PLASMID
27,207-2 19 ( 1992)
Construction
of a Sequenced Clostridium perfringensEscherichia co/i Shuttle Plasmid
JOANSLOAN,TRACYA. WARNER,PAUL T. SCOTT,TRUDIL. BANNAM, DAVID I. BERRYMAN, AND JULIAN I. ROOD’ Department of Microbiology, Monash University, Clayton 3168. Australia Received September 19, 1991; revised November 25, 1991 A new Clostridium perjkingens-Escherichia co/i shuttle plasmid has been constructed and its complete DNA sequencecompiled. The vector, pJIR4 18, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and 2uc.Zgene from pUCl8 are also present, which means that X-gal screening can be used to select recombinanta in E. coli. Roth chloramphenicol and erythromycin resistance can be selected in C. perjringens and E. coli since pJIR4 18 carries the C. perjiiingens catP and ermBP genes. Insertional inactivation of either the cutP or errnBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR4 18 and its applicability for the cloning oftoxin genesfrom C. perfn’ngenshave been demonstrated by the manipulation ofa cloned gene encoding the production-of phospholipase C. Q 1992 Academic Press, Inc.
In recent years our knowledge of the genetics of the strict anaerobe Clostridium perfiingens has expanded rapidly. These develop merits have primarily been due to the cloning and molecular analysis of antibiotic resistance genes (Abraham and Rood, 1985a, 1987; Abraham et al., 1985; Berryman and Rood, 1989; Rood er a/., 1989), the discovery of methods by which C. perj?ingens cells can be transformed with plasmid DNA (Heefner et al., 1984; Allen and Blaschek, 1988; Scott and Rood, 1989), and the elucidation of a complete genetic and physical map of the C. perfringens genome (Canard and Cole, 1989). Several shuttle plasmids capable of independent replication in both C. perfringens and Escherichia coli have been constructed. These vectors include three plasmids [pJU 12, pJUl3, and pJU16 (Squires et al., 1984)] based on the cryptic C. perfringens plasmids pJUl2 1 and pJU122 and the C. perfringens tetracycline resistance gene, tet(P) (Abraham and Rood, 1985a; Abraham et al., 1988); ’ To whom correspondence should be addressed.
pHR106 (Roberts et al., 1988), which incorporates pJU 122 and the C. perfringens chloramphenicol resistance gene, catP (Abraham et al., 1985); pAK201, which has the catP gene and the caseinase plasmid pHBl0 1 (Rim and Blaschek, 1989); pSB92A2, which has the catP gene and the cryptic plasmid pCP1 (Phillips-Jones, 1990); and pTG67, which incorporates a Staphylococcus aureus cat gene and the bacteriocinogenic plasmid pIP404 (Garnier and Cole, 1988b). The most widely used shuttle vector is pHR106 (Allen and Blaschek, 1988; Mahony et al., 1988; Scott and Rood, 1989). None of these plasmids are ideal for use in studies on the pathogenesis of C. perjiingens infections. Since there are no reports in the literature of naturally occurring strains of C. perfringens which produce B-lactamase (Williamson, 1983), it is not desirable to use vector plasmids containing the E. coli p-lactamasegene (Ma) for the cloning and manipulation of genes encoding extracellular toxins and enzymes. All of the current vector plasmids, except pAK201, carry this gene. The presence of a gene for a putative virulence factor, namely, a caseinase, makes pAK201
201
0147-619X/92 $5.00 Copyright 0 1992 by Academic Press, Inc All rights of reproduction m any form reserved.
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SLOAN
potentially unsuitable for biosafety reasons even though the gene is apparently not expressedin strains carrying this plasmid (Rim and Blaschek, 1989). None of the existing shuttle plasmids have been sequenced, utilize X-gal screening in E. coli, or have a complete multiple cloning site. With the exception of pTG67, these vectors utilized poorly studied C. perfiingens plasmids whose replication mechanisms have not been elucidated. In this paper we describe the construction, sequenceanalysis, and utilization of a new C. perfringens-E. coli shuttle plasmid, pJIR4 18. This plasmid has the replication region from pIP404; the C. perfringens catP and erythromycin resistance (ermBP) genes; the replication region, 1acZ’gene, and multiple cloning site from pUC 18; and does not have an intact bla gene.
ET AL.
Perron et al., 1985) was used as the source of the E. coli replication region, the la&Z gene, and the multiple cloning site. The recombinant plasmids pJIR62 (Abraham et al., 1985), pJIR71 (Abraham et al., 1988), and pJIR229 (Berryman and Rood, 1989) were used as the sources of the C. perfringens catP, tet(P), and ermBP genes, respectively. The plasmid pJIR 145 was a Tnl725-insertion derivative of pJIR7 1 (Abraham et al., 1988). Determination of Phospholipase C Activity
The C. perfringens strains were grown overnight in 20 ml of TPG broth containing the appropriate antibiotics. The supematant fluid was collected by centrifugation, freezedried, and resuspended in one-tenth volume of phosphate-buffered saline. Phospholipase C activity was determined using an egg yolk agar plate method (Stevens et al., 1987). SamMATERIALS AND METHODS ples (20 ~1)of concentrated supernatant fluid Strains, Plasmids, and Media were placed into 3-mm wells that had been punched into plates containing 20 ml of egg All C. perfringens strains were derivatives yolk agar. The width of the zone of precipitaof strain I3 (Mahony and Moore, 1976), extion was measured after overnight incubation cept that pIP404 was derived from CPNSO at 37°C and converted to units of enzyme (Gamier and Cole, 1988b). The plc mutant, activity by reference, on each standardized JIR367, was isolated by treatment of strain plate, to wells containing known amounts of 13 with nitrosoguanidine (Rood and WilkinType 1 phospholipase C (Sigma). The level of son, 1975) followed by screening on egg yolk enzyme activity was directly proportional to medium, which consisted of 4% (v/v) egg enzyme concentration. Protein was meayolk in nutrient agar. All E. coli strains were sured by the Bradford (1976) method using derivatives of DHSa (BethesdaResearchLabbovine serum albumin as the standard. oratories) or MC1022 (Casadaban and Cohen, 1980). C. perfringens strains were grown Plasmid Isolation and Analysis in fluid thioglycollate broth (Difco LaboratoBoth large (Abraham and Rood, 1985b) ries), TPG broth (Rokos et al., 1978) nutrient broth, or nutrient agar (Rood, 1983). and small (Roberts et al., 1986) scale plasmid C. perfingens cultures on agar media were preparations from C, perfringens cells were as incubated in anaerobic jars in an atmosphere previously described. Plasmid DNA was preof 10% Hz and 10% CO2 in Nz. Antibiotics pared from E. culi cells essentially as previwere used at the following concentrations: ously described (Bimboim and Doly, 1979; chloramphenicol (30 &ml), erythromycin Holmes and Quigley, 1981). Restriction en(25 pg/ml), and tetracycline (5 pg/ml). E. coii donucleases and other enzymes were from strains were grown aerobically in 2 YT Boehringer, Bethesda Research Laboratories, (Miller, 1972) supplemented with chloram- New England Biolabs, or Promega and were phenicol (30 pg/ml), erythromycin (50 pg/ used in accordance with the manufacturer’s ml), or tetracycline (10 &ml) where appro- directions. Transformation of C. perfringens priate. The cloning vector pUCl8 (Yanisch- strain 13 was by electroporation (Scott and
