JOURNAL OF BACTERIOLOGY, June 1993, p. 3838-38430021-9193/93/123838-06$02.00/0Copyright © 1993, American Society for Microbiology

Vol. 175, No. 12

Construction and Characterization of a Phage-Plasmid Hybrid(Phagemid), pCAK1, Containing the Replicative Form of

Viruslike Particle CAKi Isolated from Clostridiumacetobutylicum NCIB 6444

AUGUSTINE Y. KIMt AND HANS P. BLASCHEK*Department ofFood Science, University of Illinois, 580 Bevier Hall,

905 South Goodwin Avenue, Urbana, Illinois 61801

Received 21 October 1992/Accepted 15 April 1993

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Esche-richia coli plasmid pAK102 (Apr Emr) and the 6.6-kbp HaeIII-linearized replicative form of the CAKi viruslikeparticle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColElreplication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed withprotein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coliDH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNAsynthesis replication origin of CAKi and associated gene expression are functional in E. coli DH11S. Thesingle-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5ct' orDH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformationsuggests that the complementary-strand DNA synthesis replication origin of CAKi is also functional in E. coli.The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and varioussolvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Followingphagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C.perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromy.cin-resistant transformants of both C. acetobutylicum and C. pefringens suggested that it was identical to that

present in E. coli transformants.

Interest in the physiologically diverse genus Clostnidiumhas been renewed because of the presence of species withrecognized industrial potential (1, 2, 15, 31, 32) as well asanaerobic pathogenic properties (25). There currently isconsiderable industrial interest in the nontoxinogenic clos-tridia as practical biomass conversion reactors for produc-tion of solvents and organic acids (1, 2, 15). To understandclostridial metabolic pathways related to the production ofindustrial chemicals, (i.e., Clostridium acetobutylicum ABEfermentation) or anaerobic pathogenesis, (i.e., C. perfrin-gens intoxication), systems for genetic analysis (e.g., trans-formation and stable expression vectors) need to be devel-oped. C. acetobutylicum and C. perfringens represent themodel species for clostridial genetics (31, 32).

Recently, gene transfer systems for C. perfringens and C.acetobutylicum have been developed (11, 17, 22, 25, 31, 32).However, the strain-specific nature of transformation andthe instability of various shuttle vectors necessitated thedevelopment of a more versatile genetic system for clostridia(25, 31, 32).Recent identification of the CAK1 filamentous viruslike

particle from C. acetobutylicum NCIB 6444 (12, 13) pro-vided the opportunity for development of a genetic systemfor Clostndium spp. resembling that of Eschenchia colifilamentous bacteriophage M13 (8, 16, 20, 21, 30). Filamen-tous bacteriophages have been described as single-strandedDNA (ssDNA) viruses which infect male-specific gram-

* Corresponding author.t Present address: U.S. Department of Agriculture, Eastern Re-

gional Research Laboratory, Wyndmoor, PA 19118.

negative bacteria (20, 23, 33). The genome of filamentousphages contains intergenic regions which are folded intohairpin structures. The larger intergenic region contains acomplementary-strand (CS) DNA synthesis replication ori-gin, a viral-strand (VS) DNA synthesis replication origin,and multiregulatory elements (e.g., rho-dependent and -in-dependent transcription terminators) (4, 6, 10, 23, 24, 28,33). Following infection by filamentous phages, the incomingviral ssDNA is converted to the replicative-form (RF) dou-ble-stranded DNA (dsDNA) by binding RNA polymerase atthe CS replication origin (4, 6, 10, 28). The new viral DNA issynthesized by initiation of virally coded gene 2 protein atthe VS replication origin by a rolling-circle mechanism ofreplication (4, 6, 23, 28). Formation of the RF of a phagefrom newly synthesized viral DNA is controlled by theconcentration of virally encoded gene 5 proteins (4, 27, 33).The intergenic region can also be used as DNA insertionsites without impairing filamentous-phage propagation (4,21, 23, 33).The most advantageous aspect of a genetic system based

on filamentous phage is that the cell is not lysed duringpropagation of progeny phages (20, 23, 33). Consequently,this genetic system allows investigation of the membranecompartmentalization of the host cell, as well as the mech-anism of secretion of extracellular proteins (23, 33). Further-more, the characteristic plasmidlike mode of replication,together with the secretion of ssDNA, provides for a versa-tile cloning vector and the means for both site-directedmutagenesis and DNA sequencing analysis (21, 23). Such agenetic system can be used in studies which examine themechanism of viral DNA replication, since virally encoded

