JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 275-281 Vol. 163, No. 10021-9193/85/070275-07$02.00/0Copyright C) 1985, American Society for Microbiology

Cloning and Mapping of the Genetic Determinants for Microcin B17Production and Immunity

JOSE L. SAN MILLAN, CONCEPCION HERNANDEZ-CHICO, PEDRO PEREDA, AND FELIPE MORENO*

Unidad de Genetica Molecular, Servicio de Microbiologia, Centro Especial Ram6n y Cajal, Carretera de Colmenar, Km9,100, Madrid-28034, Spain

Received 4 February 1985/Accepted 8 April 1985

Plasmid pMccBl7 (70 kilobases [kb]) codes for the production of microcin B17, a peptide that inhibits DNAsynthesis, and for microcin B17 immunity. A BamHI-EcoRI fragment of 5.1 kb from pMccBl7 was cloned intopBR322 in two steps. The resulting plasmid (pMM102) overproduced microcin B17 and expressed immunityagainst microcin. Mcc- and Mcc- Imm- mutants were isolated on plasmids pMccB17 and pMM102 by deletingvarious DNA fragments and by inserting different translocatable elements. Physical and phenotypic charac-terization of these mutants showed that a DNA region of 3.0 to 3.5 kb is required to produce microcin B17,whereas an adjacent region of about 1.0 kb is required to express microcin B17 immunity.

The word microcin was proposed to designate antibioticcompounds, produced by enterobacteria, that differ fromcolicins in being able to pass through cellophane membranes(1). Another characteristic that distinguishes microcins fromcolicins is that microcin synthesis is not inducible by DNA-damaging agents (3). However, as with colicins, bacterialstrains producing a specific microcin are immune to the samemicrocin, and microcin synthesis, as well as immunity, isencoded by plasmids (4). Some 46 independent microcin-producing isolates from human feces have been classified infive groups on the basis of cross-immunity as well as on thebasis of genetic and biochemical criteria (3, 4). MicrocinB17, previously named microcin 17, is the prototype of oneof these groups, which includes four independent isolates.

Microcin B17 is a hydrophobic peptide of about 4 x 103daltons (unpublished data). The first effect of microcin B17on susceptible bacteria is the specific inhibition of DNAsynthesis. This inhibition is irreversible and leads to thedegradation of bacterial DNA, induction of the SOS systemof DNA repair, and, finally, to the killing of sensitive cells(M. Herrero and F. Moreno, submitted for publication).

Microcin B17 is produced by strains of Escherichia coliwhich carry plasmid pMccB17 (previously named pRYC17[2, 13]). This wild-type plasmid, originally found in a strainof E. coli called LP17 (2), was transferred by conjugation toE. coli K-12, in which it maintained the ability to direct theproduction of microcin and to ensure self-immunity.pMccB17 is 70 kilobases (kb) long and is found in one to twocopies per chromosome; it belongs to the incompatibilitygroup FII and does not contain any conventional antibioticresistance markers (2).We are interested in the genetic control of microcin B17

synthesis. Results indicating the involvement of the chromo-somal gene ompR in microcin production have been pub-lished previously (13). In this study we present resultsindicating that all of the plasmid information required tosynthesize microcin B17 and to ensure immunity againstmicrocin seems to be contained in a unique 5.1-kb fragmentof pMccB17. A physical and genetic map of this fragment ispresented.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains and plasmids. The source and character-istics of strains used in this study are listed in Table 1.Media and chemicals. Liquid and solid LB-rich medium

and M63 minimal medium were used essentially as describedby Miller (17). Minimal medium was supplemented withglucose (0.2%) and vitamin Bi (1 .g/ml). The followingantibiotics were used at the indicated final concentrations (inmicrograms per milliliter): ampicillin (Ap; 50), tetracycline(Tc; 20), kanamycin (Km; 20), streptomycin (Sm; 100). Theselective medium for clones immune to microcin B17 wasprepared as follows. Plates with M63 minimal medium werecovered with a cellophane membrane onto which was over-laid 3 ml of molten minimal agar containing 5 x 107 cells ofstrain BM7008. After 30 h of incubation at 37°C, the cel-lophane film was removed, and residual contaminating cellswere killed with chloroform vapors. Plates were then sup-plemented with glucose (0.2%), by spreading 0.25 ml of a20% glucose solution onto the plates.

Microcin B17 assay on plates. To determine the capacity ofa strain to produce microcin B17, 2 x 107 sensitive indicator(BM21) cells were overlaid onto M63 plates with 3 ml of softagar. Single colonies to be checked were then transferredwith a toothpick onto the seeded plates. After overnightincubation, the microcin B17-producing colonies gave aclear growth inhibition zone surrounding the colony. Theinhibition halo was normally about 1 cm in diameter whenthe producing bacteria carried the wild-type plasmidpMccB17 or one of its derivatives obtained by transposoninsertion. No inhibition zone was observed when immune orinsensitive strains were used as indicators.

