Structure-functional characterization of Lactococcus AbiA phage defense system
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ABSTRACT:
PROVIDER: PRJEB71919 | ENA |
REPOSITORIES: ENA
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Project description:Phages are viruses that specifically infect and kill bacteria. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated for the treatment or prevention of infections caused by multidrug resistant bacteria. Whether foes or allies, their importance is undeniable. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 due to a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (ORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of ORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages’ takeover of bacterial cells and provides novel information on phage-host interactions.
2019-01-29 | PXD011263 | Pride
Project description:Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which remain poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin-antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits toxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein.
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Project description:A spontaneous mutant of the lactococcal phage phi31 that is insensitive to the phage defense mechanism AbiA was characterized in an effort to identify the phage factor(s) involved in sensitivity of phi31 to AbiA. A point mutation was localized in the genome of the AbiA-insensitive phage (phi31A) by heteroduplex analysis of a 9-kb region. The mutation (G to T) was within a 738-bp open reading frame (ORF245) and resulted in an arginine-to-leucine change in the predicted amino acid sequence of the protein. The mutant phi31A-ORF245 reduced the sensitivity of phi31 to AbiA when present in trans, indicating that the mutation in ORF245 is responsible for the AbiA insensitivity of phi31A. Transcription of ORF245 occurs early in the phage infection cycles of phi31 and phi31A and is unaffected by AbiA. Expansion of the phi31 sequence revealed ORF169 (immediately upstream of ORF245) and ORF71 (which ends 84 bp upstream of ORF169). Two inverted repeats lie within the 84-bp region between ORF71 and ORF169. Sequence analysis of an independently isolated AbiA-insensitive phage, phi31B, identified a mutation (G to A) in one of the inverted repeats. A 118-bp fragment from phi31, encompassing the 84-bp region between ORF71 and ORF169, eliminates AbiA activity against phi31 when present in trans, establishing a relationship between AbiA and this fragment. The study of this region of phage phi31 has identified an open reading frame (ORF245) and a 118-bp DNA fragment that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA.
| S-EPMC179059 | biostudies-other