JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 48-55Copyright X) 1976 American Society for Microbiology

Vol. 126, No. 1Printed in U.SA.

Essential Genes in the metB-malB Region ofEscherichiacoli K-12

KAREN A. ARMSTRONGI* AND DAVID P. FANDepartment of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108

Received for publication 6 November 1975

We isolated 25 independent mutants that are deficient in essential geneslocated in the metB-malB region of the Escherichia coli chromosome. Themutations were mapped within this region by using several F' factors and werealso classified into 11 cistrons by complementation testing. There is clustering ofmutations in essential genes within the metB-malB region.

The preceding paper (2) delineated a methodfor the isolation and complementation ofstrains carrying mutations in essential geneslocated in a particular region of the Escherichiacoli chromosome. The method involved makingcells partially diploid for the region to be stud-ied by using an F' factor that was temperaturesensitive for replication. Strains carrying mu-tations in essential genes in the region of thechromosome covered by the F' factor requirethe presence of the F' factor for growth and arethus temperature sensitive. As a test and illus-tration of this method, one expected class ofessential gene mutations was screened for andfound mutations in the gene that codes for the,8 subunit of ribonucleic acid (RNA) polymer-ase. This gene, called rif (13, 24), was in theregion covered by the F' factor used, KAF1,which runs from metB-malB. This region isabout 2.5 min or 2.8% of the E. coli chromo-some. In the present study we were interestedin identifying by mutation as many essentialgenes in this region as possible. This paperdescribes the identification of 25 independentmutations in essential genes in this region. Themutations were roughly mapped by using sev-eral F' factors that have at least one end termi-nating within the metB-malB segment. Fi-nally, the mutations were classified into 11 cis-trons by complementation testing.

MATERIALS AND METHODSUnless otherwise stated, all materials and meth-

ods are as described in the accompanying article (2).Bacterial strains. The bacterial strains used in

this study are described in Tables 1 and 2.Nomenclature. The genetic abbreviations and

symbols are those proposed by Demerec et al. (9) andused by Taylor and Trotter (25). The symbol ess

Present address: Department of Biology, University ofCalifornia, San Diego, La Jolla, Calif. 92093. (Send reprintrequests to the University of Minnesota address.)

denotes a mutation in an essential gene. Alleles ofthe rifgene are signified by the convention ofAustinet al. (3): the wild-type rif- gene confers sensitivityto the drug rifampin, rif confers rifampin-resist-ance, and rif" results in the complete loss of the rifgene function. The genes conferring sensitivity andresistance to bacteriophage BF23 are denoted bfesand bfer, respectively, as used by Jasper et al. (18).

Mutagenesis with NG. Exponential-phase bacte-ria growing at 28 C in minimal medium were fil-tered and suspended in tris(hydroxymethyl)-aminomethane(Tris)-maleate buffer, pH 6.0 (1),with NG at 0, 10, 15, and 20 pg/ml. After 15, 35, and50 min at 28 C, the mutagenized cells were immedi-ately diluted with two volumes of M63 buffer-salts.Cells were collected by centrifugation, washed twicewith M63 buffer-salts, and suspended in lactoseminimal medium supplemented with methionine,threonine, and tryptophan.The procedure used for segregation of mutagen-

ized cells and the protocol for monitoring the successof mutagenesis by plating on rifampin-containingplates were described previously (2).

Bacteriophage BF23. Stocks of bacteriophageBF23 were the generous gift of H. R. Warner.Strains to be tested for phage resistance and sensi-tivity were grown to exponential phase at 37 C inMGM minimal liquid medium (20) with the excep-tion of the strain carrying KAF1, which was grownat 30 C. A 0.50-ml volume of each culture was mixedwith 2.0 ml of MGM minimal top agar (MGM mini-mal liquid medium containing 0.7% Difco agar), andthe mixtures were poured onto MGM minimal plates(MGM minimal liquid medium containing 1.2%Difco agar). When the top agar had hardened, 0.02-ml volumes of four dilutions of the BF23 stock cul-ture were spotted separately onto the plates. Thesefour dilutions contained approximately 109, 107, 10',and 103 phage particles per ml. The plates wereincubated at 37 C, except for the plate on whichKAF1/RK41O1Bfe was tested, which was incubatedat 30 C. Sensitivity or resistance to the phage wasrecognized by the clearing or the lack of clearing ofthe sections ofthe plate where the dilutions ofphagehad been spotted.

