Proc. Natl. Acad. Sci. USAVol. 81, pp. 4490-4494, July 1984Genetics

Cell-division control in Escherichia coli: Specific induction of theSOS function SfiA protein is sufficient to block septation

(plac-sfi4 fusion/inducible division inhibitor/ftsZ gene/lon gene/suLA gene)

OLIVIER HUISMANt, RICHARD D'ARIt, AND SUSAN GOTTESMANt§tInstitut Jacques Monod, Centre National de la Recherche Scientifique, Universite Paris 7, 75251 Paris Cedex 05, France; and tLaboratory of MolecularBiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205

Communicated by Evelyn M. Witkin, April 2, 1984

ABSTRACT Blocks in DNA replication cause a rapid ar-rest of cell division in Escherichia coli. We have previouslyestablished that the function SfiA (SulA), induced under theseconditions as part of the SOS response, is involved in this inhi-bition of division. To separate the effects of Sf A from those ofother SOS functions, we have constructed a plac-sfiA operonfusion, permitting specific induction of SfiA protein by addi-tion of the lac operon inducer isopropyl 3-D-thiogalactopyran-oside (IPTG). In ion mutants, in which the unstable SfiA pro-tein has a longer half-life, IPTG caused a rapid arrest of celldivision. Under these conditions, there is no concomitant in-duction of the SOS response. IPTG also caused a rapid arrestof cell division in Ion' strains. These results demonstrate thatinduction of the SfiA protein is sufficient to cause inhibition ofdivision. Mutations in the sfiB gene can suppress IPTG-in-duced SfiA-mediated inhibition of division. At higher SfiAconcentrations, however, even sfiB mutants cease division; anadditional mutation genetically inseparable from sfiB restoresnormal division. These observations reinforce the hypothesisthat the SfiB protein, probably required for cell septation, isthe target of action of the SfiA division inhibitor.

In the bacterium Escherichia coli, cell division is strictly co-ordinated with DNA replication. When cellular DNA syn-thesis is interrupted, there is rapid arrest of cell septation,resulting in filamentous growth (1). In 1967, Witkin proposeda model postulating that this arrest is due to a cell-divisioninhibitor whose synthesis is induced by blocks to DNA syn-thesis (2).

This model was supported by evidence that division-repli-cation coupling is ensured in part by the SOS response, in-duced by DNA damaging treatments (3, 4). The sfiA (orsulA) gene was originally defined by mutations suppressingSOS-associated filamentation (5-7). In vivo and in vitro anal-ysis have shown that sfiA, like other SOS operons, is undernegative control by the LexA repressor (4, 8, 9). When DNAsynthesis is interrupted, the activation of the RecA proteinleads to cleavage of LexA repressor (10), resulting in induc-tion of the SOS functions (4). These observations led to thesuggestion that the sfiA gene product may be the putativeinducible cell-division inhibitor.Recovery from septation inhibition is normally rapid once

DNA synthesis resumes, but the inhibition of cell divisionpersists and is lethal in cells carrying a mutation in Ion,which codes for an ATP-dependent protease (11, 12). Therapid recovery in lon+ cells can be explained as a conse-quence of the extremely short half-life (1.2 min) of SfiA inthese cells. In Ion- cells, the half-life of SfiA is 19 min (13).Presumably, in lon- cells, the accumulation of SfiA afterSOS induction is not counterbalanced by its rapid degrada-tion, and normal cell division cannot resume.

The sfiB (or suli) gene, like sfiA, was identified by muta-tions that suppress SOS filamentation (5-7). At least some ofthese mutations affect the FtsZ protein (ref. 14; C. Jones andI. B. Holland, personal communication), apparently an es-sential cell septation component (15, 16). It has been sug-gested that the SfiB protein is the target of action of the puta-tive SfiA division inhibitor (14, 17, 18).To study the effect of SfiA on the cell in the absence of

SOS induction, we have constructed an operon fusion plac-ing the sfiA gene under control of the lac promoter. With thisfusion, we show that induction of SfiA protein alone causescell-division arrest, even in the absence of induction of theSOS response. Division inhibition caused by the inducedplasmid is suppressed by an sfiB mutation and is made moreextreme in Ion mutant cells, as is the division inhibitioncaused by SOS induction of the chromosomal sfiA' gene.The results presented here establish that the SfiA protein isan SOS-inducible cell-division inhibitor. Furthermore, theyreinforce the hypothesis that the SfiB protein is the target ofSfiA. One can explain the effect of Ion on cell division solelythrough its effect on the stability of SfiA.