C. perfrigens-E.
co/i SHUTTLE PLASMIDS
Rood, 1989)and utilized a Gene Pulser(BioRad). Unless otherwise stated, molecular techniques were as described previously (Maniatis et al., 1982). DNA Sequencing
DNA sequenceanalysiswas carried out by the dideoxy chain termination method (Sanger et al., 1977) using plasmid DNA templatesand T7 DNA polymerasesequencing kits (Pharmacia). Oligonucleotide primers were synthesizedon an Applied Biosysterns apparatus. Sequence data were analyzed and manipulated using the MELBDBSYS program developed at the Walter and Eliza Hall Institute, the Ludwig Institute for Cancer Research,and the Howard Florey Institute (Melbourne, Australia). The GenBank accessionnumber of the complete pJIR418 sequenceis M77 169. RESULTS
Construction of the Shuttle Plasmid pJIR297
Our overall approach to the construction of a new shuttle plasmid was to start with the E. coli vector pUCl8, to delete the blu gene, to add various combinations of C. perjikgens resistancegenes,to add a C. perfringens plasmid replication region, and then to refine the plasmid by eliminating duplicated restriction sites.By using genesand plasmidswhich had been completely sequenced,we aimed to construct a shuttle plasmid whose entire sequence was known. A 1.1-kb EcoRI-DraI pUCl8 fragment which contains the replication region, but not the bla gene, was ligated to a 1.6-kb PvuII-EcoRI pJIR62 fragment which contains the C. perfringens catP gene (Fig. 1). E. co/i recombinants that were resistant to chloramphenicol were selected and were screenedfor susceptibility to ampicillin. The next step was to add the C. per$+ingenstet(P) gene from pJIR71. BecausepJIR71 has several EcoRI sites, one of which is located within tet(P), it wasdecidedto add this determinant in stages.Ligation of EcoRI-KpnI
209
fragments from pJIR274 and pJIR71 was used to construct pJIR275, which encodes chloramphenicol resistanceand carries part of tet(P) (Fig. 1). The remainder of this gene was added to pJIR275 by using an EcuRI fragment from pJIR145, a Tnl725 insertion derivative of pJIR7 1. The advantageof using the tetracycline resistanceplasmid pJIR 145 was that we were able to make use of the EcoRI site which is located at the end of the transposon, the insertion site of which had previously been mappedto within closeproximity of an SphI site located within tet(P) (Abraham et al., 1988).The appropriate 1-kb EcoRI fragment from pJIR145 was isolated from a gel and ligated with EcoRI-digested pJIR275. Selection was for both chloramphenicol and tetracycline resistance. The plasmid constructed in this experiment was pJIR282 (Fig. 1). In subsequentexperiments where we attempted to further modify and manipulate pJIR282 it was observedthat the transformation frequency obtained with this plasmid was significantly lower than frequencies obtained with pUC18, pJIR274, or pJIR275. It was assumed that the plasmid was unstable and we decided to remove the tet(P) gene in further manipulations. In the subsequent construction of pJIR292, the 0.8-kb tet(P)derived EcoRI-PstI fragment from pJIR275 wasremovedand replaced with a 1.2-kb pJIR229 fragment which encoded the C. perfringens ermBP gene (Fig. 1). High levels of erythromycinand chloramphenicol-resistantE. coli transformants were obtained using pJIR292. The next step was to add a C. perfringens replicon and therefore construct a shuttle plasmid. It was decided to use pIP404 becausethe entire plasmid had beensequenced, the minimal replication region had been identified, and pIP404 did not appearto generate SSDNAduring replication (Gamier and Cole, 1988a,b). Shuttle plasmids based on pIP404 therefore should not be subjectto the instability problems common with other gram-positive plasmids which do generate ssDNA as part of their replicative process (Gruss and Ehrlich, 1989).
SLOAN ET AL.
FJG. 1. Construction of pJIR297. The initial shuttle plasmid pJIR297 was constructed from the E. coli vector pUC18 and the recombinant plasmids pJIR62, pJIR71, pJIR229, and pIP404 as indicated. The origin of the various segmentsis indicated by the key. Only relevant genesand restriction sitesare included. Sites in parentheses were altered by the cloning step.
