Transformation of Acholeplasma laidlawii with streptococcal plasmids pVA868 and pVA920

PLASMID

21,

155-160 (1989)

Transformation

of Acholeplasma Plasmids pVA868 KEVIN

laidlawii with Streptococcal and pVA920

DYBVIG

Departments of Comparative Medicine and Microbiology, University of Alabama ar Birmingham, Birmingham, Alabama 35294 Received December 22, 1988; revised February 17, 1989 One limitation with studying mycoplasma genetics is the lack of cloning vectors. Studies were undertaken to determine whether stmptococcal plasmids could replicate in Achokplasma laidlawii, for the purpose of identifying potential vectors. Plasmids pVA868 and pVA920 contain the same origin of replication and tetracycline resistance determinant. pVA920 also contains an crythromycin msistance determinant not present in pVA868. A. laidlawii was transformed with plasmids pVA868 ( 13.7 kb) and pVA920 ( 12.2 kb), and isolated from the transformants were deletion derivatives of the parent plasmids having sizes of 3.7 and 10.3 kb, respectively. The tetracycline and erythromycin resistance markers functioned in A. laidlawii, and the deletion derivatives may bc useful for development of mycoplasma vectors. However, difficulties may arise due to plasmid instability. 0 1989 Academic

press, Inc.

Mycoplasmas are a large group of procary- Tn916 functions in several mycoplasma speotes, comprising five genera, for which no cies (Dybvig and Cassell, 1987; Dybvig and cloning vectors currently exist. There are sev- Alderete, 1988; Roberts and Kenny, 1987). In the studies described here, plasmids isoeral possible approaches for the development of vectors that will replicate in mycoplasmas. lated from gram-positive bacteria were One is to combine a selectable marker with screened in Acholeplasma laidlawii to deteran origin of replication (ori) obtained from mine whether they replicate in mycoplasmas. plasmids (Bergemann and Finch, 1988; One reason for choosing A. laidlawii is the Randhand et al., 1980) or viruses (Maniloff, availability of transformation procedures 1988) isolated from mycoplasmas. One avail- (Sladek and Maniloff, 1983; Lorenz et al., able marker is the streptococcal tetracycline1988). Another reason is that background inresistance (Tcy ’ determinant tetA4 (Burdett et formation is available regarding restriction and al., 1982), a marker known to function in my- modification systems. In particular, A. Zaidcoplasmas (Dybvig and Cassell, 1987; Dybvig lawii strain 8 195 is a restriction and modifiand Alderete, 1988; Roberts et al., 1985; Rob- cation minus derivative of strain JA 1 (Sladek erts and Kenny, 1986). Another approach for et al., 1986). Described here is transformation development of mycoplasma vectors is to uti- of A. laidlawii with streptococcal plasmids lize oris from other systems. Although my- pVA868 and pVA920 (Tobian et al., 1984). coplasmas are wall-less, they have evolved A. Iaidlawii strain 8 195 was grown in myfrom gram-positive bacteria (Maniloff, 1983). coplasma medium (Dybvig and Cassell, 1987) The feasibility of utilizing oris isolated from for most studies, but tryptose broth (Liss and gram-positive bacteria gains impetus from Maniloff, 197 1) was used to grow cells for studies showing that gram-positive transposon transformation experiments. Cells were assayed as colony-forming units (CFU) on mycoplasma agar plates (Dybvig and Cassell, ’ Abbreviations used: Tc’, tetracycline resistance; CFU, 1987). Tetracycline or erythromycin at a concolony-forming units; Em’, erythromycin resistance; Em’, centration of 2 &ml was incorporated into erythromycin sensitive; Td, tetracycline sensitive; EOP, efficiency of plating. the medium for antibiotic selection, Strepto155