Identification of polA strains. The PolA- pheno-48

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

ESSENTIAL GENES 49

TABLE 1. Episomal mapping strains

Strain designation Sex Genotypea Derivation

AB1013 F' F14-7/his-4, proA2, lacYl, str-8 A(ilvE-argH) corre- CGSCbsponding to F14

AB1206 F' F141his-4, proA2, lacYl, str-8 A(ilvE-argH) corre- CGSCsponding to F14

F14/KA17 F' F14IargE, met, pur, lac, thr, trp, recAl AB1206 x KA17 (2)F14-B/KA17 F' F14-B/argE, met, pur, lac, thr, trp, recAl AB1206 x KA17 (2)F14-7/KA17 F' F14-7/argE, met, pur, lac, thr, trp, recAl AB1013 x KA17 (2)KLF5/AB2463 F' F105IargE3, his-4, proA2, leu-6, thr-1, recA13, str- CGSC

31KLF10/JC1553 F' F110/argG6, metBI, his-i, leu-6, recAl, lacYl or CGSC

lacZ4, str-104KLF23/KL181 F' F123IpyrD34, his68, trp-45, recAl, str-118 CGSCKLF33/JC1553 F' F133IargG6, metBl, his-1, leu-6, recAl, str-104 CGSC

a Only markers relevant to the current study are listed.b CGSC, E. coli Genetic Stock Center, Department of Human Genetics, Yale University, New Haven,

Conn.TABLE 2. Chromosomal marker strains

Strain designa- Sex Genotypea Derivationtion

AB468 F- his-4, proA2, purD13, lacYl CGSCbAB1160 F- ilvC7, argE3, his-4, proA2, lacYl CGSCAB2070 F- ilvE12, metE46, his-4, trp-3, proA2, lacYl, or lacZ4, CGSC

str-8 or str-9AB2291 F- ilvDl88, metE46, his-4, trp-3, proA2, lacYl or lacZ4, CGSC

str-8 or str-9AB2569 F- argHl or argE3, metA28, his-4, proA2, lacYl CGSCAT753 F- ilv-1, argHl, metBI, rha-1, lacYl or lacZ4, str-8, str- CGSC

9, or str-1 7AT2535 F- pyrB59, argHl, str-8, -9, or -17 CGSCHMS83 F- thy, polAl, polBI, rha, lacZ C. C. Richardson (6)KA14 Rift F- argE, met, pur, lac, rifr, recAl (2)KK1000 F- metB, upp-1 J. Fuchs (11)PC0132 F- purH47, lacYl, str-117 CGSCpop 555 F- argH, his, malE15, recA, strA M. Schwartz, personal

communication; (15)RC709 F- metF63, pro-22 CGSCRK4101Bfe F- argH, leuB, proC, purE, metE, lacZ, strA, rif, tonB, R. Kadner

thy, bfer, recAa Only markers relevant to the current study are listed.b CGSC, E. coli Genetic Stock Center, Department of Human Genetics, Yale University, New Haven,

Conn.

type was detected by its sensitivity to methylmeth-anesulfonate (MMS). MMS was added to sterile,cooled lactose-tetrazolium agar at a final concentra-tion of 0.04% (8). These plates were dried overnightat room temperature. Several dilutions of strains tobe tested were plated onto lactose-tetrazolium agarwith and without MMS. A reduced plating efficiencyon the MMS-containing plates was indicative ofpolA strains (8).

