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Lactococcus Lactis Bacteriophage Lysin Encoded by Two Overlapping Genes CLAIRE A

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Lactococcus Lactis Bacteriophage Lysin Encoded by Two Overlapping Genes CLAIRE A APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3063-3073 Vol. 60, No. 9 0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology Controlled Expression and Structural Organization of a Lactococcus lactis Bacteriophage Lysin Encoded by Two Overlapping Genes CLAIRE A. SHEARMAN,* KAREN L. JURY, AND MICHAEL J. GASSON Institute of Food Research, Norwich, United Kingdom Received 10 January 1994/Accepted 20 June 1994 The +vMI bacteriophage lysin is specific for lactococci and could be used to promote enzyme release during cheese manufacture. The level of lysin expression from the cloned gene using its own upstream sequences is very low. Expression in Escherichia coli by using a synthetic hybrid lysin gene and a series of BAL 31 deletions of the original cloned DNA fragment suggested that the start of the gene had previously been incorrectly assigned. Reevaluation of homology between the lysin and Bacillus subtilis PZA protein 15 led to the identification of a new potential ribosome binding site (RBS). A 0.72-kb PCR-generated fragment including this RBS and the complete lysin gene was expressed and inducibly controlled. The translational start of the lysin gene was identified as an isoleucine codon, and this may lead to a low translation rate. During the analysis of the BAL 31 deletion fragments, two proteins of 20 and 8 kDa were shown to be expressed from the originally defined lysin gene. The DNA sequence has a second open reading frame with a good RBS and two potential start methionines. The smaller lysin protein was isolated, and the N terminus was sequenced, confirming that one methionine codon acted as the start of a second gene. The larger lysin protein has homology with lysozymes. The smaller lysin protein has some features resembling those of a holin. The possible roles of these two proteins in lysis of lactococci are discussed. Bacteriophage lysins degrade bacterial cell walls. fvML3 is controlled expression by an inducible promoter. The start of a prolate-headed bacteriophage that attacks the related Lac- the lysin protein was redefined following evaluation of its tococcus lactis strains 712, C2, and ML3 (11). The lysin is highly homology with PZA protein 15. During this study, two over- specific for lactococci. Many dairy lactococci that are not lapping genes within the lysin sequence were identified, and natural hosts for the intact bacteriophage are susceptible to their possible roles in lysis are discussed. lysis by purified lysin. The 4~vML3 lysin gene has been cloned and sequenced (49), as have other prolate-headed bacterio- phage lysins, including those from P001 (19) and c2 (61). The MATERUILS AND METHODS DNA and amino acid sequences of these lysins have high levels of homology, and the 4)vML3 lysin gene hybridizes with DNA Bacterial strains, plasmids, and culture conditions. Esche- from a wide variety of prolate-headed phages (50). richia coli strains used were MC1022 (8), TG1 (20), JM107 During cheese maturation (23, 40), starter cells lyse releas- (34), JM109 (62), and JM109(DE3). JM107 was supplied by ing intracellular peptidases which contribute to the proteolytic Life Technologies Ltd. Strains JM109 and JM109(DE3) and breakdown of the casein to peptides and amino acids (31). The the pSP73 plasmid with the T7 promoter comprising the rate at which the starter cells lyse is now recognized as an inducible T7 system (54, 55) were obtained from Promega. important factor in flavor development. Differences in the rate Induction of the T7 promoter was carried out as described by of maturation can be shown for starter cultures that lyse at Studier et al. (55). Other E. coli strains used include MC1022 different rates either because of inherent differences such as derivatives F15781 carrying pFI106 (1.2-kb EcoRI lysin frag- susceptibility to autolysis or following treatment with lysozyme ment) and F15965 carrying pFI151 (0.84-kb DraI-EcoRI lysin (30) or sublethal heat shock (13, 58). Phage-resistant strains fragment) (49). Culture conditions were as described by Sam- have been associated with the manufacture of a blander brook et al. (46), with selection on L agar (33) with, when cheese, and it has been suggested that low levels of lysis occur appropriate, 50 ,ug of ampicillin, 15 ,ug of chloramphenicol, when phage-sensitive strains are used and that this contributes 500 ,ug of erythromycin, 10 ,ug of tetracycline, 20 ,ug of to flavor (32). isopropyl-3-D-thiogalactopyranoside (IPTG), and 20 ,ug of Thus, the lysin and its gene have potential as growth and 5-bromo-4-chloro-3-indolyl-1B-D-galactoside (X-Gal) per ml. acid production inhibitors and as agents to effect early starter The tac promoter (ptac) genblock supplied as a HindIll- cell lysis and intracellular