Microcin immunity test. A few fresh colonies of a microcinB17-producing strain were suspended in M63 minimal me-dium to obtain ca. 108 cells per ml. Drops of this suspensionwere placed on the surface of M63 agar and allowed to run ina straight line on the agar. When streaks were dried, theplates were incubated at 37°C for 30 h. At this time singlecolonies were cross-streaked, and the plates were reincu-bated. Only microcin-immune cells grew in the immediatevicinity of the producing bacteria.

Plasmid DNA manipulations. Plasmid DNA was extracted

275

276 SAN MILLAN ET AL.

TABLE 1. E. coli K-12 strains and phageStrains and Genotype Source or referencephage

StrainsBM21 F- gyrA (X+) 2pop3351 F- araDJ39AlacUl69A 13

malBI rpsL relA thiARYC100Q F- araDI39AlacUI69Arib-7 J. Beckwith

rpsL relA thiA recA56BM7008 BM21(pMccBl7) 2RYC200.4 BM21(pMM4) 13SC18a F- thr leu(pSC204) J. Brevet; 15RYC888 pop3351(pMM101) This studyRYC893 pop3351(pMM102) This study

BacteriophageX467b c1857 Oam8 Pam29 b221 I. Saint-Girons; 5

rex: :Tn5a pSC204 is a derivative of pSC101 which is unable to replicate at 42°C and

which carries a copy of transposon Tn3 that confers resistance to ampicillin(15).bPhage X467 contains a copy of transposon TnS which confers resistance to

kanamycin and bleomycin (5, 10).

from overnight LB cultures. Cleared lysates were obtainedand precipitated as described previously (8). The DNA pelletwas suspended in TE buffer (10 mM Tris-hydrochloride [pH8.0] and 1.0 mM EDTA) and dialyzed against the samebuffer. When necessary, supercoiled DNA was further puri-fied by density centrifugation in a cesium chloride gradientcontaining ethidium bromide (16). This was the case forwild-type pMccBl7 and its derivatives obtained by Tn3transposon insertion. The restriction enzyme digestionswere carried out for 2 h as indicated previously (16). Whenincomplete digestion was desired, the reaction was arrestedafter 20 min. The number and the size of the DNA fragmentsgenerated by the endonucleases were analyzed in 0.8 or1.2% agarose gels. Electrophoresis buffer was Tris-EDTA-borate (89 mM Tris-hydroxide, 89 mM boric acid, 2 mMEDTA [pH 8.0]) (16), and a typical run was done at 150 V for3 to 4 h. Mapping of restriction sites was done by compari-son of the fragment sizes of single and double digests, usingas a reference the size of HindIIl and EcoRI fragments fromphage lambda DNA (22) and that of HaeIII fragments fromphage 4X174 replicative form DNA (21). Ligation of DNAfragments was carried out with T4 DNA ligase, as indicatedpreviously (24), for 16 h at 4°C. DNA concentrations wereabout 70 to 80 ,ug/ml. The ratio of pBR322 DNA andpMccBl7 DNA was 1:7 in the experiment leading to theisolation of plasmid pMM101.

Genetic methods. Transformation was carried out es-sentially as indicated previously (9). The transformationmixture was diluted 20-fold in fresh LB medium and incu-bated at 37°C for 1 to 2 h to allow expression of antibioticresistance or immunity against microcin B17 or both. Con-jugation and P1 transduction were carried out as describedby Miller (17).

Plasmid copy number determination. BM21 cells carryingplasmids were grown in M63 minimal medium supplementedwith Casamino Acids (0.4%). Radioactive labeling wasachieved by the addition of [methyl-3H]thymidine (1 ,uCi/ml;46 Ci/mM) and 2-deoxyadenosine (250 ,ug/ml) to cultures(absorbance at 600 nm, 0.1) which were incubated until theabsorbance at 600 nm was 0.5. Ten milliliters of each culturewere lysed with Sarkosyl by the method of Nisioka et al.(18), and the lysates were centrifuged to equilibrium in a

cesium chloride density gradient in the presence of ethidiumbromide. Gradients were fractionated in 20-,u samples, andradioactivity in each fraction was determined as describedpreviously (16). Copy numbers were calculated from theamount of radioactivity in the plasmid and chromosomalpeaks and the size of plasmids (pMccB17, 70 kb; pBR322,4.3 kb; pMM101, 26 kb; pMM102, 9.1 kb), although it shouldbe pointed out that the plasmid copy number is under-estimated by this method. It was assumed that the size of theE. coli chromosome is 3.8 x 103 kb (25).