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TABLE 1. Bacterial strains and extrachromosomal DNAsused in this study

Microorganism or Relevant characteristic Referenceextrachromosomal DNA or phenotypeW or source

C. acetobutylicum NCIB 6444 12C. acetobutylicum ATCC 824 Plasmid free; Ems 12C. perfringens ATCC 3624A Plasmid free; Ems 11C perfringens 13 Plasmid Free; EmsE. coli K802 Plasmid free; Aps Em' 29E. coli DH5o' Plasmid free; Aps (F-) BRLE. coli DH11Sb Plasmid free; Ap" (F') 16

CAK1 RF Viruslike particle 12pVA677 Emr 18pUC19 Apr 30pAK102 Apr Emr This studyPhagemid pCAK1 Apr Emr This study

aEm, erythrornycin resistance; Ap, ampicillin resistance; F, F plasmid.b Nuclease is present in the periplasm (3).

gene expression is precisely regulated in vivo to maintaindifferences in protein concentration over the course of theviral life cycles (23, 27). However, a limitation of clostridialgene expression in the E. coli host system (25, 31, 32) is arequirement for laborious subcloning into suitable shuttlevectors, followed by subsequent transformation into clos-tridia to examine regulatory and structural gene sequencechanges, as would occur during site-directed mutagenesiswhen the M13 genetic system is used.The objective of this study was to construct a phage-

plasmid hybrid (phagemid) for investigation of viral propa-gation in E. coli and to examine the replication of pCAK1 inclostridia in preparation for the use of this construct in thedevelopment of an M13-like genetic system for the genusClostridium.

MATERIALS AND METHODS

Bacterial strains and extrachromosomal DNAs. The bacte-rial strains and extrachromosomal DNAs used in this studyare shown in Table 1. E. coli DH5a' was purchased fromBethesda Research Laboratories (BRL; Gaithersburg, Md.).E. coli DH11S was a kind gift from F. Bloom at BRL. All E.coli strains were grown in Luria broth (19) medium at 37°C.The conditions for cultivation and maintainence of C. ace-tobutylicum and C perfringens strains were previouslydescribed (11-13). C. acetobutylicum and C. perfringenswere grown in Trypticase-glucose-yeast extract (11) at 37°C.For selection of E. coli transformants, Luria broth agarplates (1.5%, wtlvol) were supplemented with 100 tg ofampicillin per ml and/or 100 pg of erythromycin per ml. Forselection of C. acetobutylicum and C. perfringens transfor-mants, Trypticase-glucose-yeast extract agar plates weresupplemented with 25 ,ug of erythromycin per ml and incu-bated under anaerobic conditions (85% N2, 10% CO2. 5%H2) with a Coy Anaerobic Chamber (Coy Laboratory Prod-ucts Inc., Ann Arbor, Mich.).Extracbromosomal DNA isolation. Large-scale isolation of

extrachromosomal DNA from C. acetobutylicum, C. per-fringens, or E. coli was done by the modified alkaline lysismethod previously described (11-13). ExtrachromosomalDNA was purified by CsCl-ethidium bromide isopycnicbuoyant density gradient centrifugation (0.98 g of CsCl perml for C. acetobutylicum and C perfringens and 1 g of CsCl

per ml for E. coli) at 55,000 rpm for 20 h with a Beckman 50Ti rotor as previously described (11).For rapid identification of recombinant plasmids in E. coli

transformants, phenol-chloroform-isoamyl alcohol (25:24:1)extraction was used. Ampicillin-resistant E. coli transfor-mants were inoculated into 10 ml of Luria broth and incu-bated at 370C overnight. One milliliter of an overnight-grownE. coli culture was harvested and suspended in 30 1.l ofdistilled H20. An equal volume of phenol-chloroform-isoamyl alcohol was added to the cell suspension and vigor-ously vortexed for 2 min. After centrifugation, 20 1.l of theaqueous phase containing DNA was directly examined byagarose gel electrophoresis to identify the existence ofrecombinant plasmids.