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coccal plasmids pVA868 and pVA920 were isolated from Streptococcussanguis (Challis) as described by Tobian and Macrina (1982). Plasmids ( 10 pg) were transformed into A. laidlawii (lo* CFU) using the polyethylene glycol-mediated procedure described previously (Dybvig and Cassell, 1987). Transformed cells were incubated in nonselective medium for 2 h at 37°C and assayedon mycoplasma agar plates containing selective concentrations of antibiotic. Streptococcalplasmid pVA868 contains the tetA4 gene as a selectable marker and the ori from pVA380-1 (Macrina et al., 1980). A. laidlawii was transformed with pVA868 and a single Tc’ transformant was obtained. The plasmid isolated from this transformant was a deletion derivative of pVA868 and is designated pKJ 1. For most experiments, pIU 1 was isolated using a cleared lysate procedure. Cultures (500 ml) were grown maintaining antibiotic selection. Cells were harvested by centrifugation (10 min at SOOOg),washed in ice-cold phosphate-buffered saline, and resuspended in 20 ml of 5% sucrose in buffer consisting of 50 mM Tris and 100 mM EDTA, pH 8.0. Cells were gently lysed by the addition of 4 ml of 10%sodium dodecyl sulfate, and 6 ml of 5 M NaCl was added. After overnight storage at 4°C the lysate was centrifuged at 25,OOOg for 1 h at 4°C. The supernatant was collected and extracted twice with a 1:1 mixture of phenol and chloroform. The aqueous phase was extracted with ether, and nucleic acids were precipitated by the addition of 2 vol of icecold ethanol. After incubation at -20°C for 1 h, nucleic acids were recovered by centrifugation for 10 min in a microcentrifuge (Eppendorf). RNA was removed by digestion with RNase A followed by phenol extraction and ethanol precipitation. The resulting DNA was used for transformation studies. For mapping of restriction sites, chromosomal DNA was removed by banding the plasmid on cesium chloride-ethidium bromide gradients (Maniatis et al., 1982). About 70 pg of pKJ1 was obtained from 1 liter of culture. pKJ1 isolated using the cleared lysate procedure was predominantly superhelical, co-

valently closed circular DNA with some open circular DNA present as well. The size of the plasmid was only 3.7 kb which is 10 kb smaller than the parent plasmid. An alternative method employed to isolate pKJ1 was the alkaline lysis procedure designed for isolation of plasmids from Escherichia coli (Maniatis et al., 1982). Plasmid DNAs were analyzed by electrophoresis in 0.8% agarose gels stained with ethidium bromide as described (Dybvig et al., 1986). The predominant form of pKJ1 obtained by alkaline lysis migrated faster on the gels than the double-stranded, superhelical form of pKJ1 obtained by the cleared lysate procedure. This faster-migrating form was resistant to digestion with most restriction endonucleases but degraded by digestion with 10 units of Sl nuclease (Bethesda Research Laboratories) at 37°C for 30 min using previously described conditions (Dybvig et al., 1985). These data suggest that alkaline lysis resulted in isolation of a single-stranded form of pKJ 1. This supposition is supported by several reports (e.g.,te Riele et al., 1986a;de1Solar et al., 1987) describing plasmids from grampositive bacteria that have single-stranded forms with relative mobilities on agarosegels similar to the single-stranded form of pKJ1 described here. Restriction maps of pVA868 and pKJ 1 were constructed (Fig. 1A). A similar map of pVA868 hasbeen published elsewhere(Tobian et al., 1984). The ori is located between the Hind111 site at position 10.6 clockwise to the Hind111 site at position 13.7 (Tobian et al., 1984). The tetMgene is located in the vicinity of the St1 site at position 1.4 (Tobian et al., 1984). Two approaches were used to determine the relationship between pVA868 and pKJ1. The first approach was to analyze restriction endonuclease digests of these DNAs on agarosegels to identify fragments in common. The second approach was to analyze digests of pVA868 on Southern blots probed with pKJ 1 labeled with 32Pby nick translation as described (Maniatis et al., 1982). Hybridization with pKJ 1 was limited to the region of pVA868 depicted by the inner line in Fig. 1A. pKJ1 must contain the tetA4 gene because it

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A

PIG. 1. Restriction maps of pVA868 and pVA920 showing relationships to pKJ 1 and pKJ3. (A) pVA868 (13.7 kb) shown by the outer circle with pKJ1 (3.7 kb) sequencesshown by the inner line. (B) pVA920 (12.2 kb) shown by the outer circle with pKJ3 (10.3 kb) shown by the inner line. Parenthesesin (A) indicate that additional, unmapped sites for the enzyme exist in pVA868. Maps were constructed by analysis of restriction endonuclease-digestedDNAs on 0.8% agarosegels as previously described (Dybvig et al, 1986).

confers tetracycline resistance to A. laidlawii and it has retained homology to another teMcontaining plasmid, pJI3 (Burdett et al., 1982). Presumably, pKJ1 utilizes the same ori as pVA868. It is unlikely that pKJ 1 acquired an ori from its host becauseSouthern blot analysis failed to detect any homology between pKJ 1 and chromosomal DNA from A. laidlawii. Streptococcal plasmid pVA920 contains the same ori and tetM gene as pVA868. In addition, pVA920 contains an erythromycin-resistance (Em”) determinant. A. laidlawii was transformed with pVA920 and a single Tc’ transformant was obtained. The plasmid isolated from this transformant was a deletion derivative of pVA920 and is designated pKJ3. The restriction maps of pVA920 (12.2 kb) and pKJ3 (10.3 kb) are given in Fig. 1B. A similar map of pVA920 has been published elsewhere (Tobian et al., 1984). Deleted in pKJ3 are HindIII, CM, and Hue111cleavagesiteslocated within pVA920 sequencesoriginally derived from pVA380-1. The Em’ determinant of pVA920 is adjacent to these cleavage sites in the vicinity of map position 7 (Tobian et al., 1984). Since this determinant is still present in pKJ3 (seebelow), the deletion that resulted in pKJ3 was primarily limited to pVA380-1 sequences. The host strain of A. laidlawii used in these studies did not form colonies on agar contain-