Previous studies showed that the origin of replication (ori) and essential replication gene (rep) from pIP404 were located within a 3.1-kb Hind111 fragment of pIP404 (Garnier and Cole, 1988a,b). This fragment was isolated and ligated into the Hind111 site of pJIR292 to form pJIR297 (Fig. 1). This plasmid was stable in E. coli and could be used to
transform C. perfn’ngensstrain 13 to erythromycin and chloramphenicol resistance. Plasmid DNA was isolated from these C. perpingem transformants, shown to be identical to pJIR297 from E. coli, and used to transform E. coli to resistance to the same antibiotics. These E. coli transformants contained plasmids which were identical to the original
C. perjkgens-E.
coli SHUTTLE
pJIR297 preparations. Due to the difficulties in isolating plasmid DNA from C. perfringens we now routinely transform recombinant plasmids back into E. coli to check their restriction profiles. Construction of the Shuttle PlasmidpJIR418 Further manipulations of pJIR297 were necessary to remove superfluous restriction sites and reintroduce a multiple cloning region. One of the KpnI sites was removed by partial digestion of pJJR297 with Asp7 18 (an isoschizomer of KpnI) and subsequent filling in of the 5’ overhangs with the Klenow fragment of DNA polymerase I. The KpnI and EcoRI sites of the resultant plasmid, pJIR335, were then removed in a similar manner to form pJIR403 (Fig. 2). Digestion with BamHI and PstI, followed by treatment with T4 DNA polymerase, was used to remove these sites. The plasmid that was obtained, pJIR408, was partially digested with Hind111 and Klenow-filled to remove one of the two Hind111 sites (Fig. 2). The plasmid produced from these manipulations, pJIR4 15, like all of the preceding pJIR297 derivatives, could be readily transfered between C. perfringens and E. coli. To reintroduce a multiple cloning site and X-gal selection, PvuII and partial EcoRV digestion was used to replace the pUCl8 replication region with the larger pUCl8-DraI fragment (Fig. 2). Recombinants were selected in E. coli as blue colonies on X-gal plates containing erythromycin and chloramphenicol. The plasmid pJIR418 was the end product of this seriesof plasmid manipulations. This plasmid has been repeatedly passagedbetween C. perfringens and E. coli without observable changes in properties or restriction profiles. In addition, it could be transformed into C. perfringens at a comparable frequency to pHR106. Confirmation of the Value of pJIR418 as a Shuttle Vector To confirm that pJIR4 18 can be effectively used for the manipulation of virulence and
PLASMIDS
211
other genes,the C. perfringens phospholipase C gene, plc, from the recombinant plasmid pTOX6 (Saint-Joanis et al., 1989) was cloned into the Hind111 site present in the multiple cloning site of pJIR418. Erythromycinand chloramphenicol - resistant transformants were selected in E. coli DHSa and white colonies picked off X-gal plates. Screening of these clones on medium containing egg yolk led to the identification of cells producing phospholipase C and containing the predicted recombinant plasmid, designated pJIR443. This plasmid was then used to transform the wild-type C. perfingens strain 13 and a nitrosoguanidine-derived plc mutant of strain 13, JIR367 (P. Scott and J. Rood, unpublished results). C. perfringens transformants carrying pJIR443 consistently produced higher levels of phospholipase C when compared with derivatives harboring the control plasmid pJIR4 18 (Table 1). When plasmid DNA was extracted from these transformants and used to retransform E. cob, the resultant derivatives carried plasmid DNA that was indistinguishable from the original pJIR443 preparation from E. coli. Cloning experiments were also carried out to show that the EcoRV site located within catP can be used as an insertional inactivation site in C. per$ringens and E. coli. A C. perfringens chromosomal DNA preparation was digested with EcoRV and ligated with EcoRV-digested pJIR4 18. The ligation mixture was used to transform strain 13 to erythromycin resistance and then the resultant colonies were screened for their susceptibility to chloramphenicol. A number of chloramphenicol-sensitive C. perfringens recombinants were obtained. Plasmid DNA was prepared from these recombinants and used to transform DHSa cells to erythromycin resistance. Examination of the resultant plasmids in E. coli confirmed that they contained inserts at the EcoRV site (data not shown). Further experiments of a similar nature showed that the ScaI site located within the ermBP gene was also a suitable site for insertional inactivation.
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SLOAN ET AL.
Fw 2. Construction of pJIR4 18, pJIR4 10, and pJlR480. The stepsinvolved in the construction of these shuttle plasmids from pJIR297 are indicated. The origin of the various segments is indicated by the key. Only relevant genes and restriction sites are included.
C. perjkingens-E. coli SHUTTLE PLASMIDS TABLE 1 PH~SPHOLIPASEC
C. perjiingens
Acrrwn
OF
REI~~MB~ANTS
Strain
Phospholipase C Activity” (units X 104/pgprotein)
13 13(pJIR418) 13(pJIR443) JIR367’ JIR367(pJIR4 18) JIR367(pJIR443)
1.03 f 0.92 1.04 f 0.38 4.43 f 1.72 GO.43f 0.035 ~0.58 + 0.072 2.63 AZ1.04
’ Each value representsthe mean of at least three separate determinations. ’ JIR367 is a plc mutant derived from strain 13.