RESULTSIsolation of mutants defective in essential

genes. A procedure for the isolation of strainscarrying mutations in essential genes that hadbeen induced by the frameshift mutagen ICR-191 {3 -chloro- 7 -methoxy -9- (3- [chloroethyl]-

amino propylamino) acridine dihydrochloride}was described previously (2). It may be recalledthat mutants deficient in essential genes wererecognized because they formed microcoloniesat 42 C. In the present study, we used ICR-191again to induce a number of mutations in es-sential genes. We followed the same procedureas that described previously (2) with one modi-fication: in addition to examining isolates thatformed microcolonies at 42 C, we also examinedthe colonies that showed no visible growth atthe restrictive temperature. The rationale forthis modification was that some colonies withmutations in essential genes might result inmicrocolonies too small to be visible on the

VOL. 126, 1976

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

50 ARMSTRONG AND FAN

42 C replica plates. In fact, two mutants wereisolated by using this modification.Also in the present study, we isolated several

mutants deficient in essential genes after mu-tagenesis with NG. NG is a mutagen with base-substitution activity (27) and can efficiently in-duce temperature-sensitive mutations (1). Forthis reason, we expected some of the isolatesthat either formed microcolonies or did notgrow at all at 42 C after NG mutagenesis to betemperature-sensitive mutants instead of mu-tants deficient in essential genes. Tempera-ture-sensitive mutants, in contrast to ess mu-tants, do not depend upon the presence of theKAF1 episome for growth and thus should beable to substitute the F123 trp+ episome forKAF1 when mated at 28 C with strain KLF23/KL181. Consequently, our first screening proce-dure subsequent to the identification of isolatesthat formed microcolonies or did not grow at allat 42 C was to spot-mate these isolates withstrain KLF23/KL181 at 28 C. Isolates that wereunable to form viable Trp+ His+ Pyr+ merodip-loids in this mating were tentatively identifiedas containing deficiencies in chromosomal es-sential genes in the diploid region.

Genetic characterization of mapping epi-somes. As a first step in characterizing themutations in essential chromosomal genes, wewished to determine the approximate locationof these mutations within the metB-malB chro-mosomal region. For this purpose, we used sev-eral F' factors (14, 22, 23; Table 1) that carrydifferent sections ofE. coli chromosomal deoxy-ribonucleic acid (DNA). The chromosomal DNAcarried by each of these F' factors has at leastone end terminating within the metB-malB re-gion (22). For complementation purposes, an F'factor that does not carry a gene carried byKAF1 can be considered deleted for that gene.Therefore, a series of F' factors with differenttermination positions in the metB-malB regionwould effectively provide a series of differentdeletions of episomal genes. All ofthe F' factorswe used have been described previously (22)except one, F14-B, which we discovered afterconjugation between strains AB1206 and KA17,an F-RecA- recipient.

Before mapping our mutations in essentialgenes, we wished to verify that our stock cul-tures of the F' factors did in fact carry thechromosomal DNA regions described by Low(22). For this purpose, the episomes were trans-ferred into several F- strains that carry one ormore auxotrophic markers (Table 2), and mero-diploid prototrophs were selected. Chromo-somal markers are not normally transmitted bythe F' strains because they are either recA orhave a deletion that corresponds approximately

to the chromosomal DNA carried by the epi-some.The presence of the rif, rha+, and bfes genes

on the episomes was determined in two furthertests. The episomal strain was first crossedwith F-Rif', F-Rha-, and F-Bfer strains. Auxo-trophic markers were used to select againstboth the donor and the recipient strains. Proto-trophs from the selective plates were purifiedtwice to give single-colony isolates on the samemedium. The purified isolates were eithertoothpicked to rifampin-containing or rham-nose minimal medium or tested for resistanceto bacteriophage BF23 as described above. Sen-sitivity to either rifampin or phage BF23 (Rifsand BfeS, respectively) is dominant to resist-ance (Rif and Bfer); therefore, if an episomecarried the rifr or bfe; gene, the merodiploidwould be phenotypically rifampin or BF23 sen-sitive.The results of mating our F' factors into sev-

eral chromosomal marker strains are given inTable 3 and presented schematically in Fig. 1.In contrast to previous reports (8, 22), F110 wasfound not to carry the region ofDNA frompolAto metB. It can be seen that the end points ofthe F' factors divide the DNA between metBand maIB into at least three regions, which wedenoted I, II, and III. We designated these epi-somes as mapping episomes, and we used themto map the mutations in essential genes to oneof the three regions they delineate.