ing selective concentrations of either tetracycline or erythromycin; the spontaneous mutation rate for resistance to either antibiotic was
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isolated from cells transformed with pKJ 1 had restriction maps indistinguishable from the parent plasmid. To examine the stability of pKJ 1 in A. Zuidluwii, stocks of cells containing pKJ1 were passagedby daily transfer ( 1:100 dilution) into fresh medium lacking tetracycline. Passaged stocks were assayedfor CFUs on agar, and the efficiency of plating (EOP) was calculated as the ratio of the number of colonies on agar supplemented with tetracycline divided by the number of colonies on drug-free agar. The EOP of stocks passagedone, two, and three times was 1,0.5, and 0.2, respectively. Control stockspassagedin medium supplemented with tetracycline maintained an EOP of 1. To further evaluate the apparent loss of tetracycline resistance in some cells, passagedstocks were randomly subcloned. One out of six subclones from the stock passagedtwo times was Tc” while eight out of eight subclones from the stock passagedthree times were TcS. DNAs isolated from TcSsubclones were probed with pKJ 1 on Southern blots. No hybridization was observedindicating that the entire plasmid had been lost. The stability of pKJ3 in A. laidlawii was similarly examined. Cells were passagedthree times in the absence of antibiotics and subcloned. Twenty-one subcloneswere examined for antibiotic sensitivity. The phenotype of 6 subcloneswas T&m’. The other 15 subclones were TcSEm”,suggestingagain that the entire plasmid was lost. The loss of plasmids pKJ1 and pKJ3 during cell passaging suggeststhat these plasmids are not always partitioned into each daughter cell during division. Further experiments were designed to determine whether deletions in pKJ3 can occur during passaging,perhaps at a frequency lower than loss of the total plasmid. After passagingthree times in the absence of antibiotics, the stock was split and passageda fourth time in the presence of either tetracycline or erythromytin. These stocks were subcloned maintaining selection with one antibiotic, and the phenotypes of the individual subclones were examined. For the stock in which tetracycline selection was maintained, all subclones ( 12/ 12)

were Tc’Em’. For the stock in which erythromycin selection was maintained, 11 out of 12 subclones were Tc’Em’; one subclone was Tc”Em’. The finding that the ori from pVA380-1 functions in A. laidlawii was an unexpected result becausethis ori had not previously been shown to function in organisms other than streptococci. Attempts to transform Mycoplasma pulmonis using previously described methods (Dybvig and Alderete, 1988) with pVA868, pVA920, pKJ1, and pKJ3 were unsuccessful, suggesting that the host range of these plasmids among the mycoplasmas may be limited to Acholeplusma. However, plasmids pKJ 1 and pKJ3 have retained the ability to replicate in streptococci. These plasmids have been successfully transformed into Streptococcus mutans (S. Hollingshead, personal communication). Isolation of pKJ 1 using protocols involving alkaline lysis resulted in DNA with singlestranded characteristics. Perhaps pKJ 1 can exist in an alkaline-sensitive form similar to that of the relaxation complex described for colicinogenic factor E, (Clewell and Helinski, 1970). Gram-positive bacterial plasmids for which single-strandedforms have been isolated are thought to replicate via a rolling circle mechanism (Gros et al., 1987; te Riele et al., 1986b; de1 Solar et al., 1987). Instability of these plasmids is common (Gruss et al., 1987; de1Solar et al., 1987; Bron et al., 1988), perhaps as a result of recombination stimulated by single-stranded DNA (Ehrlich et al., 1986). A similar phenomenon may be occurring with the plasmids described here. When first transformed into A. laidlawii, pVA868 and pVA920 underwent deletions of an intriguing nature. The low transformation frequencies (< 10e8 per CFU) with pVA868 and pVA920 suggestthat most of the incoming molecules underwent deletions which interfered with plasmid replication or expression of antibiotic resistance;pKJ 1 and pKJ3 would have been generated by relatively rare events. Higher transformation frequencies were obtained with pKJ 1 and pKJ3 ( 1Op6per CFU), which is consistent with the lack of observed