Isolation of viruslike particles containing ssDNA. Isolationof viruslike particles containing ssDNA from C. acetobutyli-cum NCIB 6444 or from E. coli transformants containingpCAK1 was done as previously described (12). Stationary-phase cells of C. acetobutylicum NCIB 6444 containingCAK1 or E. coli transformants containing pCAK1 werecentrifuged at 10,000 x g for 15 min in an RC-2 centrifuge(Sorvall, Norwalk, Conn.). Viruslike particles were precip-itated by adding an equal volume of 20% polyethylene glycol(PEG) 8000 (Sigma Chemical Co., St. Louis, Mo.) and 2 MNaCl (Sigma) solution to the supernatant. The recoveredviruslike particles were suspended in distilled H20 or TEbuffer (19). Extracellular ssDNA was prepared by phenol-chloroform-isoamyl alcohol extraction with or without pro-teinase K (BRL) digestion of either CAK1 or pCAK1 coatproteins in TE buffer at 370C overnight. Following ethanolprecipitation, ssDNA was suspended in distilled H20.DNA manipulation. Restriction enzymes HaeIII, HindIII,

EcoRI, and PstI; Bal 31 nuclease; and T4 ligase werepurchased from BRL, and all DNA manipulations were doneas described by the manufacturer and Maniatis et al. (19).DNA was analyzed by either 0.7 or 1% agarose gel electro-phoresis with Tris-acetate running buffer, and the gel wasphotographed as previously described (11). The concentra-tion of dsDNA or ssDNA was determined by measuring theoptical density at 260 nm (1.0 for 50 pLg of dsDNA or 33 ,ugof ssDNA per ml; 12) with a DU-40 spectrophotometer(Beckman Instruments, Inc., Fullerton, Calif.).

Electroporation-induced intact cell transformation. E. colielectroporation-induced competence was achieved as de-scribed previously (11). Electroporation-competent E. colicells were stored in a freezer at -70'C until use. C. aceto-butylicum and C. perfringens electroporation-induced intactcell transformation comretence was achieved as describedbelow. Late-exponentiaF-phase cultures (optical density at600 nm, 1.2) were harvested and washed with electropora-tion solution (10% PEG 8000 in distilled H20). The cellpellets were suspended in 1/20 of a volume of electroporationsolution, and 0.8 ml of the cell suspension was mixed withgreater than 10 (for C. acetobutylicum) or 1 (for C. perfrin-gens) ,ug of DNA. Electroporation was done with a GenePulser with a Pulse Controller (Bio-Rad Laboratories, Rich-mond, Calif.) set at 2,500 V, 25 ,uF, and infinite resistance,and the pulse delivery time ranged between 30 and 40 ms.Following postelectroporation incubation (10 min on ice),the cells were diluted into 9 volumes of Trypticase-glucose-yeast extract medium and incubated at 37°C for 7 h anaero-bically (C. acetobutylicum) or 1 h aerobically (C. pefrin-gens). Cells were plated on selective Trypticase-glucose-yeast extract agar plates and incubated anaerobicallyovernight at 37°C.

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Avalvail

HindPilCISl

Haelll ~~~~~~~~~~~indNllHaslil _ z~lEcoRVRF of CAK1 Pstl

6.6 Kbp

EcoRI

EcoRV EcoRIClai

AcclII

PsinaSmal

LHindllpAK1v25Kbp

E~coilIR.OAaHandlll

/

12 34

d

E coli R.O.

Pvull

Hindill

FIG. 1. Schematic illustration of the construction of phagemidpCAK1. Plasmid pAK102 was constructed in E. coli K802, and allother plasmids were constructed in E. coli DH5cx'. The RF ofpCAK1 was constructed by inserting uniquely cut, SmaI-restrictedpAK102 into the uniquely cut HaeIII site of the RF of CAKiisolated from C. acetobutylicum NCIB 6444. The bold line onpCAK1 indicates the RF of CAK1. R.O., replication origin.

