 DORMAN ET AL.

riol. 161:673-680.

70. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

71. Tse-Dinh, Y. 1985. Regulation of Escherichia coli DNA topo-isomerase I gene by DNA supercoiling. Nucleic Acids Res. 13:4751-4763.

72. Wang, J. C. 1985. DNA topoisomerases. Annu. Rev. Biochem.54:665-697.

73. Worcel, A., and E. Burgi. 1972. On the structure of the foldedchromosome of Escherichia coli. J. Mol. Biol. 71:127-147.

74. Yamamoto, N., and M. L. Droffner. 1985. Mechanisms deter-mining aerobic or anaerobic growth in the facultative anaerobeSalmonella typhimurium. Proc. Natl. Acad. Sci. USA 82:2077-2081.

75. Yerkes, J. H., L. P. Casson, A. K. Honkanen, and G. C. Walker.1984. Anaerobiosis induces expression of ant, a new Esche-richia coli locus with a role in anaerobic electron transport. J.Bacteriol. 158:180-186.

J. BACTERIOL.

on March 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from