Isolation of pMccBl7::Tn3 mutant plasmids. Exponentiallygrowing cultures of strains RYC200.4 and SC18 were mixedand incubated overnight without shaking at 30°C. Portionswere then plated in 42°C in LB plates supplemented withampicillin and tetracycline. Several Tcr Apr transconjugantswere purified and then crossed with strain pop3351, selectingfor resistance to streptomycin, tetracycline, and ampicillin at37°C. Tcr Apr Smr clones were checked for microcin produc-tion and microcin immunity.

Isolation of pMM102::TnS mutant plasmids. A culture ofRYC893 su- (pMM102) strain was incubated and infectedwith phage X467, as indicated by Berg (5). Adsorption wasallowed for 20 min at 30°C, and then 0.05-ml samples weredistributed in tubes containing 1 ml of fresh LB medium andincubated for 2 h to allow kanamycin resistance gene expres-sion. Finally, 20 ml of LB medium containing kanamycin (20,ug/ml) was added, and cultures were incubated overnight at42°C. Cleared lysates from these cultures were prepared,and DNAs were used to transform strain pop3351 by select-ing for Apr Kmr clones, and the plasmid DNA content wasstudied in all of them. Only clones containing a unique DNAspecies of about 14.8 kb resulting from the insertion of asingle copy of TnS in pMM102 were characterized further.

Isolation of pMM102::insertion sequence mutant plasmids.The experimental basis and the method used in this selectionare indicated below.

RESULTS

Mapping of the microcin B17 determinants on plasmidpMccBl7. To obtain some insight on the location of themicrocin B17 region on plasmid pMccB17, we isolated andcharacterized mutations that impair microcin production.Mutants were isolated in plasmid pMM4, a Mcc+ Imm+Tra+ derivative of plasmid pMccB17, containing a singlecopy of transposon TnlO inserted at the coordinate at 65 kb(Fig. 1 [13]). Thirty-three independent clones with Tn3inserted in pMM4 were isolated as indicated above and wereassayed for microcin production and microcin immunity.Two of the clones were Mcc- Imm-, and one was Mcc-Imm+. When the plasmid content of these clones wasexamined it was found that all three carried a single copy ofTn3. Results of the restriction analysis with Hindlll showedthat the Mcc- Imm+ mutant (plasmid pMM22) contained asingle copy of Tn3 inserted in the small 0.85-kb HindIII Cfragment, and that the Mcc- Imm- mutants (plasmidspMM21 and pMM23) each contained a copy of the trans-poson in the 3.75-kb HindIII B fragment of pMccB17 (Fig.1). Further analysis showed that the location of the insertionwas different for pMM21 and pMM23, although both mappedto the right of the coordinate at 69 kb, between the Sall andEcoRI sites (Fig. 1). Moreover, both plasmids pMM21 andpMM23 contained a second insertion of about 1.4 kb in theBamHI-SalI fragment bounded by coordinates at 65 and 69kb. These results suggest that DNA sequences involved inmicrocin production and microcin immunity are adjacent

J. BACTERIOL.

MICROCIN B17 GENETIC DETERMINANTS 277

Mind III frgments"c~ "s"

S

E

Ligase

BamHI-EcoRl

FIG. 1. Cloning of the microcin B17 genetic determinants. Physical maps were constructed by single and double digests with restrictionendonucleases. In the mapping of pMM101 and pMM102 we took advantage of previously published information concerning the location ofrestriction sites in pBR322 (7, 23). The restriction map of pMccB17 is also based on the analysis of various fragments which were subclonedinto pBR322 (data not shown). Location of inserts MM4 (TnJO) and MM21, MM22, and MM23 (Tn3) are shown. Abbreviations: B, BamHI;E, EcoRI; H, HindIll; S, Sall; Ligase, T4 DNA ligase; b/a, ampicillin resistance gene; tet, tetracycline resistance gene.

and that plasmid pMccB17 does not contain other microcinregions.

Cloning of the microcin B17 region. The 22-kb EcoRIfragment of pMccBl7, containing the microcin-related se-quences which are affected in plasmids pMM21, pMM22,and pMM23, was cloned into the EcoRI site of pBR322 withselection for microcin immunity and for Apr and Tcr (Fig. 1).The resulting plasmid (pMM101) also conferred the ability toproduce microcin B17. A physical map of pMM101 wasmade by digesting DNA with combinations of EcoRI,HindIII, BamHI, and Sall restriction enzymes (Fig. 1).As all transposons that impair microcin-related functions

mapped in a BamHI-EcoRI fragment of pMccB17 (Fig. 1),pMM101 DNA was cleaved with both enzymes, and reli-gated DNA fragments were introduced into pop3351 cells bytransformation with selection for Apr. Ninety-eight Apr Tcsclones were checked for microcin production and immunity.They presumably had lost the small 375-base-pair (bp) EcoRI-BamHI fragment from pBR322 necessary for tetracyclineresistance expression. Forty of them were Mcc- Imm -, andthe others 48 were Mcc+ Imm+. Eight Mcc+ Imm+ cloneswere found to contain a unique 9.1-kb plasmid (pMM102)composed of the 4-kb EcoRI-BamHI fragment from pBR322and the 5.1-kb EcoRI-BamHI fragment from pMccB17.Consequently, the 5.1-kb EcoRI-BamHI fragment betweencoordinates at 65 and 70 kb of pMccBl7 was found tocontain all of the plasmid information required to synthesizemicrocin B17 and for immunity to it. Further analysis withother restriction enzymes showed that the structures of therecombinant plasmid pMM102 was as indicated in Fig. 1.

Plasmids pMM101 and pMM102 direct the overproductionof microcin B17. The antibiosis halos produced by strainsRYC888(pMM101) and RYC893(pMM102) were twice thediameter of that produced by E. coli K-12 strains carryingpMccB17 or its derivative pMM4. Plasmids pMM102 andpBR322 were present at about 22 copies per chromosome;plasmid pMM4 was present at 1 to 2 copies per chromosome,thereby confirming previous results with plasmid pMccB17

(2). Although we were unable to obtain a correct sedimenta-tion profile with pMM101 DNA in two assays, results ofexperiments in which pMM101-encoded P-lactamase wasassayed indicated that this plasmid was also present in a highcopy number (data not shown). These results suggest thatthe amount of microcin production could be more or lessrelated to plasmid copy number.

Deletion analysis of plasmid pMM102. To obtain informa-tion on the location of the genetic determinants for microcinB17 synthesis and for microcin B17 immunity on the 5.1-kbfragment from pMccB17, we prepared plasmids deleted invitro for different fragments of pMM102. We took advantageof the fact that enzymes AvaI, HindIII, HpaI, KpnI, PvuII,and Sall generate two fragments in pMM102 (Fig. 2), one ofwhich contains all of the information required for plasmidreplication and for ampicillin resistance. DNA frompMM102 was cut separately with each of these enzymes,diluted to favor intramolecular ligation, religated with T4DNA ligase, and used to transform strain pop3351. Of the 10Apr transformant clones checked in each experiment, noneproduced microcin. Plasmid DNA from two clones of eachexperiment was extracted and submitted to restriction en-zyme analysis. In all cases the plasmids had the expectedstructure. When assayed for microcin phenotypes it wasfound that plasmids pMM120 (deletion of HindlIl fragment),pMM121 (deletion of KpnI fragment), pMM122 (deletion ofAvaI), and pMM129 (deletion of PvuII fragment) were Mcc-Imm', whereas plasmids pMM130 (deletion of HpaI frag-ment) and pMM123 (deletion of Sall fragment) were Mcc-Imm-. Other deleted derivatives were obtained by partialdigestion of pMM102 with endonucleases AccI and HincII.In the first case several Apr clones were classified into twogroups which were Mcc- Imm+ (e.g., pMM138) or Mcc-Imm- (e.g., pMM137) (Fig. 2). After partial digestion withHinclI we were able to isolate four different deleted plasmidsfrom pMM102 (pMM131, pMM132, pMM133, andpMM134). Plasmid pMM134 was identical to pMM130. Thestructure and the phenotype of the other three plasmids are

VOL. 163, 1985

278 SAN MILLAN ET AL.

u

E z=""12--EI11/I I II \ 1-3.CL

U)0 4 LV x 0 x tu

...102 J =..I I IIIill I I I I I I A ..=.=

pMM2 -dS - - - 0lpMM122pMM132 -,pMM129pMU 138pUM 135 - - -_pUN 120 - - --pMU 121 _ _ _pMU 123 -_pUN 137 _ __pUN 133 _ -PUN 130 - - -

pN 131 -4p_ 142__

uEE

++

.- -- +

FIG. 2. Physical map and phenotype of deletions in the microcin B17 region. Restriction map of plasmid pMM102 was carried out asindicated in the text and in the legend to Fig. 1. The relative positions of the sites are drawn to scale. Dashed (pBR322) and solid (pMccB17)lines indicate sequences known to be present; the blank spaces indicate sequences known to be deleted. The thick line in pMM142 indicatesthat fragment BamHI-BglII is inverted. The BamHI-EcoRI microcin fragment is 5,100 bp long. Abbreviations and symbols: Mcc, Microcinproduction; Imm, immunity; C1, the left moiety of transposon Tn5 (see text).