Construction of Additional Shuttle Plasmids Several research projects in this laboratory involve the manipulation of erm or cat genes from C. perfringens. The shuttle vector pJIR4 18 is not suitable for these experiments becauseit already contains both resistancedeterminants. To facilitate these studies we constructed two additional vectors, pJIR4 10 and pJIR480, as shown in Fig. 2. Each of these shuttle vectors has only one antibiotic resistance gene. The plasmid pJIR410 only contains the ermBP gene and therefore can be used for the cloning and manipulation of cat genes. Similarly, the catP plasmid, pJIR480, can be used for the analysis of erm genes. Complete NucIeotide Sequenceof pJIR418 There are four separate DNA segments which have been joined together to form pJIR418. Since the complete sequences of pUC18 (Yanisch-Perron et al., 1985) and pIP404 (Garnier and Cole, 1988a) were known we were able to extract the sequences of the fragments present in pJIR4 18. The sequence of the EcoRI-Hind111 catP fragment from pJIR62 was also known (Steffen and Matzura, 1989). The ermBP region was sequenced in a separatestudy in our laboratory (D. Berryman and J. Rood, unpublished results). By combination of all of these sequences we were able to compile the com-
213
plete sequence of pJIR4 18. However, due to the complexity of the manipulations used in the construction of pJIR4 18 it was necessary to confirm these data by sequencing across each of the junction regions between the four segments. These regions were the sites at which all of the plasmid modifications had been carried out and would therefore be the most likely sites at which any sequencevariations introduced by our manipulations would be present. Four oligonucleotides which were located near the ends of the pUCl8-, pIP404-, catP-, and ermBP-derived regions were synthesised and used as primers to obtain sequence data from pJIR4 18. All sequence determinations were repeated several times. As expected several minor changes were observed in the junction regions and the alterations were incorporated into the final sequence (Fig. 3). Two regions of major sequence variation were also observed. Sequencing across the pIP404-catP junction consistently revealed sequencedifferences between the experimental data and the published sequence. These differences extended well beyond the junction region. Consequently, the entire EcoRIHind111 fragment from pJIR62 was resequenced on both strands in an overlapping manner. The data obtained (T. Bannam and J. Rood, unpublished results) were in complete agreement with the sequence obtained from pJIR418. The pJIR62 sequence determined in this laboratory was therefore incorporated into the pJIR418 sequence rather than the published sequence.The final variation occurred at the junction of the pUC 18 and pIP404 regions. These segments were blunt-end ligated after DraI digestion of pUC 18. To our surprise pJIR4 18 contained three copies of the 19-bp DraI fragment (pUC 18 coordinates 1563- 1582) at the junction of the pUC 18- and pIP404-derived segments (Fig. 3). These DraI fragments were present in both orientations and obviously represented separate accidental cloning events. The complete 7358-bp sequence of pJIR418 is shown in Fig. 3. Note that the
214
SLOAN ET AL. 20 40 60 70 W 10 30 50 80 !GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCT SmrI ECdlI Hiti 110 120 130 140 170 190 150 160 180 GGCGTTACCCMCTTMTCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTMTAGCGMGAGGCCCGCACC~TCGCCCTTCCCMCAGTTGCG~
100 200
290 210 220 230 240 250 260 270 280 GCCTGMTGGCGMTGGCGCCTGATGCOGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACMTCTGCTCTGA
300
380 390 310 320 330 340 350 360 370 TGCCGCATAGTTMGCCAGCCCCGACACCCGCCMCACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA~CM~TGTGACC
400
410 420 430 440 460 470 480 490 450 GTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCG~CGCGCGAGACG~GGGCCTCGTGATACGCCTATTTTTATAGGTTMTGTCA
500
510 520 530 540 560 570 580 550 TGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG~TGTGCGCGG~CCCCTATTTGTTTATTTTTCT~TACATTC~TATGTATCC
600
610 620 630 640 GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT
590
690 660 670 680 650 GAAAAAGGMGAGTATWLGTATTCMCATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG 790
710 720 730 740 750 760 770 700 GCATTTTGCCTTCCTGTTTTTGCTCACCCAWYVLCGCTGGTG~GT~GATGCT~GATCAGTTGGGTGCACGAGTGGGTTACATCGMCTG~TC 810 820 830 840 TCMCAGCGGTMWLTCCTTGAGAGTTTTCGCCCCGAAWUTTTT TT -TV
910 920 930 TTAAAAATGAAGTTTTATCAAAMAA
850
940 950 960 TTTCCMTMTCCCACTCTAGCCACAAACACOCCCTATA
1010 1020 1030 1040 1050 ACATGTMTATTACTTTACGCCCTAGTATAGTWLTAATTTTTTTACATTC~TGCCACGC 1110 1120 1130 TAACTAAAGTAAAAAATTATCTTTACAACCTCCCCAAAA