MATERIALS AND METHODSBacterial and Phage Strains, Plasmids. All strains used in

this work were derived from the K-12 strain GC4663 of geno-type F- AlIon-100 pyrD trp::TnJO his Agal rpsL. To keep thederivatives as nearly isogenic as possible, markers were in-troduced by P1 transduction: sfiA85 (5) with Pyr', supFwith Trp', AlacU169 with Pro' (after transduction toproC::TnS), lon+ with zba300::TnJO, sfiB114 (5) with ValR(ilvH613) or sfiB* with leu::TnJO. An exception was therecAl mutation, introduced by mating with HfrKL16 recAlsrlC300::TnJO (selection for tetracycline and streptomycinresistance). The F'IacI01lacz::Tn5 factor, kindly providedby H. Shuman, was crossed into all strains (selection forkanamycin and streptomycin resistance). The final step inall constructions was transformation with pGC165 orpGC165sfiA' DNA; the transformants were cultivated in thepresence of ampicillin.The sfiB* mutation was transferred from the original iso-

late (sfiB114 sfiB*; see text) into a Ion strain by cotransduc-tion with leu::TnlO. Ten transductants that were methylmethanesulfonate resistant (MeMesR) (sfiB or sfiB*) weretransformed with pTU302 DNA; all remained MeMesR(sfiB*).The phages used were Plvir for transduction, XcI857 Sam7

for detecting supF, Xp(sfiA::lac) clind (19), and XSM5 (13).XSM5 carries a 1.78-kilobase BamHI fragment carryingsulA' and the amino-terminal end of ompA inserted withinthe int gene of a XcI857 derivative of the XD69 vector (13,20).

Abbreviations: MeMes, methyl methanesulfonate; IPTG, isopropylf-D-thiogalactopyranoside; bp, base pair(s).§To whom reprint requests should be addressed.

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

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The plasmids used (other than tie F' factor) were pUC8(21), pSM3 (9), and pTU302 (22). The construction ofpGC165 (sfiAam46) is described in the text and the legend toFig. 1. The recombinant plasmid pGCJ165 sfiAt was isolatedby growing XSM5 (sfiAW) on a strain carrying pGC165, usingthe lysate to transduce a sup+/F'lacIP strain to ampicillin-resistance (resulting from XSM5::pGC165 cointegrates), andscreening the latter for filamentous growth in the presence ofisopropyl ,-D-thiogalactopyranoside (IPTG) and absence ofX immunity (the X prophage segregated rapidly).Growth Conditions. All experiments were carried out in

tryptone broth (23) at 370C. Minimal medium, used in strainconstruction, was appropriately supplemented M63 (23).Antibiotics were used at the following concentrations: ampi-cillin, 100 ,ug/ml; streptomycin, 200 Ag/ml; kanamycin, 25ttg/ml; tetracycline, 10 ,ug/ml. MeMes was used at 0.025%and IPTG was 5 mM.Recombinant DNA Techniques. Restriction endonucleases

and T4 ligase were used as recommended by the manufactur-ers. Other techniques used included rapid extraction or puri-fication of plasmid DNA, agarose gel electrophoresis, andelectroelution of DNA bands from agarose gels (24).

Miscellaneous Techniques. P1-mediated transduction (23),bacterial conjugation (23) and transformation (24), 3-galacto-sidase assays (23), and lysogenization with Xp(sfiA::lac)cIind (19) have been described. Particle counts were madewith a model ZB Coulter Counter. Photomicrographs weretaken in phase contrast with a Jenamed microscope (Zeiss).