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tucuccus. In “Microbiology-1982” (D. Schlesinger,Ed.), deletions in pKJ 1. The relatively high transpp. 155-158. Amer. Sot. Microbial. Washington, DC. formation frequency with pKJ3 suggeststhat D. B., AND HELINSKI, D. R. (1970). Properties deletions that occurred in this plasmid were CLEWELL, of a supercoiled deoxyribonucleic acid-protein relaxalimited to sequencesnot involved with plastion complex and strand specificity of the relaxation mid replication or expression of both antievent. Biockemistry 9,4428-4440. DEL SOLAR, G. H., PLTYET,A., AND EsPIN~sA, M. (1987). biotic resistance determinants. Initiation signals for the conversion of single stranded In generation of pKJ 1 and pKJ3, the same to double stranded DNA forms in the strcptococcal region corresponding to a portion of pVA380plasmid pLS1. Nucleic Acids Rex 15, 5561-5580. 1 sequenceswas deleted. This region corre- DYBVIG, K., AND ALDERETE, J. (1988). Transformation sponds to pVA868 sequences in the neighof Mycoplasma pulmonis and Mycophna kyorkinis: Transposition of Tn916 and formation of cointegrate borhood of map position 11. Whether there is structures. Plasmid 20, 33-4 I. something specific about this region that proDYBVIG, K., AND CASSELL, G. H. (1987). Transposition motes instability in A. laidlawii remains to be of gram-positive transposon Tn916 in Ackoleplasma determined. The finding that two types of laidlawii and Mycoplasma pulmonis. ScienceX35,1392deletions occurred when A. laidlawii was 1394. transformed with pKJ3, some derivatives were DYBVIG, K., NOWAK, J. A., SLADEK, T. L., AND MANILoFF, J. (1985). Identification of an enveloped phage, TcTEmSand others were Tc*Em’, indicates that mycoplasma virus L172, that contains a ICkilobase deletion events were not limited to a single single-stranded DNA genome. J. Viral. 53, 384-390. region. The transformation processitself may DYBVIG, K., SLADEK, T. L., AND MANILOFF, J. (1986). generate deletions since pKJ3 was relatively Isolation of mycoplasma virus L2 insertion variants and miniviruses. J. Viroi. 59, 584-590. stable while maintained in A. iaidlawii. pK.J1 was stable as long as antibiotic selec- EHRLICH, S. D., NOIROT, P. H., PETIT, M. A., JANNIERE, L., MICHEL, B., AND TE RIELE, H. (1986). Strueturai tion was maintained, and it may be useful as instability of Bacillus subtilis plasmids. In “Genetic Ena vector. Its single Hind111site should be useful gineering” (J. K. Setlow and A. Hollaender, Eds.), Vol. for cloning purposes becauseit corresponds to 8, pp. 7 l-83. Plenum, New York. the junction between pVA380-1 and S. mu- GROS, M. F., TE RIELE, H., AND EHRLICH, S. D. (1987). Rolling circle replication of single-stranded DNA plastans sequences in pVA868 (Tobian et al., mid pC194. EMBO J. 6,3863-3869. 1984). Thus, this site should not be involved GRUSS, A. D., Ross, H. F., AND NOVICK, R. P. (1987). with plasmid replication or expression of anFunctional analysis of a palindromic sequencerequired tibiotic resistance.It remains to be determined for normal replication of several staphIyoeoecaI plas mids. Proc. Natl. Acad. Sci. USA 84,2165-2169. whether the propensity of the plasmids described here to undergo deletions will lead to LISS, A., AND MANILOFF, J. (1971). Isolation of mycoplasmatales viruses and characterization of MVLl, instability of inserted sequences.

MVL52, and MVGS 1. Science 173,725-727. LORENZ, A., JUST, W., CARWSO, M. S., AND KLOTZ, G.

ACKNOWLEDGMENTS This work was supported by Grant RR00959 from the Animal Resources Branch of the National Institutes of Health. I thank G. Cassell for her support and assistance. REFERENCES BERGEMANN,A. D., AND FINCH, L. R. (1988). Isolation and restriction endonucleaseanalysis of a mycoplasma plasmid. Phmid 19,68-70. BRON, S., LUXEN, E., AND SWART, P. (1988). Instability of recombinant pUBll0 plasmids in Bacillus subtilis: PIasmidencoded stability function and effects of DNA inserts. Plasmid 19,231-241. BURDETT, V., INAMINE,J., AND RAJAGOPALAN, S. ( 1982).

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