Localization, cloning, and expression of genetic determinants for bacteriophage resistance (Hsp) from the conjugative plasmid pTR2030

• Published in: Applied and Environmental Microbiology Vol. 55; no. 7; pp. 1684 - 1689
• Main Authors: Hill, C. (North Carolina State University, Raleigh, NC), Romero, D.A, McKenney, D.S, Finer, K.R, Klaenhammer, T.R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-07-1989
• Subjects: BACTERIOFAGOS; BACTERIOPHAGE; Bacteriophages; Biological and medical sciences; Biotechnology; CLONACION; CLONAGE; Cloning, Molecular; Coliphages; Conjugation, Genetic; DNA, Bacterial - analysis; DNA, Bacterial - genetics; Escherichia coli - genetics; Food industries; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genes, Bacterial; Genetic engineering; Genetic technics; Heat-Shock Proteins - genetics; IDENTIFICACION; IDENTIFICATION; Lactococcus lactis - genetics; Methods. Procedures. Technologies; Milk and cheese industries. Ice creams; Mutation; PLASMIDE; PLASMIDIOS; Plasmids; Protein Biosynthesis; Restriction Mapping; STREPTOCOCCUS; Transcription, Genetic; Virus; Chemosensitivity; Bacteria; Lactobacillaceae; Genetics; Lactobacillus lactis; Phage; Lactococcus lactis; Nucleotide sequence; Determinant; Escherichia coli; Gene expression; Strain; Plasmid; Resistance; Streptococcaceae; Gene; Mutagenesis; Restriction map; Shuttle vector; Micrococcales; Genetic engineering; Molecular cloning; Escherichieae; Enterobacteriaceae
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.55.7.1684-1689.1989

Genetic determinants for a bacteriophage resistance mechanism (Hsp+) encoded by plasmid pTR2030 (46.2 kilobases [kb]) were localized by mapping an 11.5-kb deletion that accompanied the transition of Lactococcus lactis LMA12-4 transconjugants (M.E. Sanders, P.J. Leonard, W.D. Sing, and T.R. Klaenhammer, Appl. Environ. Microbial. 52:1001-1007, 1986) from phage resistance to phage sensitivity. The deleted 34.7-kb replicon (pTR2023, Hsp-) retained its conjugative ability, demonstrating that the phage resistance and conjugal transfer determinants were genetically distinct. The Hsp region of pTR2030, which was contained within a 13.6-kb BglII fragment, was cloned into the BamHI site of bacteriophage lambda EMBL3, and Hsp was subcloned into the Escherichia coli-Streptococcus shuttle vector pSA3. The recombinant plasmids pTK6 and pTK9 were recovered in E. coli HB101 and contained a 13.6-kb insert in opposite orientations. L. lactis MG1363 transformants carrying pTK6 or pTK9 exhibited a significant reduction in plaque size, in addition to a slight reduction in the efficiency of plaquing for both prolate and small isometric phages. Phenotypic reactions observed for the recombinant plasmids suggest that pTR2030-encoded Hsp acts similarly against both prolate and small isometric phages. Tn5 mutagenesis was used to define the region essential for the expression of the Hsp+ phenotype. Any of four insertions within a 3-kb region resulted in the loss of phage resistance, whereas a further 26 insertions outside this locus had no effect on Hsp expression. In vitro deletion analysis confirmed that the 3-kb region contained all the information necessary for the observed resistance