1140
1210 1220 1230 RAATAARRAAATAAAAAAATARRAAAATAAAAAARRTlUU
1240
1310 TATAAAAATLAAAAAA
1330 1320 TATAAAAATAMAAAA
1410 1420 1430 AAAATATTTTTTATTTAAAGTTTGAMAAAA
890 870 880 TTAAAAATGAAGTTT'TTCATTTTTAA
860
1060
970
980
990
1340 1350 TATAAAAATAAAAAAA TAT-T-T
800 900 1000
1070 1080 1090 1100 AAAAAAATAAAGGGGCACTATAATAAAAGTTCCTTCGGAAC
1150 1160 1170 1180 GAACAGGTACAAAGTACCCTATAATACAAGCGTAAAAAAA 1250
700
1260 1360
1270
1190 1200 TGAGGGTAAAAATAAAA
1280
1290
1370 1380 1390 AAAAAAATATAMAATAAAAAAATAAAAAAATATA
1440 1450 1460 1470 TTTTTTTATATTATATMTCTTTGMG~~TATAAACCCATTTTTT
1400
1490
1500
1550 1560 1570 1510 1520 1530 1540 1580 TTCATATACGTAATATOLCGTTCT~TGTTTTTATTGGTACTTCT~CATTAGAGT~TTTCTTTATTTTT~GCCTTTTTCTTT~GGGCTTTTATTT
1590
1600
1610 1620 1630 1640 1650 1660 1670 1680 TTTTTCTTMTACATTTMTTCCTCTTTTTTTGTTGCTTTTCCTTTAGCTTTTMTTGCTCTTGATMTTTTTTTTACCTCTMTATTTTCTCTTCTCTT
1690
1700
1710 1720 1730 1740 1750 1760 1770 1780 ATATTCCTTTTTAGAAATTATTATTGTCATATATTTTTGTTCTTCTTCTGT~TTTCT~T~CTCTAT~~GTTTCATTCTTATACTTATATTGCTTA
1790
1800
1810 1820 1830 1840 1850 1860 1870 1880 TTTTTATCTAAATMCATCTTTCAGCACTTCTAGTTGCTCTTATMCTTCTCTTTCACTT~TGTTGTCT~CATACTATTMGTTCT~~T~T
1890
1900
1910 1920 1930 1940 1950 TTAATGCCTTCTCAATGTCTTCTGTAAAGCTACAAAGATATCATA~TCTCTTAGTTC
1970
1980
1990
2000
2020 2030 2040 2070 2010 2050 2060 ACAAAGTTTTATTATGTCTTGTATTCTTCCATMTAAATTTATTTTGCTTGGTCTACCCTTTTTCCTTTCATATGGT
2080
2090
2100
2120 2130 2110 TTTPlATTCAGGTAAAAATCCATTTTGTATTTCTCTTAAG
2180
2190
2200
2210 2220 2230 2240 2250 2260 2270 2280 TTCCTGGAACTCTTAATATTCTGGTTGCATCTAAGGCTTGTCTATCTGCTCC~GTATTTT~TTGATTATAT~TATTCTT~CCGCTTTCCAT~
2290
2300
2310 2320 2330 2340 2350 2360 2370 2380 TGGTAATGCTTTACTAGGTACTGCATTTATTATCCATATT~TACATTCCTCTTCCACTATCTATTACATAGTTTGGTATAGG~TACTTTGATT~
2390
2400
2410 2420 2430 2440 2450 2470 2460 2480 TAATTCTTTTCTAAGTCCATTAATACCTGGTCTTTAGTTTTGCCAGTTTTAT~T~TCC~GTCTAT~CAGTGTATTT~CTCTTTTATATTTTCTA
2490
2500
2510 2520 2530 2540 2570 2550 2560 2580 ATCGCCTACACGGCTTATAGTATTTAGAGTTATATAGATATTTTCATCACTCATATCT~TCTTTT~TTCAGCGTATTTATAGTGCCATTGGCT
2590
2600
2610 2630 2640 2620 2650 2660 2670 2680 ATATCCTTTTTTATCTATARCGCTCCTGGTTATCCACCCTTTACTTCTACTAT~TATTATCTATATAGTTCTTTTTAT~CAGCTTT~TGCGTTTCTC
2690
2700
2140
2710 2720 2730 2740 ACTTATTCACCTCCCCTTCTGTRAAACTRAGAAAATTATRT
2150
2750
1960
1480
1300
2160
2760
2170
2770
2780
2790 2800 AAAAAATAGAGTAAGTCCC
2810 2820 2830 2840 2850 2860 2870 2880 CAATTGAAACTTAATCTATTTTTTATGTTTTAATTTTATTATTTTTATT~TATTTT~CT~TT~TGATTCTTTTT~TTTTTTACTATTTCAT
2890
2900
2940 2950 2960 2970 2980 2910 2920 2930 TCCATAATATATTACTATAATTATTTACAAATAATATTTCTTCATTTGT~TATTTAGATGATTTACT~TTTTAGTTTTTATATATT~T~TT~TG
2990
3000
FIG. 3. Complete nucleotide sequence of pJIR418. The sequence was assembled from the known se quences of the pIP404 (Gamier and Cole, 1988a;Accession number M I448 l), pUC 18 (Yanisch-Perron et al., 1985;Accession No. VBOO25),ermBP (D. Berryman and J. Rood, unpublished results; Accession No. X58285), and catP(T. Bannam and J. Rood, unpublished results; Accession No. M74769) components of
215
C. perfringens-E. coli SHUTTLE PLASMIDS 3010 3020 3030 TATMTTTATAT~TCAAAGGAGCTTATAAAT'fATTA
3080
3090
3100
3110 3120 3130 3140 3150 3160 3170 3180 TTATGTTTAAATTTMTTGTATTTTTTTCATATMT~GCCGTTGMGT~CCMTCCATTTTCCTTATGATGTTATTATT~TTTMGTTTTATM
3190
3200
3210
3220
3040
3050
3060
3070
3240 3250 3260 3270 3280 3290 AAAAAAACTAGTUUVLTTTCCGGCTTTATTAAACTTATTATTTTTAG~TTTTATTTTCATTTTCATCTTTACA
3230
TAATATCTTTATTATATTTATTGTTTTT
3310 3320 3330 3340 3350 3360 3370 3380 GWLTTTGATTATATCTTTATATGTTTTATCARATATTATTATCTTTTTCT~TTTATATATATTTTTATTATATTTATTATTATATATATTTTATTTTTA
3300
3390
3400
3490
3500
3510 3520 3530 3540 3550 3560 3570 3580 3590 TAGTTTTTAGTAAAATTMTTTCAATATTCCACRATTCCACMTATTATATTATMGCTAGCTTTGCATTGTACTTTTC~TCGCTTCAC~TGCGGTTATCTCC~
3600
3690
3700
3790
3800
3890
3900
3990
4000
4080
4090
4100
3410
3420
3430
3440
3450
3460
3470
3480
AGTTTCTTTCTAACAGCTATTAAAAAGAAACTTAAARATA
3610
3620
3630
3640
3650
3660
3670
3680
MGATAAAGTCTTTTCATCTTCCTTGATGM~TMGATTTTCTCCGTCTCCGCCGGCAGMTTGMGCGGGGTACTACGGTATCGTCTGCGT~TCTTC 3710
3720
3730
3740
3750
3760
3770
3780
CGTTGTCTGATAWLTGATAGTCATAGGCTCATTTTCTTCCGTTTCGGT~GGGGATAGGTTCGCCCTTT~GAGCAGGGCGGC~TGG~G~TTMC 3810
3820
3830
3840
3850
3860
3870
3880
TTGCTTTTCCCATCGCCCGGATCTCCCTGCMTAATTTCAC 3910 3920 3930 3940 3950 3960 3970 3980 TTGCCTTGATGATTTCMGAGGTACGCTGAAATTCATTCATTTCGTTTTCATTTAGTTTCATTTTTTCTTGTTCTCCTTTTCTCTG~TAT~CCACAG 4010
4020
4030
4040
4050
4060
4070
ATTGATACTAAAACCTTGGTTGTGTTGCTTTTCGGGGCTT~TCMGG~TCCTTGTTTTMGCCTTTC~G~CACMGGTCTTTGTACTA 4110
4120
4130
4140
4150
4160
4170
4180
4190
4200
4240
4250
4260
4270
4280
4290
4300
ACCTGTGGTTATGTATAAAATTGTAGATTTTAGGGTAACA 4210
4220
4230
