Vol. 41, No. 1INFECTION AND IMMUNITY, July 1983, P. 54-600019-9567/83/070054-07$02.00/0Copyright © 1983, American Society for Microbiology

Cloning and Analysis of the Kl Capsule Biosynthesis Genes ofEscherichia coli: Lack of Homology with Neisseria

meningitidis Group B DNA SequencesCHRISTINE ECHARTI,'t BERNARD HIRSCHEL 2t* GRAHAM J. BOULNOIS 2 JENNIFER M.

VARLEY,2 FRANCIS WALDVOGEL,3 AND KENNETH N. TIMMIS"2

Max-Planck Institute for Molecular Genetics, Berlin-Dahlem, West Germany1; and Department of MedicalBiochemistry2 and Division of Infectious Diseases,3 University of Geneva, Geneva 4, Switzerland

Received 28 December 1982/Accepted 11 April 1983

Genes coding for production of the Kl polysaccharide capsule of Escherichiacoli have been cloned. Complementation, insertion, and deletion analyses wereused to localize the Kl genes and demonstrated that a minimum of 9 kilobases ofDNA split into at least two gene blocks is involved in synthesis and assembly ofthe capsule. One of the gene blocks is responsible for biosynthesis of thepolysaccharide, and the other is responsible for extracellular appearance ofcapsular material. Using cloned Kl genes as probes in Southern blot experiments,we detected homology to DNA from strains of E. coli capsular types K92, K7, andK100. In contrast, no homology was apparent between Kl genes and DNA frommeningococcus group B, although the Kl and group B capsules are chemicallyand immunologically identical.

Bacterial meningitis is one of the most seriousinfectious diseases that attack healthy people.Despite antibiotic treatment of meningitis infec-tions, case fatality rates are above 10%, andneurological sequelae are common. Among themany bacteria populating the human body andenvironment, only a few are capable of causingmeningitis; Streptococcus pneumoniae, Neis-seria meningitidis, and Hemophilus influenzaeaccount for the majority of cases (17). Thesespecies presumably possess important virulencefactors absent from bacteria that do not causethe disease.

Bacterial polysaccharide capsules have longbeen suspected to play a critical role in theability of such bacteria to invade and proliferatein the central nervous system (for reviews, seereferences 20 and 26), since all strains causingmeningitis have a capsule, whereas many close-ly related avirulent strains do not.

Meningitis in newborns differs from meningi-tis in individuals of other age groups. The caus-ative organism is often Escherichia coli, which,in 80% of cases, produces the Kl polysaccharidecapsule. Production of this capsule is considera-bly less prevalent in E. coli strains isolated fromblood of adults with septicemia or from the fecesof normal persons, where only 10 to 20% are Kl

t Current address: Bundesgesundheitsamt, West Berlin,West Germany.

t Permanent address: Division of Infectious Diseases, Uni-versity of Geneva, Geneva, Switzerland.

positive (for review, see reference 30). In addi-tion to epidemiological evidence, results fromexperimental infections implicate the K1 capsulein pathogenesis. In mice experimentally infectedwith E. coli Kl+ strains, the fatality rate wasproportional to the concentration and persis-tence of capsular material in the cerebrospinalfluid (18). Moreover, comparison of Kl + or Kl -strains of E. coli that were otherwise isogenicrevealed that production of the Kl capsule bybacteria was correlated with a low 50% lethaldose for mice and an increased resistance tophagocytosis (2) and killing by serum (7, 29).Furthermore, the capsule was found to diminishcomplement-mediated opsonization (12), an ac-tivity in cerebrospinal fluid that seems to be ofprognostic importance in meningitis (31).The Kl capsular polysaccharide, a homopoly-

mer of ox-2-8-linked, N-acetyl neuraminic acid, isantigenically and chemically identical to thepolysaccharide of N. meningitis group B, theorganism responsible for more than 80% ofmeningococcal disease in developed countries;it is very probable, therefore, that the twocapsules have the same functional role in thevirulence of these two unrelated bacteria.