shown in Fig. 2. Finally, by carrying out digestions withBamHI and BglII together, we obtained plasmid pMM135, inwhich the BamHI-BglII fragment was deleted, and plasmidpMM142, in which the BamHI-Bgll fragment was inverted.Both plasmids pMM135 and pMM142 were Mcc- Imm+.Taken together, the results presented above indicate that

the genetic determinant(s) for immunity is located in theright arm of the cloned BamHI-EcoRI fragment. Deletion ofany of the sequences to the left of the KpnI site closest to theSalI site did not reduce the level of microcin immunity bymuch (see below), whereas deletions involving DNA locatedto the right of this KpnI site lead to a clear Imm- phenotype.Indeed, comparison of plasmids pMM121 and pMM123indicates that the 0.3-kb KpnI-SalI fragment, or a part of it,is absolutely required for immunity. The results also indicatethat the production of microcin B17 requires DNA se-quences that belong to the fragment extending from the KpnIpoint to the left of the AvaI (SmaI) site because plasmidspMM130 and pMM122 did not produce microcin. The integ-rity of the BglII site in the middle of this region was alsorequired because plasmid pMM142 did not producemicrocin. These conclusions have been verified in this study,and more precise information was gained when the mutantplasmids obtained by insertion of translocatable elementswere characterized.TnS mutagenesis of the microcin B17 region of pMM102.

Sixteen independent single TnS insertions in pMM102 (seeMaterials and Methods) were characterized with enzymesBglII, BamHI, and SmaI which cleave pMM102 DNA inonly one site and TnS DNA in one site (BamHI and SmaI)and two sites (BglII) (14). Single and double digests withthese enzymes enabled us to map the insertion site and todetermine the orientation of the insertions. Inserts mappedin 14 different sites, and all of them were inside the microcinfragment (Fig. 3). Three classes of plasmids were distin-guished. The first class (plasmids pMM206 and pMM216)retained the ability to determine both microcin productionand immunity. The second class comprised nine plasmids

that remained Imm+ but that had lost the capacity toproduce microcin. The TnS inserts in these plasmids werelocated in the central region spread along ca. 2.5 kb. Thethird class comprised five plasmids which were Mcc- Imm-.The insertions in this group were in the region to the right ofthe microcin fragment, around the HpaI site closest theEcoRI site.

In all 16 pMM102: :TnS mutant plasmids studied, theISS0-L end of the transposon was distal to the BamHI siteand proximal to the EcoRI site. This nonrandomness of TnSfor insertion orientation has been noticed previously byothers (6).

Spontaneous Mcc- mutants from pMM102. In the courseof this work we noted that E. coli recA mutants transformedwith pMM102 were unable to grow on M63 minimal mediumsupplemented with ampicillin. Transformants were obtainedin LB medium but colonies were small and translucent. Thisphenotype was due to microcin production because pMM102Mcc- derivative plasmids could transform recA strains andthe resulting transformants grew normally in any media. Wetook advantage of this phenomenon to isolate a large collec-tion of mutants with impaired microcin activity. The screen-ing method was as follows. Individual RYC1000recA(pMM102) colonies on LB medium with ampicillin werestreaked onto the same medium and incubated overnight at37°C. The plates were then maintained at ambient tempera-ture for 2 days. At this time, opaque papillae appeared on thebackground mass of translucent colonies. These papillaewere streaked onto the same medium. After incubation at37°C, some colonies exhibited a normal morphology andwere able to grow well when restreaked on M63 mediumwith ampicillin. Further characterization showed that thesemutants were immune to microcin but did not producemicrocin. To check whether the mutants were due to amutation in the pMM102 plasmid rather than in the hostgenome, plasmid DNAs were extracted from 60 mutants andused to transform strain RYC1000. In every case except one,the Apr transformants were able to grow in M63 minimal

_

J. BACTERIOL.

I----_ - - _ _

,_ _ +

MICROCIN B17 GENETIC DETERMINANTS 279

PlasmidspMM102(Pmm)

Mcc7 Imm+ Mccknm^iA

.:Tn5 a U)40

a XI8N Inco m X XIIiI'

11 II1

PlasmidspM 102 IS(pSS)

I 2J2JU }k ! L!V-

'7 N' WNC'7 N (0COCD In q

---w.1-.4- .0. 044. 4-4.