CTGMGTTMCTATTTATCMTTCCTGCMTTCGTTTAC~CGGCMATGTG~TCCGTCACATACTGCGTGAT~CTTGMTTGCC~GGMGT 4340
4350
4360
4370
4380
4390
4400
4410 4420 4430 4440 ACCACGGTATCATAGATACATTAAAAATGTTTTCCGGAGCTC
4450
4460
4410
4400
4490
4500
4550
4560
4570
4580
4590
4600
4670
4680
4690
4700
4740 4750 4760 4770 4780 4790 ACTTTAACGGTCATGCTGTATGTACAAGGTACACACTTGC~GTAGTGGTC~TACTCTTTTCTGTTCCA
4800
4310
4320
4330
ATMTTTTGTTATCTTCTTTATMTATAGTAAGGTTG
4510
4520
4530
4540
TGCTAAAAATGATTTAAAGTCAGACTTACACTCAGTCCARTTGTATAGCTTGGTATCATCTCATCA 4610
4620
4630
4640
4650
4660
TATATCCCCAATTCACCATCTTGATTGATTGCCGTCCTAA 4710 4720 TTCCCTTTTCCTTTATTTGTGT-
4730 ECORV
4810 4820 4830 4840 4850 4860 4870 4880 ACTATTTTTATCMTTTTTTCARATACCATCTMGTTCCCTCTC~TTCMGTTTATCGCTCTMTGMC~GATATTATACCACATTTTTGTGMTT
4890
4910 4920 4930 4940 TTTCAACTTGCCCACTTCGACTGCACTCCCGACTTAATRA
4990
4950
4960
4910
4980
4900
5000
TCGATCCCCG SInal 5090
5100
5190
5200
5290
5300
5310 5320 5330 5340 5350 5360 5370 5380 TCTCGATTGACCCATTTTGAC~GTACGTATATAGCTTCC~TATTTATCTGGMCATCTGTGGTATGGCGGGT~GTTTTATT~GACACTGTTTA
5390
5400
5410 5420 5430 5440 CTTTTGGTTTAGGATGAAAGCATTCCGCTGGCAGCTTAAG
5450
5460
5470
5480
5490
5500
5510 5520 5530 5540 GGTACGCTTGTAGMTCCTTCTTCAACAATCAGATAGATGGGA
5550
5560
5570
5580
5590
5600
5610 5620 5630 5640 ATACTCCCAACMTTTTATACCTCTGTTTGTTAGGGAATTTTTTTCTG
5650
5660
5670
5680
5690
5700
5010
5020
5030
5040
5050
5060
5070
5080
5140
5150
5160
5170
5180
CCGAGCGCTTAGTGGWV\TTTGTACCCCTTATCGATACRA 5110
5120
5130
TMTTTATCTACATTCCCTTTAGTRACGTGTGT~CTTTCC~TTTAC~GCGACTCATAG~TTATTTCCTCCCGTT~TMTAGATMCTATT~ 5210
5220
5230
5240
5250
5260
5270
5280
AATAWLCAATACTTGCTCATAAGTAACGGT~CGGTACTT~TTGTTTACTTTGGCGTGTTTCATTGCTTGATG~CTGATTTTTAGT~CAGTTGAC~TAT
5810 5820 5830 5840 5850 5860 5870 5880 5890 GTAAACGGTATCGGTTTCTTTTAAATTCAATTGTTTTATTGTTTTATTATTTGGTTGAGTACTTTTTCACTCGTT~GTTTTGAG~TATTTTATATTTTTGTTC
5900
the plasmid and direct sequenceanalysis as described in the text. The sequence is numbered from the unique EcoRI site in the multiple cloning site. Relevant restriction sites are included. The repeated pUC1 S-derived 19-bp DruI fragments are indicated by arrows under the sequence (866-922). See Table 2 and Fig. 4 for the location of other features. The GenBank Accession No. of the complete pJIR4 18 sequence is M77 169.
216
SLOAN ET AL. 5910 5920 5930 5940 5950 5960 5970 59EO ATGTMTCACTCCTTCTTMTTACAAATTTTTTAG~TCTMTTTMCTTCMTTCCTATTATA~TTTTM~TACTGCACTATCMCACACTCTTA
5990
6000
6010 6020 6030 6040 6050 6060 6070 6080 AGTTTGCTTCTAPlGTCTTATTTCCATMCTTCTTTTACGTTTCCGGGTAC~TTCGT~TCATGTCATAGCTGTTTCCTGTGT~TTCTTATCCGCTC
6090
6100
6110 6120 6130 6140 6150 6160 ACRATTCCACACARCATACGAGCCGGAAGCATAAAGTGTATTGCGTTGCGCTCACTGCCCG
6190
6200
6210 6220 6230 6240 6250 6260 6270 6280 CTTTCCAGTCGGGARACCTGTCGTGCCAGAAAACTTCATTCATTTTT~TTT-GGATCTAGGTGMGATCCTTTTTGATMTCTCATGACC~TCCCTT
6290
6300
6310 6320 6330 6340 AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCOTAGAC
6390
6400
6410 6420 6430 6440 6450 6460 6470 6480 AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC~GAGCTACC~CTCTTTTTCC~GGT~CTGGCTTCAGCAGAGCGCA~TACC-T
6490
6500
6510 6520 6530 6540 6550 ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCACAGTGGCTGCTG
6350
6360
6170
6370
6180
6380
6560
6570
6580
6590
6600
6610 6620 6630 6640 6650 6660 CCAGTGGCGATMGTCGTGTCTTACCGGGTTGGACTCAAGCGGGGGGTTCGTGCACACAGCC
6670
6680
6690
6700
6710 6720 6730 6740 6750 6760 6770 6780 6790 CAGCTTGGAGCWVLCGACCTACACCGAACTGAGAGATACCTACAGCGTGAGCTATGAG-GCGCCACGCTTCCCG~GGGA~GGCGGACAGGTATCCG
6800
6810 6820 6830 6840 6850 6860 6870 6880 6890 GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG-CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT~CTTG
6900
6910 6920 6930 6940 AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG
6950 6960 6970 6980 6990 7000 GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC
7010 7020 7030 7040 7050 7060 7070 7080 7090 TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT~CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC~C~CC~GCGC
7100
7110 7120 7130 7140 7150 7160 7170 7180 7190 AGCGAGTCAGTGAGCGAGGAGCGGAAGAGCGCGCCCMTACGC-CCGCCTCTCCCCGCGCGTTGGCCGATTCATT~TGCAGCTGGCACGACAGGTTTC
7200
7210 7220 7230 7240 7250 7260 CCGACTGGAAAGCGGGCAGTGAGCGCRACGCAATTAATGTTTTATGCTTCCGGCTCGTATGTT
7270
7280
7290
7300
7310 7320 7330 7340 7350 GTGTGOAATTGTGAGCGGATAACPSrTTTCACACAGGAAACAGCTATGACCATGATTAC
7370
7380
7390
7400
7360
FIG. 3-Continued
plasmid contains an additional SmaI site which is located outside the multiple cloning site. To assist future users of this plasmid the precise locations of the major features of this plasmid are listed (Table 2) and a detailed physical and genetic map is presented (Fig. 4). All of the plasmids described in this paper are available upon request.
