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Applied and Environmental Microbiology, February 2006, p. 1110-1117, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1110-1117.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

Allan L. Delisle,1* Gerard J. Barcak,2 and Ming Guo1

Department of Biomedical Sciences, School of Dentistry,1 Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Baltimore, Maryland 212012

Received 28 July 2005/ Accepted 7 November 2005

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective {lambda} holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.

* Corresponding author. Mailing address: Department of Biomedical Sciences, School of Dentistry, University of Maryland, Baltimore, Baltimore, MD 21201. Phone: (410) 706-7538. Fax: (410) 706-0193. E-mail: adelisle{at}umaryland.edu.

Applied and Environmental Microbiology, February 2006, p. 1110-1117, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1110-1117.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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Copyright © 2006 by the American Society for Microbiology. All rights reserved.