lm

1 kb

Mcc Imm+

FIG. 3. Mapping of insertion mutations in the microcin B17 region. Location of Tn5, in the upper part, and of the insertion sequences, inthe lower part of the figure, are shown. Tn5 insertions marked with stars exhibit Mcc+ Imm+ phenotype. The numbers 81, 91, 2, 8, and 27designate IS] insertions and 18 designates an IS2 insertion; the rest of the numbers designate ISIO insertions. Horizontal arrows under theinsertion sequences indicate their relative orientation; direct orientation (0) is as has been indicated previously (11, 12, 19). Abbreviations:Ac, AccI; Av, AvaI; B, BamHI; Ba, BalI; Bg, BglII; E, EcoRl; Hc, HincII; Hd, HindIII; Hp, HpaI; P, PstI; S, Sall; Sm, SmaI; Sp, SphI.

medium and did not produce microcin, although they were

immune. Consequently, the mutations were indeed locatedin the plasmid. Twenty-one of these plasmid mutants were

chosen for physical analysis and compared with pMM102.Except for one plasmid (pSS32), which was smaller, all theothers were larger than pMM102. When digested with Sallall plasmids yielded a fragment of 5.0 kb, such as pMM102(the fragment containing the sequence from pBR322 and theright end of the microcin region), and a second fragment, thesize of which was different, depending on the mutant. Thefragment of 4.1 kb from pMM102, which contains themicrocin production region, was reduced to 3.45 kb inplasmid pSS32. A detailed restriction analysis showed thatthis plasmid had lost a 0.65-kb sequence at the left end of themicrocin region, including the AvaI (SmaI) site and theHindIll and AccI sites situated around it.

In all the other mutant plasmids the 4.1-kb Sall fragmenthad increased in size. In five cases (pSS2, pSS8, pSS27,pSS81, and pSS91) the size of the insertion was estimated tobe 0.8 kb; in all the others it was 1.30 to 1.35 kb. Furtherrestriction analysis showed that 0.8-kb inserts were cleavedby PstI and BalI, each at one site, and the digestion patternsof corresponding plasmids were those expected of the 768-bpinsertion element ISJ (19). The insertion in pSS18 hadunique cleavage sites for AvaI, HindlIl, and HpaI, and thesize of fragments generated with these enzymes in single or

double digests were those expected of IS2 (11). The other 14inserts were identified as ISJO, the inverted repetitive se-

quence of transposon TnJO, because they were cleaved in a

unique site by SphI and in another one by HinclI, with bothsites being separated by 348 bp (12). E. coli K-12 usuallycarries several copies of IsJ and IS2 but no copies of ISJO.The presence of ISIO in the strain used in these studies(RYC1000) can be explained by the fact that the insertionand subsequent deletion of TnJO was carried out during itsconstruction (J. Beckwith, personal communication). It isprobable that a copy of ISIO remained in the chromosome ofRYC1000 when TnJO was deleted (20).

Figure 3 shows the mapping and orientation of theseinsertion sequence elements in the microcin B17 region. Tothe left of the site of the Tn5 insertion in pMM206 (Mcc+Imm+) there are two IS] insertions which determine theMcc- phenotype. Therefore, it seems likely that the DNAregion between the BamHI site and this TnS insertioncontains genetic information which is essential for the pro-

duction of microcin. To obtain further evidence we cleavedpMM206 DNA with BamHI, religated the fragments, andtransformed pop3351, selecting for Apr. Of the transformantclones, 90% were Mcc- Imm+, and the remaining 10% wereMcc+ Imm+. Plasmid DNA from several clones of each typewas extracted and analyzed. It was found that, whereas theMcc+ plasmids contained two BamHI fragments of sizesidentical to those present in pMM206, the Mcc- plasmidslacked the small BamHI fragment which includes the rightarm of TnS and the terminal left region of the microcinfragment. In conclusion, DNA to the left of the TnS insertionin pMM206 is indeed required for microcin production, andit is most likely that this insertion is located in anintercistronic region.

DISCUSSION

The results reported here indicate that all of the informa-tion in the plasmid required to determine the microcin B17functions, microcin production and immunity, are in a5.1-kb, unique fragment of pMccBl7. All of the Mcc- Imm+insertion mutations map in a DNA stretch of about 3.3 kb onthe left side, suggesting that this region is specifically re-quired for microcin synthesis. As these mutations are lo-cated in many different sites spread along the stretch, thewhole sequence is probably involved in the production of theantibiotic. Such a sequence could encode as much as 125,000daltons of protein. As microcin B17 is only about 4,000daltons (unpublished data), this region must contain morethan one gene involved in microcin synthesis. An alternativepossibility is that microcin is coded by a gene which is partof an operon in which the microcin gene is distal to thepromoter. This last hypothesis seems to be very unlikely inview of all the results of this study.