work. The properties of pJIR4 18 that make it ideally suited to these studies include the ability to use X-gal selection in E. coli, or insertional inactivation at the EcoRV or %a1 sites for screening in C. perfringens and E. coli, TABLE 2 LOCATIONOFMAJORFEATURE.S IN pJIR4 18 SEQUENCE
DISCUSSION
There has been a very significant expansion of interest in the genetics of C. perji-ingens in recent years and genesencoding several C. perfringens toxins and extracellular enzymes have been cloned and sequenced (Rood and Cole, 1991). It is clear from these studies that much of the future work on this microorganism will involve molecular approaches to the study of the pathogenesis of C. perfringens infections (Rood and Cole, 1991). We have constructed a shuttle plasmid that has been specifically designed for this
Location (bp) 1-57 310 1190-1419 1482 2102 4207 4830 5165 5902 6780-7000 7345
Description of Feature pUC I I-derived multiple cloning site (EcoRI-HindIII) Stop codon of 1acZ gene pIP404 replication origin (approx.) Stop codon of pIP404 rep gene Start codon of pIP404 rep gene Stop codon of catP gene Start codon of catP gene Stop codon of ermBP gene Start codon of ermBP gene pUC 18 replication origin (approx.) Start codon of IacZ’ gene
C. perj?ingens-6 coli SHUTTLE PLASMIDS
-Nae
I 3653
FIG. 4. Physical and genetic map of pJIR4 18. Internal size markers are in kilobases, and coordinates of restriction sites am in base pairs. Genetic symbols are as follows: oriCP and oriEC, origins of replication from pIP404 and pUC18, respectively; rep, replication gene from pIP404; catP and ermBP, C. perjikgens chloramphenicol and erythromycin resistance genes, respectively; lac.Z, &alactosidase a-peptide gene from pUC 18.The direction of transcription is indicated by the arrows.
and the presenceof the standard pUC 18 multiple cloning site. In addition, further manipulations of pJIR418 derivatives will be facilitated by the knowledge of its complete sequence. As with any shuttle plasmid there are some limitations to the use of pJIR4 18. The presence of an additional SmaI site located between the ermBP and catP genesmeans that it is difficult to use the SmaI site in the multiple cloning region for blunt-end cloning. However, the EcoRV and ScaI sites are acceptable alternatives for such manipulations. In the construction of pJIR418 the 18I-bp region located between the PvuII site and EcoRl site in pUCl8 (pUC 18 coordinates 447-628) was duplicated, leading to the formation of an inverted repeat. As a result the
217
standard reverse sequencing primer cannot be used for sequence analysis of pJIR4 18 derivatives. This is not a major problem since most sequencing studies are likely to be carried out in E. coli using other more suitable vectors. We have demonstrated that pJIR4 18 can be used for cloning in either C. perfringens or E. coli and can be used to freely move toxin genesbetween these organisms. The manipulations carried out with the plc gene are typical of the types of constructions that will be used in pathogenesis studies. No evidence of plasmid rearrangements was detected, even after several passagesbetween genera. The recombinant plasmid pJIR443 complemented the chromosomal plc mutation and the enzyme levels that were obtained were as expected for a gene encoded on a pIP404 replicon. It is anticipated that pJIR418 will be widely used for the manipulation of C. perfiingens genes. Together with the more specialized shuttle vectors pJIR410 and pJIR480, both of which were specifically designed for the manipulation of antibiotic resistance determinants, pJIR4 18 represents the best characterized and most versatile cloning vector yet constructed for C. perfringens research. Although these studies have only involved the transformation of derivatives of C. perfiingens strain 13 it is hoped that pJIR4 18 will have a broader use in other members of the genus Clostridium. Hopefully, the derivation of the plasmids described in this report will be followed by the construction of other well characterized vectors, such as promotor probe and expression plasmids, which will enable the function of genesfrom C. perfringens to be carefully analyzed in an homologous host system. ACKNOWLEDGMENTS We thank Stewart Cole for providing his pTOX6 recombinant plasmid; Margaret Katz, Stephen Billington, and Mike Lyristis for helpful discussions; and Pauline Howarth for her most capable technical assistance.This researchwas generously supported by the Australian Re-
218
SLOAN ET AL.
searchCouncil and the Commonwealth Serum Laboratories.