It is of interest that the fused episome KAF1derived from FllO and used to confer partialdiploidy to our parent strain carries all of thegenes that we determined that F110 carries,even though episomal fusion is believed to re-sult in deletion of at least some F' DNA (28).Mapping the mutations in essential genes.

Thus far, we have isolated 25 independentstrains with mutations in essential chromo-somal genes. Fifteen of these mutants (desig-nated by numbers less than 3000) were derivedfrom ICR-191 mutagenesis, and 10 were derivedfrom mutagenesis with NG (designated bynumbers greater than 3000).Each of the 25 mutations was mapped by

mating the mutant strains with strains carry-ing the mapping episomes F14, F14-7, F14-B,F105, F133, and FllO (Fig. 1). Because all of themapping episomes carried met+ genes and all ofthe mutant strains were Met- (2), the transferof a mapping episome into a mutant straincould be carried out by selecting Met+ recipi-ents. The F' donor strains were selected againstby using auxotrophic markers not carried bythe recipients. If a particular mapping episomecarried the ess+ gene that was deficient on thechromosome, viable Met+ merodiploids were

J. BACTERIOL.

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

TABLE 3. Genetic characterization of mapping episomes

F14/ F14-7/ F14-B/ KLF5/ KLF33/ KLF1O/ KAF1/Recipient Markers tested KA17a KA17a KA17a AB JC1553a JC1553a KA17astrain F14b F14-71' F14-Bb FlO5b F1335 Fi0b" KAF1b

AB468 purD+ -C -C + - - + +AB1160 ilvC+ + + + - + - -

argE + + + + + + + +AB2070 ilvE+ + + + - + - -

metE+ + + + - +AB2291 ilvD + + + + - + - -

metE+ + + + - +AB2569 metA+ - - - - - + +AT753 argH+ + + + + + + +

rha-1 + + + + + +AT2535 argH+ + + + + + + +

pyrBHMS83 polA NT"' NT NT NT NT NT -KA14 Rifr argE+ +' +' + + + + +

rif r' r s' r r s sKK1000 metB+ +C + + + + +

PCO132 purH+ - - + - - + +pop 555 argH+ + + + + + + +

malB+, argH+ - - - - - + +RC709 metF+ + + + + + + +RK4101Bfer bfer s s s s s s s

"Donor.bEpisome donated.In these matings, strains AB1206 and AB103 were used to donate F14 and F14-7, respectively.

"NT, Not tested.The letters s and r signify sensitivity or resistance to rifampin or phage BF23.

wI 0x <

metB metFf 9 g I cr k k Iiv rho..

(74.7) (78) (79)

FIlO, KAFI

F)05

F14, F14-7 F133

F14-B

Rgion I

maiB

Regiona

FIG. 1. metB-malB region of the E. coli chromosome. The figure shows the markers used in our geneticanalysis ofthe mapping episomes and the regions ofDNA carried by the mapping episomes, which divide themetB-malB region into three sections. The numbers in parentheses refer to minutes on the E. coli map.

formed after mating. If, on the other hand, noviable Met+ colonies were formed, we concludedthat the episome did not carry the ess + gene forwhich the chromosome was ess.The results of crossing all of our mutants

deficient in essential genes with all of the map-ping episomes are shown in Table 4. Of the 25independently isolated mutants, 2 mapped inregion I, 23 mapped in region II, and nonemapped in region Ill; thus, it is clear that es-

sential genes are not randomly distributedthroughout the diploid region.The accompanying paper (2) describes the

characterization of two of the mutations in es-sential genes as rifO mutations. We used ourmapping episomes to determine the genetic lo-cation of both of these mutations and of allother rill mutations we have isolated and char-acterized using the procedure described previ-ously (2). All of the rir mutations map in region