All of the Imm- insertion mutations mapped in a 1-kbsegment located on the right side of the 5.1-kb fragment.This segment, which is required to express immunity, couldextend from the KpnI site close to the Sall site (Fig. 2), up tothe site of the Imm+ TnS insert in plasmid pMM216 (Fig. 3).All Imm- mutants are also Mcc-, which raises the questionof whether the immunity region plays a role in microcinsynthesis. Three possible hypotheses can be proposed toaccount for these mutants. The first one is that the immunityand synthesis regions constitute an operon in which thesynthesis region is distal relative to the promoter. Insertions

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280 SAN MILLAN ET AL.

in the immunity region would exert a polar effect, therebyseverely reducing or abolishing expression of the synthesisregion. The second one is that the immunity region includesa gene whose product is required in some way for microcinsynthesis. The third hypothesis is that the Mcc- Imm-plasmids are the result of two mutational events, the firstbeing the transposon insertion in the immunity region, andthe second being the mutation lying in the synthesis region.Results in favor of the last hypothesis were provided byphysical analysis of derivative plasmids pMM21 andpMM23. As already indicated, both plasmids contained aTn3 insertion in the immunity region and a second insertionof about 1.4 kb in the BamHI-SaiI fragment on the left. Wedetected no apparent physical changes in the microcinsynthesis region of the pMM102::TnS Mcc- Imm- deriva-tives, although our methods do not allow us to detect a basepair change or short DNA deletions or additions. Anotherresult supporting the third hypothesis was provided by theimpossibility of inverting the orientation of the 4.1-kb Sallfragment on pMM102. Given that the interruption of theDNA sequence in SalI leads to the loss of immunity, thefailure to obtain the derivative with the Sall fragmentinverted is easily explained if this fragment contains suf-ficient information to synthesize microcin. Cells receivingsuch a plasmid would be killed. Indeed, the fragment couldbe inverted when it carried a Mcc- mutation (plasmidpSS91). These data also provide an explanation for ourfailure to isolate an Mcc+ Imm- mutant. In conclusion, theBamHI-SalI fragment of 3.8 kb carries all of the informationrequired to produce active microcin B17.An evaluation of the level of immunity in Mcc- Imm+

mutants suggests that optimal expression of this functioninvolves DNA sequences located in the microcin synthesisregion. When RecA- cells harboring Mcc- Imm+ plasmidswere checked by cross-streaking for immunity, it was foundthat the degree of immunity was different, depending on themutant plasmid. So, whereas recA(pMM120) cells grewnormally even in the vicinity of the streak of producer cells,the growth of recA(pMM121) cells was inhibited in the zonenearest to the streak. An even more important inhibition wasobserved with recA(pMM122) and with all other derivativesdeleted for the terminal left end of the microcin region andwith all Mcc- Imm+ insertion derivatives of pMM102. Thesimplest explanation for these data is that regulatory se-quences located between the BamHI and SmaI sites can beused to express immunity to microcin B17. This does notexclude the possibility that the immunity region has its ownindependent regulatory signals. It should be emphasized thatthese differences in immunity were only observed in RecA-cells, which are highly sensitive to microcin (Herrero andMoreno, submitted for publication).

Plasmid pMM102 inhibits growth of recA(pMM102) cells.This inhibition is total in minimal medium in which microcinproduction is optimal. Microcin overproduction, however, isnot the cause of this inhibition because RecA- cells contain-ing a low-copy-number plasmid minireplicon of pMccB17, inwhich the 5.1-kb BamHI-EcoRI fragment has been recloned,also failed to grow in minimal medium (M. C. Garrido and F.Moreno, unpublished data). Because wild-type pMccB17 donot exhibit this phenomenon, it follows that pMM102 islacking genetic information which reduces or neutralizesmicrocin effect(s) when the RecA product is absent. Thisinformation must be related to microcin production becauseMcc- pMM102 mutant plasmids do not impair the growth ofrecA hosts. Experiments devoted to understanding thisquestion are under way in our laboratory.

ACKNOWLEDGMENTS

We are grateful to F. Baquero, A. P. Pugsley, and M. Schwartz foruseful discussion and critical reading of the manuscript and to S.Jimenez and J. Talavera for excellent technical assistance. We thankI. Saint-Girons for providing X467 and J. Beckwith and J. Brevet forbacterial strains.

This work was supported by grants from Fondo de Investiga-ciones Sanitarias de la Seguridad Social (Ministerio de Sanidad) andComisi6n Asesora de Investigaci6n Cientifica y Tecnica (Ministeriode Educaci6n) and by the Programa General de RelacionesCientfficas Hispano-Francesas (Ministere des Affaires EtrangeresFrance). J.L.S.M. was a recipient of a Caja de Ahorros de Madridfellowship.

LITERATURE CITED1. Asensio, C., J. C. Nrez-Dfaz, M. C. Martinez, and F. Baquero.

