Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.774720
Title: Characterization of ToxINPa-sensitive bacteriophages of Serratia sp. ATCC 39006
Author: Chen, Bihe
ISNI:       0000 0004 7961 9235
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
Abortive infection (Abi) is an anti-phage mechanism in which a bacterium initiates its own death upon phage infection. This prevents or decreases the production of phage progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa, first found in Pectobacterium atrosepticum. ToxINPa consists of an endoribonuclease toxin and RNA antitoxin and is inactive as a heterohexameric complex until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa-mediated Abi and what mechanisms are involved. This study aimed to address this issue by first introducing ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S39006). A novel environmental S39006 phage, ΦCBH8, that is sensitive to ToxINPa-mediated Abi was isolated, characterized, and compared to its spontaneous "escape" mutants that were insensitive to ToxINPa. Characterization of ΦCBH8 led to the discovery of a new genus of T4-family phages that are all ToxINPa-sensitive. Genomic comparison of ΦCBH8 with its spontaneous ToxINPa-escape mutants led to the discovery of two distinct genetic loci that are candidates for activation of ToxINPa-mediated Abi. One such locus was a multi-gene region of the phage genome, the deletion of which enabled some ΦCBH8 mutants to escape ToxINPa. Different ToxINPa-escape mutants had variable deletions in their genomes, but this multi-gene locus was deleted in all of the deletion mutants. Experiments showed that at least two genes in this locus must be simultaneously involved in activating ToxINPa-mediated Abi and one phage gene product within the locus (ORF18) was extremely toxic to the bacterial host. Another locus was the asiA gene. This study showed that ΦCBH8 mutants with mutated versions of asiA became insensitive to ToxINPa but genetic complementation restored their sensitivity. AsiA is involved in σ-appropriation but the mutated AsiA in ΦCBH8 mutants was no longer functional. Further experiments showed that AsiA from wild type ΦCBH8 reduced ToxINPa promoter activity while mutant AsiA did not, suggesting that AsiA could be involved in activating ToxINPa-mediated Abi by perturbing toxINPa transcription. Finally, an outer membrane protein (OmpW) was defined as the ΦCBH8 receptor in S39006. Transfer of the S39006 ompW gene to other bacterial species rendered some of them sensitive to the phage thereby enabling expansion of ΦCBH8 host range. Results showed that ΦCBH8 was insensitive to ToxINPa in E.coli, suggesting that activation of ToxINPa-mediated Abi is host-dependent. The function of OmpW was explored to try to link bacterial host physiology with phage susceptibility In conclusion, this work characterized novel phage genes involved in activating ToxINPa-mediated Abi. This provided evidence that viral gene lethality and σ-appropriation might be related to the mechanism(s) for phage activation of ToxINPa-mediated Abi.
Supervisor: Salmond, George Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.774720  DOI:
Keywords: bacteriophage ; toxin-antitoxin ; abortive infection
Share: