Genetic Analysis of Escherichia coli K-12 Chromosomal Mutants

JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 60-670021-9193/80/10-0060/08$02.00/0

Vol. 144, No. 1

Genetic Analysis of Escherichia coli K-12 ChromosomalMutants Defective in Expression of F-Plasmid Functions:

Identification of Genes cpxA and cpxBJOAN McEWEN AND PHILIP SILVERMAN*

Department ofMolecular Biology, Division ofBiological Sciences, Albert Einstein College ofMedicine,Bronx, New York 10461

Two temperature-sensitive, chromosomal mutants of Escherichia coli wereselected for their inability to express deoxyribonucleic acid donor activity andother activities associated with the conjugative plasmid F. These mutants werealso auxotrophic for isoleucine and valine at 410C. Each mutant strain containedtwo altered genes: cpxA, located at 88 min on the E. coli K-12 genetic map, andcpxB, located at 41 min. Mutations in both genes were required for maximalexpression of mutant phenotypes. The parent strain of mutants KN401 andKN312 already contained the cpxB mutation that is present in both mutants(cpxBl). This mutation by itself was cryptic. The cpxA mutations representdifferent mutant alleles since they are of independent origin. A cpxA mutation byitself significantly affected the expression of plasmid functions and growth at410C in the absence of isoleucine and valine, but strains containing both a cpxAand cpxB mutation were more severely affected. Along with the observation thatboth cpxA mutations were revertable, the temperature sensitivity of cpxA cpxB+cells suggests that both cpxA alleles contain point mutations that do not com-pletely destroy the activity of the cpxA gene product.

We recently described two temperature-sen-sitive, chromosomal mutants ofEscherichia coliK-12 selected for their inability to express cel-lular properties associated with the presence ofthe conjugative plasmid F (12). The mutants failto elaborate F-pili, surface organelles requiredfor conjugal DNA transfer and F-specific bacte-riophage adsorption, and they are also defectivein the expression of surface exclusion, the prop-erty of F-bearing cells to act as poor conjugalrecipients. These effects were manifested in Hfras well as F' mutant strains and could not there-fore be attributed to the loss of F-plasmid DNAfrom mutant cells (12). Instead, the mutationsaffect the synthesis of F-plasmid tra gene prod-ucts required for the expression of DNA donorand related activities (Eoyang, Sambucetti, andSilverman, submitted for publication; see refer-ence 1 for a recent review of F tra genes andfunctions). Moreover, since both mutants exhibittemperature-sensitive growth in the absence ofisoleucine and valine, as described fully in theaccompanying manuscript (13), and since theyare identically altered in envelope protein com-position (12; J. McEwen and P. Silverman, inpreparation), the mutations apparently affectthe synthesis or function of proteins in additionto those determined by plasmid genes.We have now shown that each of these mutant

strains contains two altered genes, one of which60

we designate cpxA, located at 88 min on the E.coli K-12 genetic map, and the other, cpxB,located at 41 min. The cpxB mutation was al-ready present in the parent strain of the twomutants, and the cpxA mutations arose whenwe mutagenized this strain (12). Mutations inboth genes are required for maximal expressionof mutant phenotypes.

MATERIALS AND METHODSBacterial strains and bacteriophage. Bacterial

strains important for these studies are described inTable 1 and Fig. 1. Bacteriophages Qfi, XNK55 (b221cI857 Oam29 cIII::TnlO) (8), and P1CM clrlOO (17)were from our laboratory stocks.Media and growth conditions. Nutrient broth

and minimal medium were as previously described(12). LB medium was 1.0% Difco tryptone, 0.5% Difcoyeast extract, and 0.5% NaCl supplemented with 40,ug of thymidine per ml. LBCa medium and LBMgmedia contained, in addition, 10 mM CaCl2 or 10 mMMgCl2, respectively. XYM broth contained 1.0% Difcotryptone, 0.25% NaCl, 0.1% Difco yeast extract, and0.2% maltose. The Zwf phenotype was determined ina pgi mutant background as an inability to grow onagar medium containing glucose as carbon source (4).The Eda- and Fad- phenotypes were determined asan inability to grow on glucuronic and oleic acids,respectively (6, 15).

Genetic methods. Matings on agar media werecarried out as described by Low (11). Liquid matingsand surface exclusion experiments were carried out as

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GENES REQUIRED FOR F-PLASMID FUNCTIONS

TABLE 1. Bacterial strainsaGenotype

Fi[a] metBI cpxBl argG6F'116 (thyA+)/[a] cpxAI metBI thyA23 recAl cpxBIargG6

Same as KN401, except cpxA2F-[a] cpxAI thyA cpxBI argG6Hfr[a] metBI thyA cpxBI argG6Hfr(a] argHI rpoB cpxBISame as AE1011 except metBISame as AE1010 except cpxB+ zeb-l::TnIOSame as AE1031 except metB+ cpxA2Same as AE1010 except metB+ cpxAISame as AE1010 except metB+ cpxA2Same as AE1018 except eda-i zeb-l::TnlOSame as AE1011 except cpxAl metBI argH+F'105 (cpxA+ metB' argH+)/[a] cpxAI metBI argHlrpoB thyA cpxBI

F'105 (cpxA+ metB+ argH+)/[a] cpxA2 metB)Same as AE3149 except chromosome is cpxA+ cpxB+pAE4005 (cpxAI)/chromosome same as AE3149

pAE4005/chromosome same as AE3150F'116 (thyA+ zzf::TniO)/chromosome same as AE3149

F'116 (thyA zzf::TnlO)/chromosome same as AE3150F'116 (thyA+ zzf::TnlO)/[a] metBI thyA recAI cpxBIargG6

F'116 (thyA+ zzf::TniO)/[a] cpxA2 thyA recAI cpxB+argG6

F- hisGI pgi-2 eda-i rpsL115

F- pgi-2 zwfA2F- fadD88Same as AE2019 except zeb-l::TnlO

Same as K27 except eda-i zeb-l::TnlO

Hfr thi-1 rel-I cpxAI rpoB

Source or commentb

CGSC 4274Ref. 12

Ref. 12This study; derived from JC411Ref. 12This study; derived from AE1010This study; derived from AE1010This study; derived from AE1010This study; derived from AE1031Ref. 12Ref. 12This study; derived from AE1018This study; derived from AE1011This study; derived from JC411

This study; derived from JC411This study; derived from JC411This study; pAE4005 was derived from

F'105 by homogenotization (see Mate-rials and Methods)

This studyThis study; F'116 zzf: :TniO is described

in ref. 12This studyRef. 12

This study; derived from JC411

This study; spontaneous Mal' (A8) deriva-tive of DF1671 (CGSC 4889)

CGSC 4873R. Simons (see ref. 15)YThis study; derived from AE2019 (see

Materials and Methods)This study; derived from K27 (see Mate-

rials and Methods)This study; derived from KL14 (CGSC

4294)a All strains are derivatives of E. coli K-12. [a] denotes the genotype leu-6 his-I argG6 lacY) gal-6 xyl-7 mtl-

2 malAl rpsLi04 tonA2 tsx-i supE44. Strains were prepared by P1 transductions with the following exceptions:recAI was introduced when necessary by conjugation with KL16-99 (CGSC 4206; reference 9); some thyA andrpoB mutations were spontaneous (mutant strains were selected by trimethoprim or rifampin resistance,respectively; see reference 14).

b Strains obtained from Barbara Bachmann of the Coli Genetic Stock Center at Yale University are indicatedby their CGSC strain numbers.

'Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717.

described previously (12, 18). In interrupted matingexperiments, mating aggregates were disrupted at dif-ferent times as described by Miller (14). P1 transduc-tions were performed with P1CM clrlOO as describedbelow. Lysates were prepared in LB medium by infec-tion at 41°C or by heat induction of lysogens. Titerswere between 109 and 1010 plaque-forming units/ml.

Preparation of TnlO insertions near cpxB.Strain AE2019 (Table 1) was grown overnight in AYMbroth at 30°C and was concentrated by centrifugationand suspension in 0.5 volume ofAYM broth. The cells(0.5 ml) were then infected with XNK55 at 0.1 plaque-forming unit per cell for 30 min at 22°C and then for

30 min at 320C. Samples (0.1 ml) were then plated onLB agar containing 15,ug of tetracycline per ml and2.5 mM sodium pyrophosphate. The yield under theseconditions was 2 x 10-6 to 3 x 10-6 tetracycline-resist-ant colonies per plaque-forming unit of XNK55. Sixgroups of plates, each group containing about 1,000colonies, were flooded with LB broth to remove thecells. A sample (0.2 ml) from each pool was inoculatedinto 5 ml of LBCa broth, and these cultures wereincubated at 41°C until the cells reached an opticaldensity (600 nm) of 0.5. At this time, they were infectedwith P1 (0.5 plaque-forming units per cell) and incu-bated until visible lysis occurred (3 to 4 h). Cell debris

Strain

JC411KN401

KN312AE2008AE1010AE1011AE1012AE1031AE1061AE1018AE1019AE1046AE1020AE3156

AE3149AE3150AE3151

AE3152AE3122

AE3123AE3016

AE3125

AE2019

DF2000K27AE2030

AE2044

AE1127

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62 McEWEN AND SILVERMAN

FIG. 1. Genetic map ofE. coli K-12 calibrated in minutes oftransfer (inner circle). The outer circle indicatesthepositions ofgenes relevant to thepresent studies; these were taken from reference 3, except for thepositionsof cpxA and cpxB, which are described in this communication. The filled arrowheads indicate the originsand directions ofDNA transfer from the indicated Hfr strains (10, 11). The outermost segments indicate thechromosomal DNA contained in the respective F plasmids; the arrowheads indicate the position andorientation of the FDNA of these plasmids (taken from reference 10).

was removed by centrifugation, 5 mM MgCl2 was

added, and the lysates were stored at 4°C over chlo-roform.

These lysates were used to transduce the zwfA2strain DF2000 to a Tetr Zwf4 phenotype as follows. A5-ml culture of DF2000 was diluted to 2 x 10' cells perml in LBCa broth and was mixed with an equal volumeof LBMg broth containing 2 x 107 plaque-formingunits per ml of one of the P1 lysates. The mixture wasincubated for 40 min at 32°C. The cells were thencollected by centrifugation, suspended in 5 ml of nu-

trient broth containing 3 iLg of tetracycline per ml, andincubated for an additional 40 min at 32°C. The cells

were collected again by centrifugation and suspendedin 0.4 ml of LB broth. Portions (0.1 ml) were plated onLB agar containing 5 jig of tetracycline per ml andincubated at 32°C. To detect Tetr Zwfl transductants,Tetr colonies were replica plated to minimal glucose

plates containing 10 ug of tetracycline per ml. A grad-ual increase in tetracycline concentration from 3,ug/ml to 10 ug/ml markedly increased the yield of trans-ductants, presumably because the lower concentra-tions induced a component(s) necessary for resistanceto tetracycline (19). Several Tet' Zwf* transductantswere backcrossed to strain AE2019 by P1 transduction.Selection was for tetracycline resistance and for theeda-l allele of AE2019. One of these derivatives,AE2030, was used to map the cpxB gene as describedin Results. The position of the TnlO insertion inAE2030 was determined by P1 transduction withstrain K27 (eda+ fadD88) as the recipient. Analysis ofTetr transductants for Fad and Eda phenotypes andof Fad' transductants for Tet and Eda phenotypesshowed that the TnlO insertion was 87% linked to edaand 18% linked to fadD. Furthermore, the insertiondecreased the cotransduction frequency of fadD and

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eda from 16% to 8%, demonstrating that it lies betweenthem (shown below in Fig. 4). This insertion is desig-nated zeb-l::TnlO, in accordance with current nomen-clature (8). The TnlO insertion in strain AE2030 wasused as a positive selection to construct strains AE2044(cpxA+ eda-I zeb-1::TnlOcpxB fadD88) and AE1046(cpxAl eda-l zeb::TnlO cpxBl) (see Table 1).Homogenotization. Single colonies of strain

AE3156, an F'105 (cpxA )/cpxA1 cpxBl recA+ strain(see Table 1), were replica plated to minimal agarlacking isoleucine and valine, and the plates wereincubated at 41°C. Approximately 0.5% of the coloniesfailed to grow. This frequency is consistent with theformation of F'105 (cpxA1 )/cpxA1 homogenotes (14)since it is far higher than the frequency ofspontaneouscpxA mutations (18). The F' plasmids in these strainsretained the metB and argH alleles of F'105. Oneplasmid, pAE4005, was used for complementationstudies reported below.

RESULTS

Growth ofmutant cells. Strains KN401 andKN312 were isolated as temperature-sensitive,phage Q-resistant mutants of AE3087, an F'strain of E. coli K-12 (12). The QJ resistance ofboth mutants could be attributed to chromo-somal mutations that prevent the expression ofcellular properties associated with the presenceof F-plasmid DNA (12).In the complete growth medium employed in

our previous studies, mutant and parental cells

grew at comparable rates: the doubling times ofboth were 30 min or 25 min at 340C or 410C,respectively. In contrast, both mutants exhibitedtemperature-sensitive growth on miniimal me-

dium containing only those nutritional supple-ments required by AE3087. This growth defectwas manifested as an inability of mutant cells toform colonies on agar medium at 410C. Whenmutant cells were shifted from 340C to 410C inliquid minimal medium, their growth rate grad-ually declined over an interval corresponding toseveral nornal doubling times (13). This growthdefect of KN401 and KN312 results from theirinability to synthesize isoleucine and valine at410C (13).Two observations indicated that the 1v- phe-

notype and the inability of mutant cells to ex-press F-plasmid functions in nutrient broth(Cpx- phenotype) are caused by the same mu-tation(s). First, two independently isolated, tem-perature-sensitive Cpx- mutants (KN401 andKN312) (12) were 11v-. Second, spontaneous llv+revertants of KN401 and KN312 regained sen-sitivity to QJ, indicating that the reversionevent(s) also restored F-plasmid functions.Therefore, we mapped the mutations in KN401and KN312 using the 1lv- phenotype as a con-

venient and easily scorable property of mutantcells.Two niutations in KN401 and KN312 re-

quired for temperature-sensitive growth.Matings on agar media with several Hfr strainswhose points of origin are distributed aroundthe E. coli genome (Fig. 1) were used to localizethe mutation(s) in an F- recA+ derivative ofKN401. Hfr strains Ra-2 and KL209 promotedthe heaviest patches of recombinant growth onminimal agar plates incubated at 410C. Theorigins and directions of DNA transfer fromthese strains suggested that a mutation inKN401 lies in a narrow interval of the linkagemap that contains the metB, argH, and rpoBgenes (Fig. 2). In liquid matings with KL209 asdonor, the frequency of Ilv+ recombinants rela-tive to the frequencies of Met', Arg+, His', andLeu+ recombinants indicated that a mutant genein KN401 is close to metB (data not shown).Significant Ilv+ recombinant formation also oc-curred in patch matings with Hfr strains KL16,KL983, KL96, and KL99, whose origins of trans-fer are also shown in Fig. 1, and liquid matingswith KL16 as donor suggested the presence of asecond mutation in KN401, in a gene close to his(data not shown).The distribution of unselected markers among

the llv+ recombinants obtained from liquid mat-ings with llv+ donors confirmed the presence oftwo mutant genes in KN401. Of 50 Ilv+ recom-binants derived from a cross with KL209 asdonor, 36 were Met' and none was His', Leu+,or Arg+. Similarly, of 98 llv+ recombinants de-rived from a cross with KL16 as donor, 75 wereHis+, one was Leu+, and none was Met+ or Arg+.Further evidence for two mutant genes was theobservation that the Ilv+ recombinants from thetwo crosses differed in colony morphology: thosefrom the KL16 cross formed small colonies,whereas those from the KL209 cross formednormal colonies.

Full expression of the Ilv- phenotype ofKN401 thus requires mutations in two widelyseparated genes. We designated the gene linkedto metB as cpxA and the gene linked to his ascpxB (12). Though KN401 is a double mutant,its isolation required only one mutation, in thecpxA gene, because the parent strain of KN401(AE3087) already carried a cpxB mutation (seebelow). Furthermore, as shown below, KN312also contains a cpxA mutation, and since KN401and KN312 were both derived from AE3087,they must both contain the same cpxB mutation,which we designated cpxBl (12).Gene cpxA. The location of the cpxA gene

near metB was determined by P1 transduction.The donor strain was KN401 or its metB deriv-

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moet

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- 35~~30 0.

FIG. 2. Gene order and cotransduction frequencies of cpxAl (A) or cpxA2 (B) and metB, argH, and rpoB(derived from the data in Table 2).

TABLE 2. Pl transduction analysis of cpxAaTransductant genotype % of transductantsb

Expt Selected do- KN401 KN312nor allele cpxA metB argH rpoB (cpxAl) (cpxA2)

donor donor

Cross ID: cpxA metB argH' rpoB+ argH+ R R D D 57.4 48.3R: cpxA+ metB+ argHrpoB R D D D 20.6 19.7

R R D R 11.8 15.3R D D R 5.4 8.0D D D R 2.9 6.4D D D D 2.0 1.4D R D D/R 0.5 0.8

Cross IID: cpxA metB+ argH+ rpoB+ argH+ R R D D 55.6 44.3R: cpxA+ metB argHrpoB R D D D 18.5 22.5

R R D R 13.0 12.6R D D R 3.7 14.6D D D R <1 4.3D D D D 7.4 <1D R D D/R 1.8 0.8

metB+ D D R R 35.4 33.3R D R R 29.0 31.5R D D D 22.0 24.1R D D R 9.2 7.4D D D R 2.9 1.8D D D D 1.6 1.8

aD, Allele derived from the donor; R, allele derived from the recipient. For experiment I, 204 argH+transductants from donor KN401 were examined and 360 from donor KN312. For experiment II, 54 argH+transductants and 54 metB+ transductants derived from a spontaneous metB+ derivative of KN401 wereexamined; 253 argH+ and 314 metB+ transductants derived from a spontaneous metB+ derivative of KN312were examined. The recipient strains AE1011 (cross I) and AE1012 (cross II) are described in the text and inTable 1.

b Recombinant classes that were not observed were omitted from the table.

ative (Table 1). The recipients were strains (Table 2). The data indicate the gene order andAE1011 (cpxBl cpxA+ metB+ argH rpoB) and cotransduction frequencies shown in Fig. 2A.AE1012 (cpxBl cpxA+ metB argH rpoB). Se- This analysis places the cpxA gene at about 88lection was for Arg+ or Met+ transductants. min on the current linkage map of E. coli K-12These were scored for three unselected markers (1). A similar analysis of KN312 (Table 2) indi-

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cated that this mutant also carries a cpxA mu-tation (Fig. 2B), which we confirmed by comple-mentation studies (see below). Accordingly, wedesignate the allele in KN401 as cpxAl and thatin KN312 as cpxA2.Both the cpxAI (not shown) and cpxA2 (Ta-

ble 3) mutations are recessive to the cpxA + alleleof F'105, which carries DNA from the region ofthe E. coli chromosome containing the cpxAgene (Fig. 1). pAE4005, a homogenotized F'105derivative that carries the cpxAI mutation (seeMaterials and Methods), was used to confirnthat the cpxAl and cpxA2 mutations are in thesame gene. As shown (Table 3), strain AE3151,the F' cpxAI/cpxA2 merodiploid, was Cpx- andllv-, whereas AE3152, the F' cpxAl/cpxA+ mer-odiploid, was Cpx+ and Ilv+.

Several experiments provided direct evidencethat the 1lv- and Cpx- phenotypes are bothcaused by a cpxA mutation in a cpxB mutantcell. A P1 lysate from a metB+ derivative ofmutant KN401 was used to prepare 120 metB+transductants of Hfr strain AE1010. These wereanalyzed for growth on minimal agar at 410C(flv phenotype) and for sensitivity to QB (Cpxphenotype). Thirty-eight transductants wereCpx- 1lv- and 82 were Cpx+ Ilv+; the two mutantphenotypes were never separated. We reachedthe same conclusion from an analysis of spon-taneous llv+ revertants of Hfr strain AE1019. Inmost cases, the reversion event had occurred inthe cpxA gene (see below). These cpxA+ revert-ants invariably regained full sensitivity to Q/. Inaddition, the revertability of the cpxA2 (strainAE1019) and cpxAl (strain AE1018) alleles sug-gest that both are point mutations. In contrast,we have not obtained a cpxB + revertant.Gene cpxB. The cpxBI mutation in KN401

and KN312 was already present in the parentstrain, AE3087, and in strains related to it. Thus,40% of the metB+ transductants of JC411, therecA + ancestor of AE3087 (12), were 1lv- whenthe P1 donor strain was cpxAl metB+. As shownbelow, this result is possible only if the recipientis a cpxB mutant. Strains AE1010, AE1011, andAE1012 (see above), which were derived from

JC411 without mutagenesis (Table 1), also con-tain the cpxBl mutation.An interrupted mating between KL96 (Hfr

his' cpxB+) and AE2008 (F- his-i cpxBIcpxAI) placed the cpxB gene about 5 min coun-terclockwise from the his genes' (data notshown). However, neither oftwo F-plasmids thatcarry bacterial DNA from this region, F'148 andF'150 (Fig. 1), complemented the cpxBl muta-tion. Chromosomal DNA of F'150 extends fromthe his genes through the zwf gene, but thelocation of its endpoint between zwfand fadD isuncertain (5). If the cpxBl mutation is recessiveto the cpxB+ allele, these observations implythat the cpxB gene lies beyond the endpoint ofF'150 and within the region that is deleted inF'148. These arguments indicate that cpxB islocated at 40 to 41 min on the E. coli geneticmap.This map position for cpxB was confirmed by

two transductional crosses. In the first, a P1lysate of AE2044 (cpxA + eda-1 ze&-1: :TnlOcpxB+ fadD88) was used to transduce the recip-ient AE1018 (cpxAl eda+ cpxBl fadD+) to tet-racycline resistance. The cotransduction fre-quencies of zeb-1: :TnlO and, respectively, eda-1,cpxB, and fadD were determined from 179 Tetrtransductants to be 84%, 80%, and 15%. In thesecond, Eda+ transductants were selected froma cross between AE1127 (cpxAl eda+ cpxB+) as

the donor and AE1046 (cpxAI eda-1 zeb-l: :TnlOcpxBI) as the recipient. An analysis of 224 trans-ductants (Table 4) suggests the gene ordershown in Fig. 3. These data place cpxB at 41min on the linkage map, closely linked to eda.

Partial phenotypes associated with cpxAand cpxB mutations. We noted above thatcpxA + cpxBl recombinants formed normal-sized colonies on solid media at 410C, whereascpxAl cpxB+ recombinants formed small colo-nies. This difference in growth rate was alsoobserved during exponential growth in liquidmedia (13). To examine whether a similar rela-tionship exists between the individual mutationsin cpxA and cpxB genes and the overall inabilityof mutant cells to express F-plasmid functions,

TABLE 3. Complementation analysis of the cpxAl and cpxA2 mutations

Strain Chromosomal geno- F' plasmid genotype QJ sensitivity' Donor activity, Growthtype at 41"CcAE3150 cpxA+ cpxB+ F'105 (cpxA+) 2.0 x 1010 1.1 X 106 +AE3149 cpxA2 cpxBl F'105 (cpxA+) 2.8 x 1010 3.8 X 106 +AE3152 cpxA+ cpxBI pAE4005 (cpxAl) 1.7 x 1010 0.9 X 106 +AE3151 cpxA2 cpxBl pAE4005 (cpxAl) <108 8.5 x 104 -

a Plaque-forming units per milliliter, average of two determinations.b Transconjugants per milliliter in 60-min liquid matings; average of two determinations. The recipient was

AE2004 (nalA metBI thyA23; see reference 12). Selection was for Nalr Met+ transconjugants.COn minimal agar.

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we constructed sets of essentially isogenic Hfrand F' strains that were cpxA+ cpxB+, cpxA2cpxBl, cpxA2 cpxB+, or cpxA+ cpxBl. DNAdonor activity or surface exclusion was measuredin each strain of the F' or Hfr set, respectively.The results (Table 5) show that the cpxA2cpxB+ strains were significantly defective inboth donor activity and surface exclusion, butnot as defective as the cpxA2 cpxBl doublemutant strains. The cpxA + cpxBl strains, onthe other hand, were essentially as proficient inboth activities as the cpxA + cpxB + strain. Thus,the cpxA mutation causes defects in F-plasmidfunctions (12) and isoleucine and valine biosyn-thesis (13) that are exacerbated by a mutationin cpxB. In contrast, the cpxBl mutation aloneis cryptic.Genetic and functional analyses ofcpxA +

revertants. The average frequency of sponta-neous llv+ revertants in five overnight culturesof AE1019 was 2.0 ± 0.5 x 10-5 revertants perviable cell. To determine whether reversionevents could occur at the cpxA locus, at thecpxB locus, or at some other locus, we used P1lysates from independently isolated Ilv+ coloniesto prepare Met+ or Eda+ transductants of cpxAcpxB mutant strain AE1020 or AE1046, respec-tively. Individual transductants, usually 50 fromeach cross, were examined for their ability to

TABLE 4. Pl transduction analysis cpxB0Transductant

genotype % ofDonor and recipient tranrduc-

zeb-l:: tantsTnio cpxB

D: eda+ cpxB+ D D 62.5R: eda-I zeb-l::TnlO D R 19.6

cpxBl R R 17.4R D 0.4

a D, Allele derived from the donor; R, allele derivedfrom the recipient. The donor and recipient strains aredescribed in the text; 224 eds+ transductants wereanalyzed.

£If-*::TnIOId II fadD

FIG. 3. Gene order and cotransduction frequenciesof cpxBl, eda, zeb-l::ThlO, and fadD (derived fromthe data in Table 3).

TABLE 5. Effect of cpxA and cpxB mutations on theexpression of F-plasmid function

Genotype' DNA donor Surface exclusion'activity'cpxA + cpxB + 28 2,454cpxA2 cpxBl 0.2 2.4cpxA2 cpxB+ 5.0 142cpxA+ cpxBl 36 1,080

a The F' strains AE3123, AE3122, AE3125, andAE3016 were used to measure DNA donor activity.The Hfr strains AE1031, AE1019, AE1061, andAE1010 were used to measure surface exclusion.

b Measured at 41°C; expressed as (transconjugantsper donor cell) x 100. The recipient was AE2004 (12);selection was for Nalr Tetr Thy+ transconjugants.

'Measured at 410C; expressed as described in Ma-terials and Methods. The surface exclusion of an F-strain is defined as 1. The donor strain was HfrH (leu+rpsL+); Leu+ Stre recombinants were selected.

grow on minimal agar at 410C. Based on ourgenetic analysis of cpxA cpxB mutants, we ex-pected about 40% of the Met+ transductants togrow if the revertant was cpxA+, about 70% ofthe Eda+ transductants to grow if the revertantwas cpxB+, and none of the transductants togrow if the reversion event occurred at a locusthat is not linked by P1 transduction to eithercpxA or cpxB (pseudorevertants). By these cri-teria 8 of 14 revertants that we tested werecpxA +, 6 were pseudorevertants, and none wasa cpxB+ revertant, even though several of therevertants exhibited a small-colony morphology.

DISCUSSIONWe exploited the Ilv- phenotype of strains

KN401 and KN312 to show that both strainscarry mutations in two genes, cpxA and cpxB.The cpxA gene is linked by P1 transduction tometB (37 to 40%) and to argH (6 to 7%), placingit at about 88 min on the linkage map of E. coliK-12. The cpxB gene lies 1800 from the cpxAgene, between eda and fadD. Our data place itcloser to the former than to the latter, at about41 min. The cpxB mutation (cpxBl) was resi-dent in the cpxA + parent strain of KN401 andKN312, where it was cryptic. The cpxAl andcpxA2 mutations occurred when KN401 andKN312 were isolated (12).The occurrence of reversions in the cpxA gene

and not in the cpxB gene suggests that cpxAland cpxA2 are point mutations, whereas cpxBlmay be a deletion. If so, the former must beresponsible for the temperature sensitivity ofmutant cells. This is fully supported by theobservation that cpxA cpxB + cells were signifi-cantly defective at 410C both for growth in min-imal medium and for expression of F-plasmidfunctions. Since the cpxBl mutation in a cpxA+

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cell was cryptic, it is possible that both genesencode functionally equivalent or identical geneproducts, but the cpxA gene product is normallysynthesized in greater quantity. The two EF-Tugenes in E. coli also appear to be expressed atdifferent levels (16). Furthermore, the fact thatboth cpxA mutants isolated in a cpxBl cell weretemperature sensitive suggests that the cpxAgene can sustain only limited genetic damage inthe absence of the cpxB gene product. Thisargument implies that at least partial functionof either the cpxA or the cpxB product may beessential for viability.Both KN312 and KN401 were selected for

resistance to QB and were subsequently shownnot to express other cellular properties associ-ated with the presence of F-plasmid DNA (12).Since none of these properties is related directlyto isoleucine and valine biosynthesis, the lv-phenotype of both mutants is surprising. Nev-ertheless, both the Ilv- and Cpx- phenotypesare jointly determined by the cpxA and cpxBmutations, since (i) spontaneous cpxA+ revert-ants or cpxA+ transductants of a cpxA cpxBmutant strain were both Ilv+ and Cpx+, (ii) cpxAcpxB+ cells exhibited partial lv- and Cpx- phe-notypes, and (iii) both KN401 and KN312, in-dependently isolated cpxA mutants selectedonly for their Cpx- phenotype, were Ilv- as well.We argue from these observations that the cpxAand cpxB mutations alter a cellular process thataffects a specific set of functionally unrelatedproteins. This process may be related to theformation of the cell envelope, since the cpxAand cpxB mutations selectively alter envelopeprotein composition (12; McEwen and Silver-man, in preparation). The role of the cell enve-lope in the expression of conjugative plasmidfunctions has been well documented (1, 7), andits possible role in the synthesis of isoleucineand valine is discussed in the accompanyingpaper (13).

ACKNOWLEDGMENTSWe are indebted to Barbara Bachmann of the E. coli

Genetic Stock Center and Robert W. Simons for generouslyproviding bacterial strains. P.S. is an Established Investigatorof the American Heart Association.

This work was supported by Public Health Service grantsCA 13330, HDO-7154, and GM 11301 from the National Insti-tutes of Health and by grant 74-144 from the American HeartAssociation.

LITERATURE CITED1. Achtman, M., P. A. Manning, C. Edelbluth, and P.

Herrlich. 1979. Export without proteolytic processing

of inner and outer membrane proteins encoded by F sexfactor tra cistrons in Escherichia coli minicells. Proc.Natl. Acad. Sci. U.S.A. 76:4837-4841.

2. Achtman, M., and R. Skurray. 1977. A redefinition ofthe mating phenomenon in bacteria. In J. Reissig (ed.),Microbial interactions, series B, vol. 3, p. 234-279, Re-ceptors and recognition. Chapman and Hall, London.

3. Bachmann, B., and K. B. Low. 1980. Linkage map ofEscherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56.

4. Fraenkel, D. G. 1968. Selection of Escherichia coli mu-tants lacking glucose-6-phosphate dehydrogenase orgluconate-6-phosphate dehydrogenase. J. Bacteriol.115:1267-1271.

5. Fraenkel, D. G., and S. Banerjee. 1971. A mutationincreasing the amount of a constitutive enzyme in E.coli, glucose-6-phosphate dehydrogenase. J. Mol. Biol.56:183-194.

6. Fraenkel, D. G., and S. Banerjee. 1972. Deletion map-ping of zwf, the gene for a constitutive enzyme, glucose-6-phosphate dehydrogenase in Escherichia coli. Ge-netics 71:481489.

7. Kennedy, N., L Beutin, M. Achtman, R. Skurray, U.Rahmsdorf, and P. Herrlich. 1977. Conjugative pro-teins encoded by the F sex factor. Nature (London)270:580-585.

8. Kleckner, N., J. Roth, and D. Botstein. 1977. Geneticengineering in vivo using translocatable drug-resistanceelements. New methods in bacterial genetics. J. Mol.Biol. 116:125-159.

9. Low, K. B. 1968. Formation of merodiploids in matingswith a class of Rec- recipient strains of Escherichia coliK-12. Proc. Natl. Acad. Sci. U.S.A. 60:160-167.

10. Low, K. B. 1972. Escherichia coli K-12 F-prime factors,old and new. Bacteriol. Rev. 36:587-607.

11. Low, K. B. 1973. Rapid mapping of conditional andauxotrophic mutations in Escherichia coli K-12. J. Bac-teriol. 113:798-812.

12. McEwen, J., and P. Silverman. 1980. Chromosomalmutations of Escherichia coli that alter expression ofconjugative plasmid functions. Proc. Natl. Acad. Sci.U.S.A. 77:513-517.

13. MeEwen, J., and P. Silverman. 1980. Mutations ingenes cpxA and cpxB of Escherichia coli K-12 cause adefect in isoleucine and valine syntheses. J. Bacteriol.144:68-73.

14. Miller, H. J. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,New York.

15. Nunn, W. P., and R. W. Simons. 1978. Transport oflong-chain fatty acids by Escherichia coli: mapping andcharacterization of mutants in the fadL gene. Proc.Natl. Acad. Sci. U.S.A. 75:3377-3381.

16. Pedersen, S., R. Blumenthal, S. Reeh, J. Parker, P.Lemaux, R. A. Laursen, S. Nagarkatti, and J. D.Frieson. 1976. A mutant of Escherichia coli with analtered elongation factor Tu. Proc. Natl. Acad. Sci.U.S.A. 73:1698-1701.

17. Rosner, J. L 1972. Formation, induction, and curing ofbacteriophage P1 lysogens. Virology 48:679-689.

18. Silverman, P., K. Nat, J. McEwen, and R. Birchman.1980. Selection of Escherichia coli K-12 chromosomalmutants that prevent expression of F-plasmid functions.J. Bacteriol. 143:1519-1523.

19. Tait, R. C., R. L. Rodriguez, and H. W. Boyer. 1977.Altered tetracycline resistance in pSC101 recombinantplasmids. Mol. Gen. Genet. 151:327-331.

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Genetic Analysis of Escherichia coli K-12 Chromosomal Mutants