The genomes of six Streptococcus thermophilus bacteriophages were compared to identify genes that could be targeted by engineered phage defense systems with potentially widespread efficacy. The genes associated with the S. thermophilus phage Sfi21-prototype genome replication module, including a putative primase and a putative helicase, were found to be among the best candidates due to their frequency of distribution in industrial phage isolates, striking sequence conservation between independent isolates, and intrinsic strategic importance in early phage development. Fourteen antisense RNA cassettes targeting the phage k3-derived helicase (hel3) or primase (pri3) genes were expressed in S. thermophilus NCK1125. These constructs consistently reduced the efficiency of plaquing (EOP) of phage k3 to between 5 x 10-1 and 2.0 x 10-3 depending on the (i) gene targeted and (ii) region of the gene that was targeted. The largest antisense RNAs were generally found to confer the largest reductions in EOP, however shorter antisense RNAs designed to the 5' region of the gene retained much of the inhibitory function, especially if they contained sequences complementary to the ribosome binding site. Expression of antisense RNAs correlated with decreased levels of phage encoded primase transcripts, likely due to increased degradation of the dsRNA complex. This, in turn, correlated with diminished phage genome replication and aborted phage development. In a separate study, invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutation within the S. thermophilus phage k3-encoded putative primase. PCR products containing the desired mutation(s) were cloned and expressed in S. thermophilus NCK1125. The majority of the examined constructs remained sensitive to phage k3, however four constructs conferred strong phage resistance to the bacterial host. The mutated residues resided within a putative ATPase/helicase domain suspected to be critical for primase function in vivo. The co-expression in trans of the K238(A/T) or RR340-341AA mutant proteins suppressed the function of the native, phage-encoded primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. According to this model, the plasmid-encoded mutant primase subunits are structurally intact and form stable interactions with the native, phage-encoded primase subunits, thus inhibiting their activity. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing more that nine log cycles. Given the magnitude of the resistance conferred, it was concluded that the putative primase is essential for genome replication in S. thermophilus Sfi21-type phages. Further, it was also clear that host-encoded factors were unable to complement the resultant deficiency. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and phage sensitivity of the host, indicating that translation was required to confer phage resistance. Residues within a critical oligomerization domain were also identified through genetic analysis. Introduction of an E437A mutation downstream of the transdominant K238T mutation completely suppressed phage resistance, indicating that the E437A mutation precluded the association of the mutant and native subunits. To our knowledge, this is the first use of subunit poisoning to inhibit phage replication in the lactic acid bacteria.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-12152003-034319 |
| Date | 15 December 2003 |
| Creators | Sturino, Joseph Miland |
| Contributors | ROBERT M. KELLY, TODD R. KLAENHAMMER, DAHLIA M. NIELSEN, ERIC S. MILLER |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-12152003-034319/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0029 seconds