I . .

r-- -I 1- I 1-% I

VOL. 126, 1976 ESSENTIAL GENES 51

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

52 ARMSTRONG AND FAN

TABLE 4. Mapping mutations in essential genes

AB1206b AB103b F14-B/ KLF5/ KLF33/ KLF10/ RegionRecipienta F14r F14-7 KA9' AB2463b JC1553b JC1553b MappedF14-EB F105r F133r FllO ind

+

+

+

+

+ Is+ IIS+ IIr+ IIS+ IIS+ lIr+ IS+ lIr+ IIS+ Ilr+ IIS+ IIS+ IIS+ . IIS+ Ihr+ IIr+ IIS+ IIS+ IIS+ IIS+ IIS+ IIS+ IIS+ Ius+ IIs

a The genotype of the recipient strains is KAFless+/KA17ess.bDonor strain.c Episome donated.d The superscripts s and r denote sensitivity and resistance to rifampin, respectively.' The symbols + and - denote ability or inability to form viable Met+ merodiploid colonies.

II, the chromosomal region to which the rifgene has been mapped previously (3, 4). Thisfinding supports the validity of our episomalmapping procedure. The other nine comple-mentation groups mapping in region II do notseem to have mutations in the rif gene since allof the mutants in these groups are phenotypi-cally rifampin sensitive.

Classification of mutants into complemen-tation groups. After the mapping of the muta-tions in essential genes into one of the threeregions between metB and malB, we wished toestablish the number of cistrons in which themutations were located. This was done by thecomplementation procedure described next.Recombination was used to transfer different

ess chromosomal mutations to episomes as de-scribed in the preceding paper (2). The ess epi-somes were then transferred into all ofthe chro-mosomal mutants by conjugation. Because theepisome (FllO met+ ess) in the donor straincarried a met+ allele and the chromosomal ess

recipient was Met-, chromosomal mutants thathad received an F110 met+ ess episome could beselected as Met+ prototrophs. The results of

these complementation tests are shown in Ta-ble 5 and summarized in Table 6. As expected,episomes that carry mutations mapping in sep-arate regions do complement. For example,F110 met+ ess(49) in region I complements thechromosomal recipient carrying the ess(260)mutation that maps in region II.

DISCUSSIONUsing a partially diploid strain of E. coli K-

12, we isolated 25 independent strains carryingmutations in essential genes located in themetB-malB region. It should be recalled thatsome functions of the cell can be performed byproducts of two or more genes all on the chro-mosome. An example is the elongation factorfor protein synthesis called EF-Tu (16). It isunlikely that we would find mutants withdefects in these genes if the functional productfrom one of the genes is sufficient for viability.We used a collection of F' factors whose ends

terminate between metB and malB to map our25 mutations into 11 complementation groupsin three regions of the E. coli genetic map. Wefound that some of the mutations in region II

+

+ +

ess(49)ess(230)ess(237)ess(254)ess(258)ess(260)ess(261)ess(363)ess(542)ess(795)ess(842)ess(101 1)ess(1047)ess(1076)ess(1202)ess(3122)ess(3484)ess(4003)ess(4075)ess(4151)ess(4160)ess(41 79)ess(4186)ess(4282)ess(4294)

+++++++++++++++++++++++++

J. BACTERIOL.

-C

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

TABLE 5. Complementation tests

ess chromo- FlIO met+ ess episomal donora

somal reclP- ess(49) ess(230) ess(258) ess(260) ess(542) ess(842) ess(1047) ess(1076) ess(3484) ess(4160) ess(4179)ientb

ess(49) -C +' + + + + + + + + +

ess(230) + - + + + + + + + + +

ess(237) + + + - + + + + + + +ess(254) + + + + - + + + + + +ess(258) + + - + + + + + + + +ess(260) + + + - + + + + + + +ess(261) - + + + + + + + + + +

ess(363) + - - - - + - - - +ess(542) + + + + - + + + + + +ess(795) + + + - + + + + + + +ess(842) + + + + + - + + + + +