In this paper, we report the cloning of the E.coli Kl capsular biosynthesis genes in E. coli K-12, their localization by insertion mutagenesis,and their use to search for homologous se-quences in DNA from other capsule-producingstrains of E. coli and N. meningitidis. Homologyamong plasmids carrying Kl genes and DNA

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CAPSULE GENES OF E. COLI Kl AND N. MENINGITIDIS 55

from E. coli producing K7, K92, and K100capsules was readily apparent, whereas no sig-nificant homology was detected with N. menin-gitidis DNA.

MATERIALS AND METHODS

Bacterial strains, bacteriophages, and plasmids. TheKi antigen-producing, wild-type strain E. coliBi7509/41, serotype 07:K1:H- (19), served as asource for the Kl antigen biosynthesis genes. Strain E.coli U9/41, serotype 02:K1 :H4, was used to propagateKl-specific bacteriophages A, B, C, D, and E (8). Kantigen test strains Pus 3432/41 (07:K7:H4), C 1407-67(073:K92:H34), and F147 (075:K100:H5) were kindlyprovided by F. 0rskov. E. coli K-12 strain LE392 (F-hsdRSJ4 (rk- mk ) supE44 supF58, lac, galK2,galT22, metBi, trpR55) was obtained from J. Collins.E. coli K-12 C600::Tn5 has been described previously(6). Strains of N. meningitidis representing serogroupsB and C and isolated from patients with meningitis,were a gift from J.-J. Picq. Cosmid pHC79 (4) wasused as the plasmid cloning vector.

Media. E. coli strains were grown in L-broth or M9medium supplemented with 0.5% Casamino Acids(Difco Laboratories). Where appropriate, antibioticswere added at the following concentrations: ampicil-lin, 50 ,jig/ml; kanamycin, 20 ,ug/ml; and tetracycline,10 ,ug/ml: Trypticase soy broth agar contained Trypti-case soy broth (BBL Microbiology Systems) and 0.5%agarose. N. meningitidis strains were grown on choco-late agar plates in 5% CO2. For DNA extraction about100 colonies were washed off the plates with phos-phate-buffered saline.DNA isolation and cloning and analysis of plasmid

molecules. Chromosomal DNA was extracted as de-scribed by Saito and Miura (24), and plasmid DNAwas prepared by the method of Timmis et al. (28).Reaction conditions for restriction endonucleasecleavage, ligation, and agarose gel electrophoresishave been described previously (1). The cloning of theKl biosynthesis genes was achieved by generating agene bank of Bi7509/41 DNA sequences in E. coliLE392 by using cosmid pHC79 in conjunction with theprocedure for in vitro packaging of ligated DNA intophage particles (4, 10).

Plasmid contour lengths were determined by elec-tron microscopy (16, 22). At least 30 molecules werephotographed and measured, and their sizes werecomputed by comparison with those of pBR322.

Restriction endonuclease cleavage maps were pro-duced by standard procedures and by BAL31 exonu-clease mapping (15).

Sensitivity of Kl+ bacteria to Kl-specific bacterio-phages. Samples (0.1 ml) of appropriate phage dilu-tions were mixed with 0.1 ml of the bacterial culture,incubated at 37°C for 15 min (preadsorption), mixedwith soft agar, and overlaid on solid agar plates.Plaques in the bacterial lawn were apparent afterovernight incubation of plates at 37°C.

Nick translation and DNA-DNA hybridization. DNAfragments in agarose gels were denatured in situ andtransferred to nitrocellulose filters as described bySouthern (27). Nick translation of plasmid DNA, hy-bridization, and washing of filters was performed asdescribed by Jeffreys and Flavell (13).