1976. A new family of low molecular weight antibiotics fromEnterobacteria. Biochem. Biophys. Res. Commun. 69:7-14.

2. Baquero, F., D. Bouanchaud, M. C. Martinez, and C.Fernindez. 1978. Microcin plasmids: a group of extrachro-mosomal elements coding for low-molecular-weight antibioticsin Escherichia coli. J. Bacteriol. 135:342-347.

3. Baquero, F., and F. Moreno. 1984. The microcins. FEMS Lett.23:117-124.

4. Baquero, F., F. SAnchez, V. Rubio, and A. Tenorio. 1981.Genetical bases of microcin classification, p. 578. In S. B. Levy,R. C. Clowes, and E. L. Koenig (ed.), Molecular biology,pathogenicity, and ecology of bacterial plasmids. Plenum Pub-lishing Corp., New York.

5. Berg, D. 1977. Insertion and excision of the transposablekanamycin resistance determinant Tn5, p. 205-212. In A. I.Bukhari, J. A. Shapiro, and S. L. Adhya (ed.), DNA insertionelements, plasmids, and episomes. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

6. Berg, D. E., and C. M. Berg. 1983. The prokaryotic transposableelement Tn5. Biotechnology 1:417435.

7. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L.Heyneker, and H. B. Boyer. 1977. Construction and characteri-zation of new cloning vehicles II. A multipurpose cloningsystem. Gene 2:95-113.

8. CIewell, D. B., and D. R. Helinski. 1969. Super-coiled circularDNA-protein complex in Escherichia coli: purification andinduced conversion to an open circular form. Proc. Natl. Acad.Sci. U.S.A. 62:1159-1166.

9. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation incalcium-chloride improves the competence of E. coli cells. Gene6:23-28.

10. Genifloud, O., M. C. Garrido, and F. Moreno. 1984. Thetransposon TnS carries a bleomycin-resistance determinant.Gene 32:225-232.

11. Ghosal, D., H. Sommer, and H. Saedler. 1979. Nucleotidesequence of the transposable DNA-element IS2. Nucleic AcidsRes. 6:1111-1122.

12. Halling, S. M., R. W. Simons, J. C. Way, R. B. Walsh, and N.Kleckner. 1982. DNA sequence organization of TnlO's ISIO-right and comparison with IS1O-left. Proc. Natl. Acad. Sci.U.S.A. 79:2608-2612.

13. Hernindez-Chico, C., M. Herrero, M. Rejas, J. L. San Millan,and F. Moreno. 1982. Gene ompR and regulation of microcin 17and colicin E2 syntheses. J. Bacteriol. 152:897-900.

14. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. Arestriction map of TnS and location of a region encodingneomycin resistance. Mol. Gen. Genet. 177:65-72.

15. Kretschmer, P. J., and S. N. Cohen. 1977. Selected translocationof plasmid genes: frequency and regional specificity oftranslocation of the Tn3 element. J. Bacteriol. 130:888-889.

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

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

18. Nisioka, T., M. Mitani, and R. C. Clowes. 1970. Molecular

J. BACTERIOL.

MICROCIN B17 GENETIC DETERMINANTS

recombination between R-factor deoxyribonucleic acid mol-ecules in Escherichia coli host cells. J. Bacteriol. 103:166-177.

19. Ohtsubo, H., and E. Ohtsubo. 1978. Nucleotide sequence of aninsertion element, ISL. Proc. Natl. Acad. Sci. U.S.A.75:615-619.

20. Ross, D. G., J. Swan, and N. Kleckner. 1979. Structures ofTnO-promoted deletions and inversions: role of 1400 bp in-verted repititions. Cell 16:721-731.

21. Sanger, F., A. R. Coulson, T. Friedmann, G. M. Air, B. G.Barrell, N. L. Brown, J. C. Fiddes, C. A. Hutchi$on III, P. M.Slocombe, and M. Smith. 1978. The nucleotide sequence ofbacteriophage 4~X174. J. Mol. Biol. 125:225-246.

22. Sanger, F., A. ,R. Coulson, G. F. Hong, D. V. Hill, and G. B.PeterSen. 1982. Nucleotide sequence of bacteriophage lambdaDNA. J. Mol. Biol. 162:729-773.

23. Sutcliffe, J. G. 1978. Complete nucleotide sequence of theEscherichia coli plasmid pBR322. Cold Spring Harbor Symp.Quant. Biol. 43:77-90.

24. Tanaka, T., and B. Weisblum. 1975. Construction of a colicinE1-R factor composite plasmid in vitro: means for amplificationof deoxyribonucleic acid. J. Bacteriol. 121:352-362.

25. Twigg, A. J., and D. Sherratt. 1980. Trans-com,plementablecopy-number mutans of plasmid ColEl. Nature (London)283:216-218.

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