RESULTS

Construction of a plac-sfiA Fusion. The precise role of theSfiA protein in SOS-associated division inhibition has been

F-

difficult to evaluate, because induction of SfiA could only beachieved by inducing the entire SOS response. To study theaction of the SfiA protein apart from effects due to otherSOS-induced functions, we constructed an operon fusion byplacing the sfiA gene under control of the lac promoter andpermitting induction of SfiA synthesis by IPTG, a gratuitousinducer of the lac operon.As a cloning vector, we chose the plasmid pUC8 (21). It is

a pBR322 derivative that carries (i) a bla gene, conferringampicillin-resistance on its hosts, (ii) the lac promoter andoperator and the initial part of the lacZ gene, and (iii) aunique site for the restriction endonuclease Sma I just down-stream from the lac regulatory region (Fig. 1).Our source for the sfiA gene was the multicopy plasmid

pSM3 (9), which carries an sfiA-amber mutant gene and theentire sfiA regulatory region (Fig. 1).The DNA sequence of the sfiA gene was determined by

Beck and Bremer (the unidentified open reading frame in ref.25), and subsequently identified as the sfiA gene by Mi-zusawa and Gottesman (13). From the sequence of sfiA andthe pBR322 sequence (26), the restriction endonuclease RsaI is expected to cut at two sites within the sfiA promoter, at athird site in the second codon of the sfiA gene, and at threeadditional sites outside the sfiA gene (Fig. 1). Three frag-ments of -800 base pairs (bp) (indicated in Fig. 1 as I, II, andIII) were purified from a partial Rsa I digest of EcoRI-cutpSM3 DNA and ligated to Sma I-digested pUC8 DNA.

Since pUC8 is a multicopy plasmid, it was possible thatthe basal level of SfiA from the clone we were seeking mightbe lethal to the cell. Our use of the sfiAam allele in the start-ing plasmid allowed us to determine this directly by screen-ing for the desired insertion in two steps. First, a nonsup-pressing (sup+) strain, in which the sfiAam gene is inactive,

mUI #H 1847 bp

-#------l 841 bpIII-I/----l791 bp

Eco RVRsal f Eco RI

160 170 1180 930 tI ~ ~ ,,i

Eco RI Smal

RV

FIG. 1. Construction of pGC165. pSM3 DNA was digested completely with EcoRI, then partially with Rsa I, permitting the separation on anagarose gel of the three "800-bp fragments indicated as I, II, and III at the top of the figure. These fragments were electroeluted from the geland ligated with Sma I-digested pUC8 DNA. Transformants obtained from the mixture were screened. The structure shown for pGC165 isbased on the following observations: (i) there are no EcoRV sites in the pUC8 sequence, a unique one in pSM3 just downstream from the sfiAgene, and a unique one in pGC165; (ii) double digestion of pGC165 with EcoRV and EcoRI released two bands of 800 bp and 2700 bp; (iii) whenthe EcoRV/EcoRI double digest was further treated with Rsa I, the 800-bp band disappeared, indicating the presence of an Rsa I site in thisfragment. The numbering of bases in the sfiA BamHI/EcoRV piece at the top of the figure is that used by Beck and Bremer (25), starting withthe BamHI site as 1. Heavy line in pGC165 is material from pSM3, including the sfiA gene.

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was transformed with the ligated mixture of pUC8 and theRsa I fragments containing sfiA. Of 230 transformants, 13contained plasmids that had increased in size by inserts of-800 bp. These hybrids were then tested for their ability toexpress sfiA.The sfiA+ allele can be distinguished from sfiA- in a lon

genetic background; lon-sfiA+ mutants, defective in thedegradation of the highly unstable SfiA protein (13), are hy-persensitive to SOS-inducing treatments, whereas lon-sfiA-strains regain wild-type resistance (5, 6). We-introduced the13 hybrid plasmids into the lon-sfiA- supF/F'lacIQ amber-suppressing strain, in which the sfiAam mutation is sup-pressed and expression from the lac promoter is repressed.The parental strain is resistant to the mild SOS inducerMeMes, because it carries a sfiA missense mutation; itwould- become MeMes sensitive if a functional SfiA proteinwere provided. Since we were looking for insertions thatplace sfiA under lac promoter control, and it was not knownwhether SfiA alone kills ion cells, we tested the transfor-mants for their sensitivity to IPTG (lac operon inducer) inthe presence of MeMes (SOS inducer). One of the plasmids,pGC165, conferred sensitivity and thus expressed sfiA. Itwas characterized further.