REFERENCES ABRAHAM,L. J., AND ROOD,J. I. (1985a). Cloning and analysis of the Clostridium perfingens tetracycline resistance plasmid, pCW3. Plasmid 13, 155-162. ABRAHAM, L. J., AM) ROOD, J. I. (1985b). Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens. J. Bacterial. 161, 636-640. ABRAHAM,L. J., AND ROOD,J. I. (1987). Identification of Tn445 1 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens. J. Bacteriol. 169, 1579-1584. ABRAHAM,L. J., WALES,A. J., AND ROOD,J. I. (1985). Worldwide distribution of the conjugative Clostridium pefiingens tetracycline resistance plasmid, pCW3. Plasmid 14, 37-46. ABRAHAM, L. J., BERRYMAN,D. I., AND ROOD, J. I. (1988). Hybridization analysis of the class P tetracycline resistancedeterminant from the Clostridiumperfringens R-plasmid, pCW3. Plasmid 19, 113- 120. ALLEN, S. P., AND BLASCHEK,H. P. ( 1988).Electroporation-induced transformation of intact cells of Clostridium perfringens. Appl. Environ. Microbial. 54, 23222324. BERRYMAN,D. I., AND ROOD,J. I. (1989). Cloning and hybridization analysis of ermP, a macrolide-lincosamide-streptogramin B resistance determinant from Clostridium perftringens.Antimicrob. Ag. Chemother. 33, 1346-1353. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. I, 1513- 1523. BRADFORD,M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein binding dye. Anal. Biochem. 12,248-254. CANARD,B., AND COLE,S. T. (1989). Genome organization of the anaerobic pathogen Clostridium perfringens. Proc. Natl. Acad. Sci. USA 86,6676-6680. CASADABAN,M. J., AND COHEN,S. N. (1980). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138, 179-207. GARNIER, T., AND COLE, S. T. (1988a). Complete nucleotide sequenceand genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens. Plasmid 19, I34- 150. GARNIER, T., AND COLE, S. T. (1988b). Identification and molecular genetic analysis of replication functions ofthe bacteriocinogenic plasmid pIP404 from Clostridium perfringens. Plasmid 19, 151- 160. GRUSS,A., AND EHRLICH, S. D. (1989). The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbial. Rev. 53,23 l-24 1.
HEEFNER,D. L., SQUIRE.V, C. H., EVANS,R. J., KOPP, B. J., AND YARUS, M. J. (1984). Transformation of Clostridium perfringens. J. Bacterial. 159,460-464. HOLMES,D. S., AND QUIGLN, M. (198 I). A rapid boiling method for the preparation of bacteria1 plasmids. Anal. B&hem. 114, 193-197. KIM, A. Y., AND BLASCHEK,H. P. (1989). Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens. Appl. Environ Microbial. 55, 360-365. MAHONY, D. E., AND MOORE, T. J. (1976). Stable Lforms of Clostridium perfn’ngens and their growth on glass surfaces. Can. J. Microbial. 22,953-959. MAHONY, D. E., MADER,J. A., AND DUBEL,J. R. (1988). Transformation of Clostridium perfringens L forms with shuttle plasmid DNA. Appl. Environ. Microbial. 54,264-267. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK,J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. PHILLIPS-JONES, M. K. ( 1990). Plasmid transformation of Clostridium perfkingens by electroporation methods. FEMS Microbial. Lett. 66, 22 1-226. ROBERTS,I., HOLMES, W. M., AND HYLEMON, P. B. (1986). Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum. Appl. Environ. Microbial. 52, I97- 199. ROBERTS,I., HOLMES, W. M., AND HYLEMON, P. B. (1988). Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens. Appl. Environ. Microbial. 54, 268-270. ROKOS,E. A., ROOD,J. I., AND DUNCAN, C. L. (1978). Multiple plasmids in different toxigenic types of Clostridium perjiiingens. FEMS Microbial. Lett. 4, 323326. ROOD, J. I. (1983). Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin. Can. J. Microbial. 29, 1241-1246. ROOD, J. I., AND COLE, S. T. (199 1). Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol. Rev., 55, 621-648. ROOD,J. I., ANDWILKINSON,R. G. (1975). Isolation and characterization of Clostridium perjiiingens mutants altered in both hemagglutinin and sialidase production. J. Bacterial. 123,419-427. ROOD, J. I., JEFFERSON,S., BANNAM, T. L., WIWE, J. M., MULLANY, P., AND WREN, B. W. ( 1989). Hybridization analysis of three chloramphenicol resistance determinants from Clostridium perfringens and Clostridium dt$icile. Antimicrob. Ag. Chemother. 33, 1569-1574. SAINT-JOANIS,B., GARNIER, T., AND COLE, S. T. (1989).
C. perfringens-E. coli SHUTTLE PLASMIDS Gene cloning shows the alpha toxin of Clostridium perfn’ngensto contain both sphingomyelinase and lecithinase activities. Mol. Gen. Genet. 219,453-460. SANGER,F., MICKLEN, S., AND COU~SON,A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Scan, P. T., AND ROOD, J. I. (1989). Electroporationmediated transformation of lysostaphin-treated Clostridium perjiiingens. Gene 82, 327-333. SQUIRES,C. H., HEEFNER,D. L., EVANS,R. J., KOPP, B. J., AND YARUS, M. J. (1984). Shuttle plasmids for Escherichia coli and Clostridium perjiiingens. J. Bacteriol. 159, 465-47 1. STEFFEN,C., AND MATZURA, H. (1989). Nucleotide sequence analysis of a chloramphenicol-acetyltransfer-
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ase coding gene from Clostridium perjringens. Gene 75,349-354. STEVENS,D. L., MITTEN, J., AND HENRY, C. (1987). Effects of a and 0 toxins from Clostridium perjiringens on human polymorphonuclear leukocytes. J Infect. Dis. 156,324-333. WILLIAMSON,R. (1983). Resistance of Clostridium perfringens to Blactam antibiotics mediated by a decreased affinity of a single essential penicillin-binding protein. J. Gen. Microbial. 129,2339-2342. YANISCH-PERRON,C., VIEIRA, J., AND MESSING, J. (1985). Improved M 13 cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC 19 vectors. Gene 33, 103- 119. Communicated by Francis L. Macrina