Self-splicing of the bacteriophage T4 group I introns requires efficient translation of the pre-mRNA in vivo and correlates with the growth-state of the infected bacterium

Department of Molecular Biology & Functional Genomics, Stockholm University, Sweden

^* To whom correspondence should be addressed. Email: britt-marie.sjoberg{at}molbio.su.se.

	Abstract

Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (thymidylate synthase, td, and ribonucleotide reductase, nrdB and nrdD). Their presence in these genes has fuelled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing we inhibited translation in T4 infected bacteria, as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after chloramphenicol addition during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. Splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase, and splicing of all three introns was reduced upon T4 infection of stationary phase bacteria. Our results implicate that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to regulation of splicing of phage introns.