FIG. 2. Agarose gel (0.7%) electrophoresis of pCAK1 isolatedfrom E. coli DH11S transformants. Lanes: 1, pCAK1 isolated byalkaline lysis; 2, ssDNA following phenol-chloroform extraction ofPEG-NaCl precipitate of E. coli culture supernatant; 3, Bal 31nuclease-treated ssDNA; 4, PEG-NaCl precipitate of E. coli culturesupernatant. The arrow indicates the position of intracellular (lane 1)and extracellular (lane 2) ssDNAs of pCAK1. d, dimer form; m,monomer form.

RESULTS

Construction of pCAKI phagemid. A schematic represen-tation of the construction of phagemid pCAK1 is shown inFig. 1. To investigate the presence of a functional intergenicregion of the CAK1 filamentous viruslike particle in E. colias a characteristic feature of filamentous phages of gram-negative microorganisms (e.g., M13, fd, and fl; 4, 20, 21, 23,33), we attempted to insert antibiotic marker resistancegenes (e.g., those for Apr, Cmr, and Emr) into the RF ofCAK1. Since these initial attempts were unsuccessful, wesubsequently constructed a gram-positive replication selec-tion vector, designated pAK102 (Fig. 1), by ligation ofHindIII-linearized pUC19 and a 2.3-kb HindIII erythromy-cin resistance gene fragment from plasmid pVA677 in E. coliK802. Plasmid pAK102, digested with suitable restrictionenzymes, was subsequently inserted into different sites ofpartially restricted (e.g., EcoRV, EcoRI, and HindIII) or theuniquely cut RF of CAK1 (e.g., AvaI and HaeIII) followinga strategy used by Messing et al. (21). The ligation mixtureswere transformed into either E. coli DH5at' or K802, andonly the insertion of SmaI-restricted pAK102 into a uniqueHaeIII site on the RF of CAK1 successfully producedampicillin- and erythromycin-resistant transformants withthe production of intracellular pCAK1 ssDNA (data notshown). The resultant 11.6-kbp presumptive pCAK1 phage-mid was isolated from E. coli transformants. The orientation

of plasmid pAK102 inserted into the RF of CAK1 wasdetermined by restriction enzyme digestion analysis (Fig. 1).These results suggested that replication of pCAK1 dsDNAdepends upon the ColEl replication origin derived frompAK102, although E. coli transformants containing pCAK1can produce intracellular ssDNA.

Isolation of viruslike particles from E. coli transformantscontaining phagemid pCAK1. To investigate the productionof viruslike particles by E. coli transformants containingpCAK1, pCAK1 was transformed into E. coli DH11S (16), astrain which has been used for preparation of highly purifiedssDNA (free of dsDNA) from phagemid vectors. Because ofthe nuclease associated with the periplasmic space of E. coliDH11S (3), culture supernatants harboring pCAK1 containssDNA complexed with protein that is very readily purified.On the other hand, transformation of pCAK1 into E. coliDH5at' resulted in the presence of dsDNA contamination,including chromosomal DNA in the supernatant. Culturesupernatants of E. coli DH11S transformants containingpCAK1 were subjected to PEG-NaCl precipitation. Theprotein-DNA complex was recovered in the absence of celllysis following PEG-NaCl precipitation and did not migrateinto agarose during agarose gel electrophoresis (Fig. 2, lane4). The proteins associated with ssDNA of pCAK1 demon-

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strated sensitivity to proteinase K and various solvents (i.e.,phenol or chloroform), similar to the results obtained previ-ously with CAK1 (12). ssDNA was subsequently purified byphenol-chloroform-isoamyl alcohol extraction, with or with-out proteinase K treatment, and ethanol precipitated (Fig. 2,lane 2). Purified pCAK1 ssDNA was subjected to digestionby Bal 31 nuclease, which has a specificity for linear dsDNA,as well as circular and linear ssDNAs (14). ExtracellularssDNA was removed by Bal 31 digestion (Fig. 2, lane 3) butnot when dsDNA-specific endonucleases HaeIII, EcoRI,HindIII, and PstI were used. These results indicate that theviruslike particles from E. coli contain ssDNA. The relativesize of pCAK1 (11.6 kb) was determined as described earlier(12). These results suggest that, at the very least, the CAK1VS replication origin, which is related to ssDNA generationby the rolling-circle replication mode (4, 6, 23, 28), is fullyfunctional in E. coli. Virally encoded proteins (e.g., gene2-like, gene 5-like, and coat proteins) related to viral propa-gation may be precisely expressed and regulated in E. colivia CAK1 promoters in the absence of cell death. Unregu-lated expression of the genes of gram-negative filamentousviruses in gram-negative microorganisms has been reportedto lead to host cell lysis because of blockage of the cellularmembrane (23, 26).