Restriction analysis of PGC165 confirmed the expectedpresence of an EcoRV site, -800 bp from the single EcoRIsite. The orientation of the cloned fragment puts the sfiAgene under lac promoter control (Fig. 1). The presence of anRsa I site in the small EcoRI/EcoRV fragment of pGC165shows that the Rsa I fragment cloned retains the Rsa I siteearly in the sfiA structural gene, so the sfiA coding sequenceshould be intact.

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Effects of an Excess of SfiA Protein. The above analysisestablishes that pGC165 carries the desired plac-sfiA fusionand further shows that the basal level of SfiA activity is notlethal to the cell. This plasmid was used for studying the ef-fects of the SfiA protein on cell division and viability.We introduced pGC165 into the ion sfiA supF/F'lacIQ and

ion sfiA sup+/F'1acJQ strains, which differ by the presenceor absence of the amber suppressor supF. In the absence ofIPTG, both strains showed normal cell division and cell size(Fig. 2 A and C). In the presence of IPTG, the supF cellsrapidly stopped dividing and formed long filaments (Fig. 2B).This cell division inhibition was clearly due to expression ofSfiA protein, because the nonsuppressing sup+ cells contin-ued dividing normally in the presence of IPTG (Fig. 2D).Plasmid DNA extracted from the sup+ culture and reintro-duced into the ion sfiA supF/F'lacIQ strain again causedIPTG-induced filamentation, showing that the sfiA insert inthe plasmid remains functional.The long-term expression of cell division inhibition is le-

thal. On solid media the supF cells were unable to form colo-nies in the presence of IPTG (plating efficiency, 10- to10-5), whereas the sup' strain was unaffected by IPTG inthe plating medium. It is thus clear that induction of SfiAprotein inhibits cell division and causes cell death.IPTG induction of sfiA does not result in general induction

of the SOS response. We measured the level of SOS induc-tion in the presence or absence of IPTG by means of a sfiA:lac operon fusion, which places 3-galactosidase synthesisunder direct LexA negative control (8, 9). This fusion and achromosomal lac deletion were included in the ion sfiAsupF/F'lacIQ/pGC165 genetic background, and UV induc-

20

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1 X

20a)a)20 o.

C)10

4

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1

Time, hr

FIG. 2. Division arrest by specific induction of SfiA. Cultures growing exponentially in tryptone broth plus ampicillin at 370C were diluted attime zero into medium with (B, D, and F) or without (A, C, and E) 5 mM IPTG. Particle number (0) and OD6, (0) were measured periodically.After 3 hr, unfixed samples were photographed (Insets, same magnification for all). The strains used were: Ion sfiA supF/F'lacI2/pGC165sfiAam46 (A and B); Ion sfiA sup /F'lacI1/pGC165sfiAam46 (C and D); lon+ sfiA sup+/F'lacIQ/pGC165sfiA+ (E and F). Three independentIon /F'IacI2 strains in different strain background gave results similar to those shown in E and F.

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ibility of p-galactosidase was verified (8). The uninducedspecific activity of 8-galactosidase was about 30 units/mgdry weight, typically the basal level of sfiA expression (8),and no induction was observed after addition of IPTG, al-though cell division was inhibited. We conclude that repres-sion by LexA is intact under these conditions. Thus the celldivision-DNA replication coupling mechanism works onlyin one direction: interruption ofDNA synthesis leads to SOSinduction and cell division inhibition, but the SfiA inhibitionof cell division does not induce the SOS response.

