New shuttle vectors for Actinobacillus actinomycetemcomitans and Escherichia coli

Gene, 169 (1996) 139-140 ©1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

139

GENE 09513

N e w shuttle vectors for Actinobacillus actinomycetemcomitans and Escherichia coli * (Recombinant DNA; plasmid; cosmid; lacZo~; pVT736-1; P15A ori; pSC101 replicon)

Yoshio Nakano, Yasuo Yoshida, Yoshihisa Yamashita and Toshihiko Koga Department of Preventive Dentistry, K yushu University Faculty of Dentistry, Fukuoka 812-82, Japan Received by J.W. Messing: 28 July 1995; Revised/Accepted: 25 September/12 October 1995; Received at publishers: 2 November 1995

SUMMARY

A series of shuttle vectors for Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was developed. These vectors carry a multiple cloning site, the lacZo~ reporter gene, the replication origin for Aa derived from pVT736-1 and a replicon for Ec originated from pUC, P15A or pSC101. With these vectors, cloning and expression can be effectively performed in Aa and Ec.

Actinobacillus actinomycetemcomitans (Aa) is a facultative Gram- rod. The organism is considered to be associated with localized juvenile (Zambon, 1985) and adult periodontitis (Petit et al., 1993). Molecular analysis of virulence factors is required for an understanding of the pathogenicity of Aa. pVT736-1, a rolling-circle plasmid, was isolated from Aa (Sreenivasan et al., 1991; Galli and LeBlanc, 1994) and an Aa/Escherichia coli (Ec) shuttle plasmid was constructed from pVT736-1 and pUC19 (Sreenivasan and Fives-Taylor, 1994). Some genes, however, are unstable or lethal in hcn vectors, such as pUC vectors in Ec. In such cases, lcn vectors (e.g., pSC101 derivatives) or icn vectors (e.g., pACYC derivatives) are Correspondence to: Dr. T. Koga, Department of Preventive Dentistry, Kyushu University Faculty of Dentistry, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan. Tel. (81-92) 641-1151; Fax (81-92) 641-3206; e-mail: [email protected] * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. The plasmids are available also upon request. Abbreviations: Aa, Actinobacillus actinomycetemcomitans (or actinomycetum-comitans); bp, base pair(s); Ec, Escherichia coli; hcn, high copy number; icn, intermediate copy number; kb, kilobase(s) or 1000 bp; Km, kanamycin; lcn, low copy number; MCS, multiple cloning sites; nt, nucleotide(s); Pollk, Klenow (large) fragment of Ec DNA polymerase I; g, resistance/resistant; Sp, spectinomycin; XGal, 5-bromo-4-chloro-3indolyl-D-~-galactopyranoside.

SSDI 0378-1119(95)00811-X

useful because this instability is presumably due to toxicity of these gene products synthesized at high level in host cell. We constructed three shuttle vectors based on hcn, icn and lcn replicons. Construction of pAES19, a hcn shuttle vector, is described in the legend to Fig. 1. Aa strain Y4 was transformed with pAES19 as described previously (Sreenivasan et al., 1991) and plasmids were isolated from several colonies. Restriction analysis indicated that these plasmids were consistent with the structure of pAES19. To clone and subclone genes which are unstable in hcn plasmids, an icn plasmid containing P15A replicon was constructed, pAES15 (Fig. 1) consists of P15A replicon and lacZ~ from pMBLcos (Nakano et al., 1995), and the Sp R gene and pVT replicon from pDL282. A chromosomal gene library was constructed with the shuttle plasmid and 2-10-kb EcoRI fragments of Aa chromosomal DNA. The gene library was stable in Ec, and Aa Y4 was able to be transformed with those plasmids. They were stably maintained in Aa cells. An lcn shuttle vector was also constructed. At first we tried to construct an lcn shuttle plasmid with the SpR gene and pSC101 replicon. However, no SpRplasmid with pSC101 replicon was obtained from unknown reasons. pAEK18 (Fig. 1) consists of the pSC101 replicon from pGD103 (Deich et al., 1988), the Km R gene from

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Fig. 1. The restriction maps of pAES19, pAES15 and pAEK18. The ori sites are the origins of DNA replication, pVTrep is the gene for the putative Rep protein derived from pVT736-1, repl01 is the gene encoding Repl01 that is essential for replication of pSC101. For construction of pAES19, plasmid pDL282 (Sreenivasanet al., 1991) was digested with BspHI + XhoI. The linearizedplasmid was blunt-endedwith PolIk and self-ligated.The resultant plasmid was digested with XbaI and blunt-endedwith Pollk. The linearized plasmid was digested with HindIII and ligated with the 516-bp AatII-HindIII fragment of pUC19 (Yanisch-Perronet al., 1985). The plasmid was isolated from the SpR Lac+ colonyand designated pAES19. pResKmMCS10 (Shiroza and Kuramitsu, 5993), pVT replicon from pDL282, and l a c Z ~ and MCS from pUC18. Although pAEK18 is maintained stably in Ec and Aa, the copy number of the plasmid in A a is lower than those of pAES19 and pAES55. The reason for this is unknown. The pSC505 replicon does not work in Aa because Aa Y4 is not transformed with pGD503. The vectors described here have the following useful properties: (i) They contain many more unique cloning sites than other A a / E c shuttle vectors. (ii) They harbour MCS and the lacZ~ gene allowing rapid colour screening for inserts in Ec on XGal indicator plates and plasmids purified from Ec or Aa can be sequenced with commercial primers. (iii) They were constructed with three types of replicons. Generally, lcn plasmids are more stable in Ec than in Aa. pAES15 and pAEK18 are useful for cloning or subcloning genes which are unstable and/or toxic in hcn vectors. In addition, these replicons are compatible with each other. This compatibility is useful for genetic complementation studies.

ACKNOWLEDGEMENTS We thank Dr. Paula Fives-Taylor for the gift of pDL282. This work was supported in part by a Grantin-Aid for Developmental Scientific Research 07557536 from the Ministry of Education, Science, and Culture of

Japan and Funds for Comprehensive Research on Aging and Health.

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