RESULTS

Cloning of the Kl biosynthesis genes in cosmidpHC79. A gene bank of the E. coli Kl+ strainBi7509/41 was established in E. coli LE392 bythe cosmid cloning system of Collins and Hohn(4, 10). Plasmid pHC79 was completely cleavedand chromosomal DNA was partially cleavedwith BamHI endonuclease. The two DNAs inequal amounts and at high concentrations (great-er than 150 ,ug/ml) were mixed, ligated, andpackaged into phage heads. The phage particlescontaining plasmid and chromosomal DNA werethen used to infect LE392 bacteria. Ampicillin-resistant (Ap') infectants were selected andtransferred to Trypticase soy broth agar platessupplemented with 5% horse serum and contain-ing antibodies against the cross-reacting N. men-ingitidis group B capsular antigen (generouslyprovided by J. B. Robbins). Three Kl antigen-producing clones (containing plasmids pKT169,pKT171, and pKT172) were identified by theappearance of precipitation haloes around thecolonies (21). The Kl+ clones were tested forsensitivity to several Kl-specific bacteriophages(Table 1). Surprisingly, reaction with the phageswas variable. Only one clone (containingpKT172) was sensitive to all phages, whereasthe others were sensitive to only one or two. AllKl + clones of LE392 gave titers of phage stocksthat were considerably higher than those ob-tained with Bi7509/41. Hybrid plasmid pKT172was selected for further study.

Characterization of the pKT172 plasmid. Thesize of pKT172 was determined by summing thesizes of restriction endonuclease-generated

TABLE 1. Sensitivity of clones producing Kl capsular material to Kl-specific phagesTiters of bacteriophages (PFU/mI)

E. coli strain40K1A K1B +K1C 4K1D +K1E

Bi7509/41 3.6 x 108 8 x 108 3.4 x 107 1.15 x 1010 1.2 x 109LE392 oa 0 0 0 0LE392(pKT169) 0 1.32 x 1010 0 0 0LE392(pKT171) 0 1.03 x 1010 8.3 x 108 0 0LE392(pKT172) 1.5 x 1010 2.2 x 1010 2.8 x 109 4.3 x 10'° 1.8 x 101

a No plaques were observed.

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56 ECHARTI ET AL.

DNA fragments separated by agarose gel elec-trophoresis and by contour length measure-ments. Both methods indicated a size of about42 kilobases (kb).A restriction endonuclease cleavage map of

pKT172 was obtained by BAL31 exonucleasemapping of the plasmid after its linearization bycleavage with the SstI endonuclease (Fig. 1).After inactivation of the exonuclease withEDTA, the DNA was digested with BamHI,HindIII, PstI, EcoRI, or Sall. Cleavage sitesnearest the SstI site were lost first, allowingcleavage maps for the relevant enzymes to beconstructed (15). The positions of sites wereconfirmed by double digestion with variouscombinations of enzymes.

Localization of the Kl genes by insertion anddeletion analysis. Mutagenesis of plasmidpKT172 by transposon TnS (which specifiesresistance to kanamycin [Kmrj) was performedas follows. The plasmid was introduced into E.coli strain C600::TnS by transformation, and 24transformant clones that were initially Kl + wereseparately propagated for about 100 generations.Plasmid DNA was prepared from each of theseclones and used to transform E. coli LE392. Of31 kanamycin-resistant (i.e., containing mutantplasmid) clones obtained, 20 were Kl- and 11were Kl . Restriction endonuclease analysisdemonstrated that Tn5 insertions causing a Kl -phenotype were clustered in a DNA segment ofapproximately 2.7 kb within fragment E3 ofpKT172 (Fig. 2). This region is therefore essen-tial for production of Kl capsule, although, asthe results described below show, it is notsufficient.

;fR ...-'t ._

BE&~...........

,t:

To delete cloned DNA segments not encodingKi production, pKT273, a TnS-containing deriv-ative of pKT172 that continues to direct thesynthesis of Kl capsular material (Fig. 2) waspartially digested with EcoRI, religated, andused to transform LE392. Since Tn5 contains noEcoRI cleavage sites (14), transformant clonesharboring plasmids containing the E3::TnS frag-ment could be directly selected by their Kmrphenotype. Of 93 Kmr transformants examined,64 produced the Kl capsular polysaccharide.The smallest plasmid coding for Kl capsulebiosynthesis, pKT274, consisted of the follow-ing three EcoRI fragments: El, which containsthe cloning vector; E3::TnS; and either E5a orE5b (Fig. 3a). The latter two EcoRI fragments,which bracket the E3 fragment of pKT172 (Fig.2) could be distinguished by digestion ofpKT274with EcoRI and either HindIII or PstI. Since thefragment in question was cleaved by HindIII,but not by PstI, we concluded that pKT274contains E5b, which in the original plasmidpKT172 was located between El and E3. Thus,EcoRI fragments E2, E4, E5a, E7, and E8 arenot required for the production of Kl antigen.To obtain the smallest recombinant plasmid

capable of directing Kl capsule production, sub-clones of pKT274 were constructed. A fragmentextending from the BamHI site of the TnS ele-ment to the BamHI site at coordinate 8.6 kb ofpKT274 (Fig. 3b) was cloned in both orienta-tions in the vector plasmid pACYCl84 (3) toyield pGB12 and pGB13. Although this fragmentspans the region identified by TnS insertions asbeing essential for capsule production, bothpGB12 and pGB13 failed to direct the produc-

FIG. 1. SstI-BamHI digestion patterns of pKT172 and localization of cleavage sites by BAL31exonuclease mapping. Plasmid pKT172 DNA was linearized with SstI endonuclease and then incubated with theexonuclease BAL31. At the times indicated, samples were withdrawn, and the digestion was terminated by theaddition of an excess of EDTA. The DNA was then cleaved with BamHI. Fragments bracketing the SstI site arethe first to decrease in length, followed by adjacent fragments, etc.

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270;7~~~~~~~~~~7

FIG. 2. Cleavage and TnS insertion map of pKT172. Restriction fragments are labeled according to size.There are two small EcoRI fragments (E7 and E8) located between E4 and E2 that are not indicated. Opentriangles indicate the positions of TnS insertions that prevent Ki antigen production, and filled triangles signifyinsertions that have no influence on Ki production. In the enlarged part of the diagram the HindIII site is markedby an arrow.

tion of capsular material. Kl genes not carriedby pGB12 or pGB13 were localized in pKT274by exploiting pGB11, an Apr Kms derivative ofpKT274 composed of only the large EcoRI frag-ment and constructed by cleavage of pKT274with EcoRI and circularization of the productsby the addition of ligase. When pGB11 wasintroduced into bacteria already harboringpGB12 or pGB13, Kl capsule production wasrestored, which showed that the large EcoRIfragment of pKT274 carries genes essential forcapsule production. This conclusion was con-firmed by the properties of deletion derivativesof pKT274 that were constructed by partialcleavage of pKT274 with BamHI and circular-ization of the products (pGB14 and 15) (Fig. 3b).

Clearly the Tn5 insertions described above donot define all cistrons involved in Kl produc-tion. In an attempt to locate these more precise-ly, further mutants were generated by insertionof the gamma-delta insertion sequence intopKT274. This involved a conjugation experi-ment with an F+ strain carrying pKT274 as thedonor and LE392 as the recipient. Selection oftranscipients that inherited the drug resistancemarkers of pKT274 identified derivatives of this

