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Journal of Bacteriology, December 2002, p. 6532-6544, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6532-6543.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Lactococcus lactis Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor

Evelyn Durmaz,1 Søren M. Madsen,2 Hans Israelsen,2 and Todd R. Klaenhammer1*

Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624,1 Biotechnological Institute, Horsholm, Denmark2

Received 28 February 2001/ Accepted 2 September 2002

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage {phi}31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage {phi}31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage {phi}31 was inhibited to an efficiency of plaquing (EOP) of 10-6 in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the {phi}31 EOP to <10-7. Phage {phi}31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the {phi}31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.

* Corresponding author. Mailing address: Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 515-2971. Fax: (919) 515-7124. E-mail: Klaenhammer{at}ncsu.edu.

Journal of Bacteriology, December 2002, p. 6532-6544, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6532-6543.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Durmaz, E., Klaenhammer, T. R. (2007). Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis. J. Bacteriol. 189: 1417-1425 [Abstract] [Full Text]  
  • Azcarate-Peril, M. A., McAuliffe, O., Altermann, E., Lick, S., Russell, W. M., Klaenhammer, T. R. (2005). Microarray Analysis of a Two-Component Regulatory System Involved in Acid Resistance and Proteolytic Activity in Lactobacillus acidophilus. Appl. Environ. Microbiol. 71: 5794-5804 [Abstract] [Full Text]  


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