enzyme release in dairy fermenta- BamHI cassette, plasmid MC1871, which contains a truncated tions. The expression of the cloned lysin in a lactococcal E. coli ,3-galactosidase gene (48), and pUC18 (SmaI digested) background caused lysis when cells reached stationary phase were all supplied by Pharmacia. Lactococcal strains used were (51). However, to exploit the lysin fully, it is necessary to obtained from a collection maintained at the Institute of Food control lysin gene expression in lactococci. Here we report the Research and included L. lactis MG1363 (17) and F17197, an further characterization of the lysin gene and demonstrate its MG1363 derivative carrying pFI145 which expresses lysin from its own upstream sequences (51). Lactococcal strains were grown at 30°C on M17 medium (57) with 0.5% glucose as the * Corresponding author. Mailing address: Institute of Food Re- carbon source (GM17). The E. coli-Lactococcus shuttle vectors search, Norwich Research Park, Colney, Norwich NR4 7UA, United pTG262 (A. Mercenier, Transgene) and pCK1 (18) were Kingdom. Phone: (0603) 255243. Fax: (0603) 507723. selected on S ,ug of chloramphenicol per ml in lactococci. 3063 3064 SHEARMAN ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains and plasmids produced in this study Strain Parental strain Plasmid Derivation FI5982 MC1022 pFI152 Synthetic 130-bp PstI-HincII cloned into pFI140 FI5886 MC1022 pFI140 0.5-kb HincII-EcoRI C-terminal lysin fragment FI6093 MC1022 pFI210 0.49-kb BAL 31 deletion in pTG262 SmaI-EcoRI FI6094 MC1022 pFI211 0.48-kb BAL 31 deletion in pTG262 SmaI-EcoRI FI6095 MC1022 pFI212 0.47-kb BAL 31 deletion in pTG262 Smal-EcoRI FI6096 MC1022 pFI213 0.71-kb BAL 31 deletion in pTG262 Smal-EcoRI FI6097 MC1022 pFI214 0.5-kb BAL 31 deletion in pTG262 SmaI-EcoRI FI6099 MC1022 pFI216 0.65-kb BAL 31 deletion in pTG262 SmaI-EcoRI FI7001 MC1022 pFI218 0.26-kb BAL 31 deletion in pTG262 SmaI-EcoRI FI7002 MC1022 pFI219 0.64-kb BAL 31 deletion in pTG262 SmaI-EcoRI F17003 MC1022 pFI220 0.58-kb BAL 31 deletion in pTG262 SmaI-EcoRI F17007 MC1022 pFI224 0.66-kb BAL 31 deletion in pTG262 Smal-EcoRI FI7008 MC1022 pFI225 0.63-kb BAL 31 deletion in pTG262 SmaI-EcoRI F17009 MC1022 pFI226 0.62-kb BAL 31 deletion in pTG262 Smal-EcoRI F17010 JM107 pFI190 HindIII-BamHI ptac genblock cloned into pFI152 F17254 JM109(DE3) pFI308 0.71-kb HindIII-EcoRI fragment from pFI213 in pSP73 FI7255 JM109(DE3) pFI309 0.63-kb HindIII-EcoRI fragment from pFI225 in pSP73 F17256 JM109(DE3) pFI310 0.5-kb HindIII-EcoRI fragment from pFI140 in pSP73 F17257 JM109(DE3) pFI311 0.26-kb HindIII-EcoRI fragment from pFI218 in pSP73 FI7263 JM109(DE3) pFI313 0.84-kb HindIII-EcoRI fragment from pFI151 in pSP73 FI7550 MC1022 pFI507 0.34-kb DraI-HincII fragment from pFI151 in pMC1871 FI7633 MC1022 pFI549 3.3-kb PstI fragment from pFI507 in pCK1 PvuII F17657 MG1363 pFI549 3.3-kb PstI fragment from pFI507 in pCK1 PvuII F17816 TG1 pFI664 0.71-kb BamHI-EcoRI fragment from pFI213 into pFI190 F17818 TG1 pFI662 0.72-kb PCR fragment cloned into pUC18 SmaI F17819 TG1 pFI663 As pFI662 but in reverse orientation F17876 JM109(DE3) pFI699 0.72-kb EcoRI fragment from pFI662 into pSP73 FI7885 MC1022 pFI703 0.84-kb DraI-EcoRI fragment in pTG262 SmaI-EcoRI FI7886 MG1363 pFI703 0.84-kb DraI-EcoRI fragment in pTG262 Smal-EcoRI Other bacterial strains and plasmids produced during the database has been altered accordingly (accession number course of this work are described in Table 1. X16178). The amino acid residue numbers of the lysin protein Molecular techniques. Standard techniques were carried out are started from the isoleucine codon at position 505, based on as detailed by Sambrook et al. (46). Restriction enzymes and data presented in this report. BAL 31 exonuclease (Life Technologies), T4 DNA ligase and DNA primer synthesis. Primer 20 and oligomers 6 and 7 Klenow polymerase (Pharmacia), Taq polymerase (Promega), were synthesized on an Applied Biosystems Inc. model 392 and calf intestinal alkaline phosphatase (Boehringer Mann- DNA synthesizer. The trityl residue of oligomers 6 and 7 was heim) were all used as instructed by the manufacturers. PCR left on at the end of the synthesis. Oligonucleotide purification was used to generate a structural lysin fragment, using primer cartridges (Applied Biosystems) were used after the oligomers 20 (5'TAATTGGAGGTGGTAAT3', positions 488 to 504) were cleaved from the support column and deprotected by and the SP6 primer (5'A'TTTAGGTGACACTATA3') exter- using normal procedures. This allowed the full-length oli- nal to the cloned lysin fragment. PCRs were performed with 1 gomers to be isolated free from shorter oligomers. ,ug of template DNA in a 50-,I volume of PCR mixture (1x Protein gels. Total proteins were prepared from the E. coli Taq buffer supplemented with 0.1 mM each deoxynucleoside strains carrying various plasmids both with and without induc- triphosphate) containing 100 ng of each primer and 1 U of Taq tion.
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