Just as sfiA induction by IPTG does not lead to inductionof the SOS response, mutations that cannot express the SOSresponse do not block sfiA-dependent filamentation. A recAderivative of the supF strain containing pGC165, unable toinduce all SOS functions, still filaments in the presence ofIPTG (not shown).We have hypothesized that Ion mutants are hypersensitive

to SOS-inducing treatments because of the increased stabil-ity of SfiA protein in Ion- strains. If this is so, one should beable to mimic the effect of increased SfiA stability by consti-tutive synthesis of SfiA in ion+ cells. On the other hand, ifion regulates some other SOS function, overproduction ofSfiA in a ion' cell may not lead to filamentation. It has beenshown that the ion mutation stabilizes not only the SfiA pro-tein (13) but also various types of abnormal proteins or pro-tein fragments (27, 28) and several X proteins (29). In addi-tion, the ion mutation increases the transcriptional level ofcertain operons (ref. 30; unpublished observations). Thus, itremained possible that there exist other SOS functions in-volved in division inhibition, which normally have to be in-duced but are already sufficiently amplified in Ion mutants.To examine these alternatives, we introduced the pGC165

plasmid into a ion+ sfiA supF/F'iaclQ strain and studied theeffect on cell division of IPTG-induced expression of SfiA.No filamentation was observed. It was known, however,that the activity of the sfiAam gene product in supF cells islower than that of the wild-type sfiA gene, because a IonsflAam supF strain is less sensitive to MeMes than Ion sfiA'strains (unpublished observations).We increased the amount of SfiA protein synthesized from

the plasmid by crossing a sfiA' allele onto the pGC165 plas-mid by recombination with a XsfiA' phage (see Materialsand Methods). The resulting plasmid, pGC165sfiA', carriesa sfiA' gene fused to the lac promoter.The ion+ sfiA/F'iacl strain was transformed with

pGC165sfiA', and the effect of SfiA amplification was ex-amined. In the absence of IPTG, cell division and cell sizewere normal, whereas addition of IPTG to the culture causedan immediate shift to filamentous growth (Fig. 2 E and F).Furthermore, this filamentation resulted in cell death; thestrain was unable to form colonies on plates containing IPTG(efficiency of plating 10-4 to 10-5).These observations show that synthesis of SfiA protein

from a high copy number plasmid is in itself sufficient toinhibit cell division in E. coli. The SfiA protein is thus aninhibitor of cell division.

Effect of sfiB Mutations on SfiA-Mediated Cell Division In-hibition. The sfiB (or suiB) mutations suppress SOS-associ-ated filamentation. To examine the effect of sfiB mutationson SfiA-mediated filamentation in the absence of inductionof the SOS response, we introduced the sfiB114 allele intothe ion supF/F'iacI2/pGC165 genetic background. It pre-vented the filamentous growth and lethality observed previ-ously in the presence of IPTG (Table 1, lines 1 and 2). Simi-larly, in the ion+ supF/F'iacI/pGC165 sfiA+ strain, thesfiB114 mutation completely suppressed IPTG-induced fila-mentation and lethality (lines 3 and 4). In both cases, theplasmids remained capable of killing sfiB+ strains, as shownby reextraction and introduction into appropriate sfiB+strains. Thus, induction of the SOS response is not needed

Table 1. Effect of sfiB mutations on SfiA-promotedfilamentation

Relevant genotype IPTG effect onIon sfiA t sfiB cell growth

lon-100 sfiAam46/supF sfiB+ Filamentationion-100 sfiAam46/supF sfiB114 No effectIon+ sfiA + sfiB+ Filamentationion+ sfiA+ sfiB114 No effection-100 sfiA + sfiB+ NDlon-100 sfiA+ sfiB114 Filamentationtion-100 sfiA+ sfiB* No effect

IPTG was added to a final concentration of 5 mM to cells growingin tryptone broth. Filamentation was assayed at 3 hr by observationin a Zeiss microscope. ND, not done (strain cannot be constructed,even in the absence of IPTG).tsfiA allele carried by the pGC165 plac::sfiA plasmid.tStrain shows some filamentation without IPTG; filamentation ismore uniform with IPTG.

for efficient suppression of SfiA-mediated filamentation by asfIB mutation.The sflB mutations suppress filamentation at normal in-

duced levels of SfiA synthesis (5). If SfiB is the target of thedivision inhibitor, the SfiB mutant protein could conceivablyretain some affinity for the inhibitor and permit filamenta-tion at sufficiently high SfiA concentrations. To obtain high-er SfiA concentrations, we constructed a ion sfiB/F'lac-IL/pGC165sfiAW strain. The Ion mutation, by stabilizing theSfiA protein, permits greater accumulation, and the multi-copy plasmid pGC165sfiA' should increase the amount ofSfiA above that found in single copy in the chromosome.The resulting strain exhibited normal growth and morpholo-gy in the absence of IPTG but filamented and died in its pres-ence (Table 1, compare lines 4 and 6). Thus, SfiA protein canultimately inhibit cell division even in the sfiB mutant, al-though apparently more SfiA is needed to inhibit a sfiBiJ4strain than to inhibit sfiB+ strains. It is worth noting that wewere unable to construct a lon/F'1acIQ/pGC165sfiA+ strainunless the sfiB mutation was present (line 5).

In a similar experiment, we found that the presence of themulticopy plasmid pTU302 (22), carrying a sfiA' gene withits own operator and promoter, made a Ion sfiB114 strainsensitive to MeMes. Resistant mutants could be selected. Inone such derivative, the mutation conferring resistance wasinseparable by P1 transduction from the sfiB1i4 allele and isthus probably located in the sfiB gene. This mutation, calledsfiB*, also suppressed IPTG-mediated filamentation in aIon/F'IacIQ/pGC165sfiAW strain (Table 1, line 7). This sug-gests that the mutant SfiB proteins still play a role in SfiAcell division inhibition.

DISCUSSIONThe SfiA (or SulA) protein of E. coli is an SOS function in-volved in SOS-associated cell division inhibition. The pres-ent work establishes that cell division is rapidly inhibited bySfiA protein, induced artificially by means of a plac-sfiAoperon fusion in the absence of induction of the SOS re-sponse. Thus, the SfiA protein is an inducible cell-divisioninhibitor, synthesized at high levels when DNA replication isperturbed, in agreement with Witkin's model (2).The SfiB (or SulB) protein is clearly involved in SOS-asso-

ciated filamentation. It has recently been shown that thisprotein is in fact the product of the ftsZ gene (ref. 14; C.Jones and I. B. Holland, personal communication). Since anftsZ (temperature-sensitive) mutant forms aseptate filamentsat nonpermissive temperatures, it was suspected that theFtsZ protein may play a role in the septation process (15,16).

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In the present work, we show that unless SfiA is a tran-scriptional activator of sfiB, no induction of SfiB is neededto permit (in sfiB+ strains) or prevent (in sfiB- strains) SfiA-mediated filamentation. Furthermore, even sfiB mutantstrains are subject to SfiA-mediated cell-division inhibitionat high SfiA concentrations, and genetic evidence suggeststhat this division inhibition involves the mutant SfiB protein.

It has been suggested that the SfiA division inhibitor mayprevent the action or expression of the SfiB protein (14, 17,18), and although other roles for SfiB cannot be rigorouslyruled out, the above observations are readily interpreted interms of this hypothesis.The SfiA-dependent mechanism of cell-division inhibition

has been shown to play a rather specialized physiologicalrole (31), tightly coupling cell division to DNA replicationwhenever the latter is interrupted. Furthermore, E. coli hasbeen shown to possess other sfiA-independent couplingmechanisms (31-34). Nevertheless, of the various modelsproposed to explain replication-division coupling (35), onlythe hypothesis of an inducible cell-division inhibitor hasbeen confirmed.

We are very grateful to Bdnedicte Michel for her generous andefficient help throughout this work. We are indebted to RogerBrent, Albert Goze, Frank Kunst, and Michael Maurizi for stimulat-ing and fruitful discussions; to Max Gottesman and Sankar Adhyafor their comments on the manuscript; and to Philippe Bouloc for hisparticipation in the work on sfiB*. This work was financed in part bygrants from the Centre National de la Recherche Scientifique (LP003601 and ASP PIRMED "Antibiotiques").

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