Transformation ofdsDNA and ssDNA ofpCAK1 into E. coliby electroporation. Because of the noninfective nature ofCAK1 in E. coli or C. acetobutylicum (12), the dsDNA andpurified extracellular ssDNA of pCAK1 were transformedinto E. coli DH5a' and DH11S by electroporation. Thisapproach enabled us to analyze the functionality of theCAK1 CS replication origin in E. coli. The transformationefficiency (number of transformants per microgram of DNA)of pCAK1 ssDNA was ca. 102, while that of pCAK1 dsDNAwas ca. 103. The ability to select ampicillin-resistant E. colitransformants following transformation of pCAK1 ssDNAsuggests that the CS replication origin sequence of CAK1 isrecognized by E. coli RNA polymerase I for initiation ofconversion of ssDNA into the RF of dsDNA (4, 23, 28, 33).E. coli RNA polymerase I forms an RNA primer from viralssDNA prior to dsDNA synthesis by DNA polymerases inthe absence of virally encoded protein expression (23, 33).

Transformation of phagemid pCAK1 into C. acetobutylicumand C. perfringens. To investigate the replication functional-ity of CAK1 in clostridia, pCAK1 isolated from E. colitransformants was transformed into C. acetobutylicumATCC 824 and C. perfringens 13. C. acetobutylicum and C.perfringens transformants were screened for resistance toerythromycin (25 jig/ml). Phagemid pCAK1 was trans-formed into C. acetobutylicum ATCC 824 and C. perfringens13 at an average transformation efficiency of 103 transfor-mants per ,ug ofDNA. pCAK1 was recovered predominantlyin the dimer form from either C. acetobutylicum (Fig. 3, lane3) or C. perfringens (data not shown). The fragments gener-ated following HindIII and PvuII digestion of pCAK1 iso-lated from E. coli, C. acetobutylicum, and C perfringenswere identical (data not shown). The results of HindIIIdigestion of pCAK1 isolated from both E. coli and C.acetobutylicum can be seen in Fig. 3, lanes 4 and 5,respectively. These results suggest that the replication originfrom the RF of CAK1 is functional in both C. acetobutyli-cum and C. perfringens. Viruslike particles containingpCAK1 ssDNA were not recovered, although results ob-tained with E. coli suggested that insertion of pAK102 intothe RF of CAK1 did not affect encoding sequences or CS andVS CAK1 replication origins. The high proteolytic andnucleolytic enzyme activities associated with the cell super-

1234 5

FIG. 3. Agarose gel (1%) electrophoresis of the HindIll-digestedRF of pCAK1. Lanes: 1, molecular size markers (HindIII-digested XDNA); 2, undigested pCAK1 isolated from E. coli DH5a' transfor-mants; 3, undigested pCAK1 isolated from C. acetobutylicumATCC 824 transformants; 4, HindIII-digested pCAK1 from E. coliDH5a' transformants; 5, HindIII-digested pCAK1 from C. aceto-butylicum ATCC 824 transformants.

natant of C. perfringens 13 may contribute to the inability torecover viruslike particles from this bacterium (data notshown). The mechanism of replication of pCAK1 in C.acetobutylicum and C. perfringens requires further exami-nation.

