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Applied and Environmental Microbiology, March 2001, p. 1128-1139, Vol. 67, No. 3
Biotechnological Institute, Department of Lactic Acid
Bacteria, 2970-Hørsholm, Denmark1;
Department of Viticulture and Enology, University of
California, Davis, California 956162;
Universal Preservation Technologies, Inc., San Diego,
California 921213; and Department
of Food Science, North Carolina State University, Raleigh, North
Carolina 276954
Received 29 August 2000/Accepted 18 December 2000
The DNA sequence of the replication module, part of the lysis
module, and remnants of a lysogenic module from the lytic P335 species
lactococcal bacteriophage
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1128-1139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Analysis of the Genetic Switch and Replication Region of a
P335-Type Bacteriophage with an Obligate Lytic Lifestyle on
Lactococcus lactis
31 was determined, and its regulatory elements were investigated. The identification
of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that 31
was derived from a temperate bacteriophage. Regulation of the
two early promoters was analyzed by primer extension and
transcriptional promoter fusions to a lacLM
reporter. The regulatory behavior of the promoter region
differed significantly from the genetic responses of
temperate Lactococcus lactis phages. The
cro gene homologue regulates its own production and is an
efficient repressor of cI gene expression. No detectable
cI gene expression could be measured in the presence of
cro. cI gene expression in the absence of
cro exerted minor influences on the regulation of the two
promoters within the genetic switch. Homology comparisons revealed a
replication module which is most likely expressed from the promoter
located upstream of the cro gene homologue. The
replication module encoded genes with strong homology to
helicases and primases found in several Streptococcus
thermophilus phages. Downstream of the primase
homologue, an AT-rich noncoding origin region was identified. The
characteristics and location of this region and its ability to reduce
the efficiency of plaquing of 31 106-fold when present
at high copy number in trans provide evidence for
identification of the phage origin of replication. Phage 31 is an
obligately lytic phage that was isolated from commercial dairy
fermentation environments. Neither a phage attachment site nor an
integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a
temperate phage ancestor. Several regions showing extensive DNA and
protein homologies to different temperate phages of
Lactococcus, Lactobacillus, and
Streptococcus were also discovered, indicating the likely
exchange of DNA cassettes through horizontal gene transfer in
the dynamic ecological environment of dairy fermentations.
*
Corresponding author. Mailing address: Department of
Food Science, Box 7624, Schaub Hall, North Carolina State University, Raleigh, NC 27695. Phone: (919) 515-2971. Fax: (919) 515-7124. E-mail:
Klaenhammer{at}ncsu.edu.
Paper FSR-00-19 of the Journal Series of the Department of Food
Science, North Carolina State University, Raleigh.
Applied and Environmental Microbiology, March 2001, p. 1128-1139, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1128-1139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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