plasmid carrying the gamma-delta sequence of F(9). The effect of such insertions on capsuleproduction was determined in a number (about150) of derivatives, and the sites of insertionwere mapped by cleavage of partially purifiedplasmid DNA preparations (11) with either Sall,HindIII, or BamHI. Figure 3b shows the loca-tions of gamma-delta insertions in representativeplasmids. The majority of the insertions (51%)fell in the interval between the TnS element andthe EcoRI site at coordinate 14.0 kb (Fig. 3b).All insertions between coordinate 8.3 kb andTnS (Fig. 3b) prevented capsule production andthereby defined a 9-kb region involved in cap-sule biosynthesis. Few gamma-delta insertionswere found in the segment of pKT274 locatedbetween coordinates 0 and 8.0 kb. The reasonfor the very asymmetrical distribution of inser-tion sites in pKT274 and pKT172 is presentlyunclear. Bacteria harboring pGB14 and pGB15failed to produce precipitation haloes in agarplates containing Kl antiserum. However, cap-sular material was detected in an Ouchterlonyassay when these bacteria were lysed by twocycles of freeze-thawing.

Hybridization of cloned Kl genes to DNA from

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E. coli and N. meningitidis. The capsules of E.coli Ki and N. meningitidis type B are chemical-ly and immunologically identical (for review, seereference 23). The possibility of homology be-tween the genes of the capsule biosynthesisenzymes in these two organisms and in E. coliproducing different capsules was therefore ex-amined by DNA-DNA hybridization as detailedin the legend to Fig. 4. As described above,pKT274 is the smallest plasmid constructedwhich codes for production of the Kl capsules.It is a convenient probe for sequences related tothe E. coli Kl genes.The probe hybridized to the expected DNA

fragments from E. coli Bi7509/41, the strain fromwhich the Kl genes were cloned (Fig. 4, lanes 4and 9). However, no homology betweenpKT274 and N. meningitidis DNA was apparent(Fig. 4, lanes 6 through 8), even when washingconditions of low stringency, allowing about18% mismatch (1 x SSC [0.15 M NaCl plus 0.015M sodium citrate], 65°C; data not shown; 13),were employed.

Interestingly, little homology was detectedbetween pKT274 and chromosomal DNA ofLE392 (Fig. 4, lane 3), which suggests that thisE. coli K-12 derivative does not contain any ofthe DNA (25 kb) from strain Bi7509/41 that ispresent in pKT274. In contrast, considerablehomology was observed between pKT274 andDNA from E. coli isolates that produce the K7,K92, or K100 capsules (Fig. 4, lanes 10 through12). In the case of DNA from the K92 strain,homology with fragments corresponding in size

to E3 and E5b of pKT172 was demonstrated(compare lanes 2 and 11 in Fig. 4). The DNA ofthe K7 and K100 strains, however, exhibitedhomology in fragments that did not correspondin size to fragments of pKT172.

DISCUSSIONA recombinant plasmid, pKT172, containing a

DNA segment about 36 kb in length from E. coliKl, has been constructed and shown to conferupon E. coli K-12 the ability to produce the Klpolysaccharide. That this material is organizedinto a capsule is suggested by the sensitivity ofbacteria harboring pKT172 to phages that specif-ically absorb to the Kl capsule (Table 1). Theproperties of subclones and deletion and inser-tion derivatives of pKT274 show that a 15-kbregion of the plasmid encodes functions for thebiosynthesis and assembly of the capsular mate-rial. This region is divided into at least two geneclusters.One cluster appears to be a contiguous seg-

ment of DNA extending from coordinates 8.3 to18 kb (Fig. 3b) that may encode enzymes for thebiosynthesis and polymerization of N-acetylneuraminic acid. Mutations in this segment pre-vent production of Kl polysaccharide. In con-trast, some mutations between coordinates 0and 9 kb prevent extracellular appearance of Klcapsular material. However, accumulation ofKi polysaccharide was detected after cell lysis.These mutations may therefore interfere withfunctions needed to transport the polysaccha-ride to the cell surface and organize it into a

10

B H

-- 1*i..i .JjJ. .YT -

-- ----1 r ---

H-- K1.

bFIG. 3. (a) Elimination of EcoRI fragments of plasmid pKT273 nonessential for Kl antigen production.

Plasmid pKT273, a Tn5 insertion derivative of pKT172 (see Fig. 2) and pKT274 were cleaved with EcoRI, andthe fragments were separated by electrophoresis in 0.8% agarose, pKT274 was the smallest plasmid isolated thatwould direct synthesis of the Kl capsule. (b) Mapping of regions essential for Kl antigen production: y-8insertion analysis. The drawing represents a linearized map of pKT274. Insertions which abolished Klproduction are indicated by open circles, and insertions that did not are indicated by filled circles. PlasmidspGB11, -12, -13, -14, and -15 contain the DNA segments delineated by the solid lines.

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r-

_

..j~ ~ .r

1 2 3 4 5

v ..

6 7

8.

8 9 10 11 12

FIG. 4. Southern blot of chromosomal DNAs of strains of E. coli and N. meningitidis with pKT274 as a

probe. E. ccli Kl (lanes 4 and 9) is Bi7509I41; E. coli Kl1' (lane 5) is another isolate obtained from a patient in

Germany. E. coli K7, K92, and K100 are clinical isolates produci'ng the corresponding capsules. Chromosomal

DNAs were digested by EcoRI, electrophoresed in 0.8% agarose, transferred to nitrocellulose, and hybridizedwith -radioactive pKT274 DNA.

capsule. Alternatively, they may reduce theamount of Kl capsular material to a level onlydetectable in the Ouchterlony test, which ismuch more sensitive than immunoprecipitation.

Silver et al. constructed a plasmid pSR23 (25)that also codes for biosynthesis of the Kl cap-sule. Plasmid pSR23 consists of four BamHIfragments; whereas subclones carrying the sepa-rate fragments failed to direct the synthesis ofthe Kl capsule, those carrying the largest frag-ment (23 kb) did cause host cells to producesialyl transferase (25). The largest BamHI frag-ments of pKT172 and pSR23 are of similar size,and the former carries the 9-kb Kl gene clusterinvolved in polysaccharide biosynthesis (Fig. 2and 3b).A derivative of pKT172 that is deleted of

EcoRI fragments not involved in capsule pro-duction has been constructed. About one-half ofthis derivative, pKT274, is required for capsuleproduction, and hence the plasmid is a conve-nient probe for examination of the relatedness ofKl genes to other capsule biosynthesis genes.Homology was found between this plasmid andDNA from E. coli strains producing differentcapsules (Fig. 4). In the case of an E. coli strainproducing the K92 capsule, DNA fragmentshomologous to the probe were found to be ofsimilar size to those carrying genes for Klbiosynthesis (namely, E3 and E5b), suggestingthat at least some of the sequences coding forcapsule production in strains of E. coli produc-ing the Kl and K92 capsules may be similar.This presumably reflects the fact that the K92polysaccharide, like Kl, is a polymer of N-

acetyl neuraminic acid (5). Although the K7 andK100 capsules are not composed of N-acetylneuraminic acid, homology between the geno-mic DNA of strains producing these capsulesand the probe carrying Kl genes was detected,which suggests some relatedness of these cap-sule genes.Although the capsules of E. coli Kl and N.

meningitidis group B are chemically and immu-nologically identical, there appears to be little orno homology in their capsular biosynthesisgenes. Studies are currently in progress to deter-mine whether the difference in polynucleotidesequence of these genes is reflected in theirproducts.

ACKNOWLEDGMENTS

We are indebted to J. B. Robbins for gifts of N. meningitidisB antiserum, B. Rowe for providing Kl-specific phages. J.-J.Picq for providing strains of meningococci, and Bayer-Lever-kusen, for gifts of ampicillin (Binotal), and we are grateful toM. Rekik for excellent technical assistance and to F. Rey andJ. Timmis for typing the manuscript.

This study was supported in part by the Swiss NationalResearch Foundation. G.J.B. and J.M.V. are postdoctoralfellows of the European Molecular Biology Organisation andthe Royal Society, respectively, whose support is gratefullyacknowledged.

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