DISCUSSION

The results of this study suggested that the CAK1 virus-like particle is a defective filamentous phage closely relatedto Ff coliphages in terms of its genetic regulation for prop-agation of its progeny. However, CAKi also demonstrateddifferences which may reflect the genetic systems of eithergram-positive C. acetobutylicum or C. perfringens, andgram-negative E. coli. Generation of ssDNA following intro-duction of pCAK1 into E. coli, together with characteriza-tion of a shuttle vector which functions between E. coli andC. acetobutylicum or C. perfringens may be used in thedevelopment of M13-like genetic system for clostridia.The inability to select CAK1-based E. coli transformants

containing only antibiotic markers suggested that the repli-cation of the RF of pCAK1 dsDNA is dependent upon theexistence of ColEl replication machinery associated withpAK102. Although both the CS and VS replication origins ofCAK1 were functional in E. coli, it is possible that expres-sion of virally encoded proteins, especially gene 2-like

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protein (nickase) (4-6, 23, 33), is not sufficient to initiaterolling-circle DNA replication. The RF mode of replicationof pCAK1 may initially depend on the more efficient ColElreplication origin until there has been expression of a suffi-cient amount of gene 2-like proteins as a consequence of thehigh copy number of gene 2-like encoding sequences in E.coli (i.e., gene amplification).The VS replication origin of CAKi is more problematic in

E. coli than is the CS replication origin, which does notrequire virally encoded proteins for conversion of ssDNAinto dsDNA (4, 6, 23, 27). VS replication of CAKi forgeneration of pCAK1 ssDNA in E. coli may occur in thefollowing ways: (i) a fully functional protein encoded byCAK1, such as the gene 2-encoded protein of E. colifilamentous phages (4, 6, 23, 33), may bind to the VSreplication origin of CAKi and initiate viral replication bycleavage of specific sequences, and/or (ii) viral DNA repli-cation of phagemid pCAK1 in E. coli is coupled with aviruslike particle extrusion which requires an ssDNA-bind-ing protein and coat proteins, such as the gene 5- and gene8-encoded proteins of E. coli filamentous phages, respec-tively (4, 23, 24, 27). Proteins encoded by CAKi in E. colitransformants may be expressed by their own promoters andbe precisely regulated during ssDNA production. Improperor nonfunctional expression of ssDNA-binding protein andgene 5-like and gene 8-like coat proteins can lead to celldeath in E. coli (23, 26).Although the mode of acquisition of the CAKi filamentous

viruslike particle by C. acetobutylicum NCIB 6444 is notclear, the existence of homology between a natural phage-mid in gram-negative microorganisms (5) and ssDNA-gener-ating plasmids in gram-positive microorganisms (7) whichreplicate via the ssDNA replication mode (i.e., rolling-circlereplication) may be significant evidence for an evolutionarytransition between phages and plasmids. It is possible thatthe transition between phages and plasmids occurred as aconsequence of the high recombinatory ability of ssDNA-generating plasmids over time in bacteria (7). The inability ofmost ssDNA-generating plasmids from gram-positive micro-organisms to replicate in E. coli (7) may reflect a differentrate of evolutionary change at the DNA sequence level.Another interesting result of this study is the development

of simple electroporation protocols for C. acetobutylicumand C. perfringens with a 10% PEG solution. We suggestedearlier (11) that the cell wall structure represents an addi-tional barrier to DNA uptake in C. perfringens. The pres-ence of 10% PEG and the low ionic strength of the electro-poration solution result in a significant increase of the pulseduration time (30 to 40 ms), which may overcome the cellwall barrier. During electroporation-induced transformation,PEG may limit cytoplasmic leakage and, via exclusion of theaqueous phase, help to push the plasmid DNA into a cell andconsequently increase cell viability and transformation effi-ciency, as previously reported for transformation ofSchizosaccharomyces pombe (9). The difference in expres-sion periods following electroporation of C. acetobutylicumand C. perfringens (7 vs. 1 h) suggests that recovery of cellviability is related to the specific growth rate.The functionality of pCAK1 in the E. coli host system,

especially in generating ssDNA, in the absence of impair-ment of E. coli cell viability, together with successful intro-duction of pCAK1 into C. acetobutylicum and C. perfrin-gens, may be the basis for the construction of an M13-likegenetic system for the genus Clostridium and thereby allowmore sophisticated molecular genetic analysis of this genus.

ACKNOWLEDGMENTSThis work was supported in part by grant ICMB 91-0059-02 from

the Illinois Corn Marketing Board, State of Illinois CompetitiveValue-Added grant 1-1-11963, DOE Energy Bioscience grant DOEDEFG 02-91ER20046, University of Illinois Agricultural Experi-ment Station Hatch grant 50-313, and a UIUC Foundation Univer-sity Scholar grant to H.P.B.

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