ABSTRACT
In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , DNA-Binding Proteins , Gene Expression Regulation, Viral , Helix-Turn-Helix Motifs , RNA-Binding Proteins , Viral Proteins , Bacteriophages/chemistry , Bacteriophages/genetics , Bacteriophages/metabolism , Bacteriophages/ultrastructure , Binding Sites , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , CRISPR-Associated Proteins/metabolism , Cryoelectron Microscopy , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , Genes, Viral , Models, Molecular , Nucleic Acid Conformation , Pectobacterium carotovorum/virology , Protein Biosynthesis/genetics , Protein Domains , Ribosomes/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Messenger/ultrastructure , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , RNA, Viral/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Substrate Specificity , Transcription, Genetic , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/ultrastructureABSTRACT
While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in Pectobacterium carotovorum phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.