ess(1011) + + + + + - + + + + +ess(1047) + + + + + + - + + + +ess(1076) + + + + + + + - + + +

ess(1202) + + + - + + + + + + +

ess(3122) + + + - + + + + + + +

ess(3484) + + + + + + + + - + +ess(4003) + + - + + + + + + + +ess(4075) + + + + + + + + + - +ess(4151) + + + + + + + + - + +ess(4160) + + + + + + + + + - +

ess(4179) + + + + + + + + + +

ess(4186) + + - + + + + + + + +ess(4282) + + + + + + + + + - +ess(4294) + + - + + - + + - + +

a The genotype of donor strains if F110 met+ essimet thy recAl ess+ KA14 (2).b The genotype of recipient strains is KAF1IrecA1 ess KA17 (2).'The symbols + and - represent presence and absence of viable Met+ merodiploids, respectively.

TABLE 6. Complementation groups

Region Complementation group

I 1. 49,a 261II 2. 230, 363II 3. 542, 254, 363II 4. 258, 4003, 4186, 4294, 363II 5. 3484, 4151, 4294, 363II 6. 842, 1011, 4294II 7. 4160, 4075, 4282II 8. 4179, 363II 9. 1047, 363II 10. 1076, 363II 11. 260, 237, 795, 1202, 3122, 363

a The first mutation listed for each complementa-tion group was carried by the strain from which anF110 ess episome was isolated.

affect the rif gene. However, there are also nineother complementation groups in region II. Allof the mutant strains in these latter groups arerifampin sensitive. One of these complementa-tion groups could well correspond to the genethat codes for the 8' subunit of RNA polymer-ase because this gene is known to be located inthe same operon as the rif gene (10).

It has been suggested that the gene coding for

a, another subunit of RNA polymerase, mightnot be located near the 8 and f3' genes (12). Thiswas recently verified by Nomura and co-work-ers (S. R. Jaskunas, R. R. Burgess, L. Lindahl,and M. Nomura, In R. Losick and M. Chamber-lin [ed.], RNA Polymerase, in press), whomapped the gene coding for the a subunit toapproximately 64 min on the E. coli geneticmap. Therefore, the genes coding for the sub-units of RNA polymerase core enzyme are notunder the coordinate control of a single operon.

Recently, several genes have been identifiedthat are near the genes for the 83 and 3' sub-units at 79 min on the genetic map. These genescode for five proteins of the 50s ribosomal sub-unit (21, 26), for a transfer RNA (tRNA) that iseither tRNAglU or tRNAI1e (Jaskunas et al.,RNA Polymerase, in press), for the elongationfactor EF-Tu (16), and for the 5s, 16s, and 23sspecies of ribosomal RNA (Jaskunas et al.,RNA Polymerase, in press; M. Nomura and S.R. Jaskunas, in Control ofRibosome Synthesis.Alfred Benzon Symposium IX, in press). Al-though it is known that the latter four genesare redundant (7, 16, 29), it is not knownwhether this is also true for the five genes

ESSENTIAL GENES 53VOL. 126, 1976

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

54 ARMSTRONG AND FAN

coding for the ribosomal proteins or the geneencoding the tRNA. Because the latter genesmay be unique and because they are probablyessential, it is possible that some of our muta-tions are in these genes.Two of our mutants, ess(363) and ess(4294),

are each deficient in more than one cistron andcould be either deletions or polar mutations. Onthe basis of their complementation patterns,the mutants in region II fall into four groups:those that complement both F110 ess(363) andF110 ess(4294), those that complement onlyF110 ess(363), those that complement neither,and those that complement only F110ess(4294). Because there are some mutantsthat complement neither, ess(363) andess(4294) could well be deletions or polar muta-tions that affect some common gene. Our lackof success in obtaining spontaneous tempera-ture-resistant revertants of KAF1/ess(363)KA17 (unpublished data) suggests thatess(363) might be a deletion. This possibility isalso supported by the failure ofess(363) to com-plement 8 ofthe 10 ess mutations in region II.We probably obtained mutations in almost

