Inhibition of bacteriophage replication in Streptococcus thermophilus by subunit poisoning of primase.
Invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutations within the putative primase of Streptococcus thermophilus phage kappa3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the kappa3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.
Sturino
Joseph M
JM
Genomic Sciences Program, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624, USA.
Klaenhammer
Todd R
TR
Genomic Sciences Program, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624, USA.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D018389:Codon, Nonsense; D019915:DNA Primase / Q000037:antagonists & inhibitors* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics; D017108:Streptococcus Phages / Q000254:growth & development*; D048249:Streptococcus thermophilus / Q000821:virology*; D010948:Viral Plaque Assay; D014779:Virus Replication / Q000502:physiology*
D018389:Codon, Nonsense; D021122:Protein Subunits; D019915:DNA Primase