all essential genes in the metB-malB regionbecause most of the complementation groupswe identified contain more than one independ-ent mutation. However, 4 of the 11 complemen-tation groups contain only one independentmutant, excluding the two multicistronic mu-tants. Therefore, it is probable that there arestill some genes in the metB-maiB region forwhich mutants were not isolated.We extrapolated from our data as follows to

estimate the total number of essential genes inthe E. coli genome. The ,3 subunit of RNApolymerase, coded by the rif gene, weighs ap-proximately 150,000 daltons (5) and is composedof about 1.4 x 103 amino acids. Thus, the size ofthe rif gene is about 4.1 x 103 nucleotide pairs.Because 5 of the 23 mutations we identified thataffected a single complementation group aredeficient in the rif gene, we estimate that 1.9 x104 nucleotide pairs in the metB-malB regioncode for essential genes. The metB-malB regionis about 2.8% of the genetic map or 1.1 x 105nucleotide pairs, assuming that the E. colichromosome contains 4.0 x 106 nucleotide pairs(17). Therefore, 17% of the metB-malB regionencodes essential genes. If this region is repre-sentative ofthe entire E. coli chromosome, then6.8 x 10W nucleotide pairs of the total genomecode for essential genes. Assuming that an av-erage protein is coded by 103 nucleotide pairs(17), we estimate that the total number of es-sential genes in E. coli is about 700. We wouldlike to emphasize that this estimate is depend-

ent upon the validity of assuming that themetB-malB region is representative of other re-gions on the chromosome.

ACKNOWLEDGMENTSWe thank R. K. Herman for his helpful suggestions and

reading of this manuscript, W. Gall for excellent technicalassistance, M. Nomura for making data available beforepublication, and all who provided the bacterial strains usedin this work. We express special thanks to B. Bachmann ofthe E. coli Genetic Stock Center for her ready compliancewith our many requests for strains.

This work was supported by Public Health Service grantGM20308 from the National Institute of General MedicalSciences. One of us (K.A.A.) was supported both by a Na-tional Science Foundation Traineeship awarded undergrants GZ 1628, GZ 2035, GZ 2396, and GZ 2733, and also bya Public Health Service training grant GM 01156 from theNational Institute of General Medical Sciences.

LITERATURE CITED

1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965.Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli K12.Biochem. Biophys. Res. Commun. 18:788-795.

2. Armstrong, K. A., and R. K. Herman. 1976. Method forthe isolation of Escherichia coli K-12 mutants defi-cient in essential genes. J. Bacteriol. 126:38-47.

3. Austin, S., and J. Scaife. 1970. A new method for select-ing RNA polymerase mutants. J. Mol. Biol. 49:263-267.

4. Austin, S. J., I. P. B. Tittawella, R. S. Hayward, and J.G. Scaife. 1971. Amber mutations ofEscherichia coliRNA polymerase. Nature (London) New Biol.232:133-136.

5. Burgess, R. R. 1969. Separation and characterization ofthe subunits of ribonucleic acid polymerase. J. Biol.Chem. 244:6168-6176.

6. Campbell, J. L., L. Soil, and C. C. Richardson. 1972.Isolation and partial characterization of a mutant ofEscherichia coli deficient in DNA polymerase II.Proc. Natl. Acad. Sci. U.S.A. 69:2090-2094.

7. Davies, J., and M. Nomura. 1972. The genetics of bacte-rial ribosomes. Annu. Rev. Genet. 6:203-234.

8. De Lucia, P., and J. Cairns. 1969. Isolation of an E. colistrain with a mutation affecting DNA polymerase.Nature (London) 244:1164-1166.

9. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E.Hartman. 1966. A proposal for a uniform nomencla-ture in bacterial genetics. Genetics 54:61-76.

10. Errington, L., R. E. Glass, R. S. Hayward, and J. G.Scaife. 1974. Structure and orientation of an RNApolymerase operon in Escherichia coli. Nature (Lon-don) 249:519-522.

11. Fuchs, J. A., and H. R. Warner. 1975. Isolation of anEscherichia coli mutant deficient in glutathione syn-thesis. J. Bacteriol. 124:140-148.

12. Hayward, R. S., S. J. Austin, and J. G. Scaife. 1974.The effect of gene dosage on the synthesis and stabil-ity of RNA polymerase subunits in Escherichia coli.Mol. Gen. Genet. 131:173-180.

13. Heil, A., and W. Zillig. 1970. Reconstitution of bacterialDNA-dependent RNA polymerase from isolated sub-units as a tool for the elucidation of the role of thesubunits in transcription. FEBS Lett. 11:165-168.

14. Hirota, Y. 1965. Genetical studies of Escherichia colisex factor. Jpn. J. Genet. 40:377-385.

15. Hofnung, M. 1974. Divergent operons and the geneticstructure of the maltose B region in Escherichia coliK12. Genetics 76:169-184.

16. Jaskunas, S. R., R. R. Burgess, L. Lindahl, and M.

J. BACTERIOL.

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

VOL. 126, 1976 ESSENTIAL GENES

Nomura. 1975. Identification of two copies of thegene for the elongation factor EF-Tu in Eswherichiacoli. Nature (London) 257:458-462.

17. Jaskunas, S. R., R. R. Burgess, and M. Nomura.1975. Identification of a gene for the a-subunit ofRNA polymerase at the str-spc region ofEscherichiacoli chromosome. Proc. Natl. Acad. Sci. U.S.A.72:5036-5040.

18. Jasper, P., E. Whitney, and S. Silver. 1972. Geneticlocus determining resistance to phage BF23 and coli-cins E,, E,, and E3 in Escherichia coli. Genet. Res.19:305-312.

19. Kornberg, A. 1974. DNA synthesis, p. 16-17. W. H.Freeman and Co., San Francisco.

20. Lanni, Y. T. 1960. Invasion by bacteriophage T5. II.Dissociation of calcium dependent and calcium inde-pendent processes. Virology 10:514-529.

21. Lindahi, L., S. R. Jaskunas, P. P. Dennis, and M.Nomura. 1975. Cluster ofgenes in Escherichia coli forribosomal proteins, ribosomal RNA, and RNA polym-erase subunits. Proc. Natl. Acad. Sci. U.S.A.72:2743-2747.

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

23. Pittard, J., J. S. Loutit, and E. A. Adelberg. 1963. Genetransfer by F' strains of Escherichia coli K-12. I.

Delay in initiation of chromosome transfer. J. Bacte-riol. 85:1394-1401.

24. Rabusay, D., and W. Zillig. 1969. A rifampicin-resist-ant RNA polymerase from E. coli altered in the 8subunit. FEBS Lett. 5:104-106.

25. Taylor, A. L., and C. D. Trotter. 1972. Linkage map ofEacherichia coli strain K-12. Bacteriol. Rev. 36:504-524.

26. Watson, R. J., J. Parker, N. P. FiiP, J. G. Flaks, and J.D. Friesen. 1975. New chromosomal location forstructural genes of ribosomal proteins. Proc. Natl.Acad. Sci. U.S.A. 72:2765-2769.

27. Whitfleld, Jr., H. J., R. G. Martin, and B. N. Ames.1966. Classification of aminotransferase (C gene) mu-tants in the histidine operon. J. Mol. Biol. 21:335-355.

28. Willetts, N., and F. Bastarrachea. 1972. Genetic andphysiochemical characterization of Eacherichia colistrains carrying fused F' elements derived fromKLF1 and F57. Proc. Natl. Acad. Sci. U.S.A. 69:1481-1485.

29. Yanofsky, S. A., and S. Spiegelman. 1962. The identifi-cation ofthe ribosomal RNA cistron by sequence com-plementarity. II. Saturation of and competitive inter-action at the RNA cistron. Proc. Natl. Acad. Sci.U.S.A. 48:1466-1472.

55

on May 13, 2018 by guest

http://jb.asm.org/

Dow

nloaded from