Phage-dependent gene expression dynamics in host-phage systems

• Applicant: Dr. Claudia Steglich
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2013 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 245857825

The marine cyanobacterium Prochlorococcus strain MED4 is prone to infection by the cyanophage P SSP7 and previous studies showed an induction of host rne (coding for RNase E) during phage infection (Lindell et al., 2007). In uninfected host cells, the level of RNase E is regulated via a negative feedback mechanism, in which a regulatory stem loop in the rne 5’UTR provides an entry site for RNase E-mediated cleavage of the mRNA. In the previous funding period we provided evidence that, however, during phage infection, rne transcription proceeds from the alternative transcription initiation site Pi, resulting in a short mRNA variant that lacks the regulatory 5’UTR. Thus, the negative feedback control mechanism is bypassed, leading to the concomitant increase in RNase E protein level. Furthermore, genome-wide transcription of phage asRNAs drives the formation of extensive duplex RNA complexes that along with polysomal translation protect from RNase E activity, while host transcripts are only partially covered with asRNAs, thus rendering the host transcriptome vulnerable to RNase E-mediated cleavage. Consequently, host-derived ribonucleic acids are fragmented to ribonucleotides and subsequently reduced to deoxynucleotides, very likely leading to a stimulation of phage reproduction. The aim of the second funding period is to fully understand the molecular mechanism of induced RNase E activity during lytic infection. Though we could show that a truncated RNase E isoform bypasses the negative feedback regulation leading to elevated RNase E protein levels, it remained unclear, which factors trigger a preferential usage of the Pi promoter. Second, we would like to gain deeper insights into structural properties and molecular cleavage characteristics of Prochlorococcus RNase E as cleavage assays suggest differences in the molecular recognition of RNase E targets between the Prochlorococcus isoforms and that of other bacteria. For most tested RNAs Prochlorococcus RNase E is insensitive to the phosphorylation state of the 5’ end and cleaves with comparable efficiency mono- and triphosphorylated substrates. For those substrates the substitution of isoleucine with valine at position 128 in the recombinant Prochlorococcus protein did not alter enzyme activity, contrarily to what has been observed for the E. coli homolog (Callaghan et al., 2005). Third, an alternative approach to the two-hybrid system will be applied to search for potential protein interaction partners of Prochlorococcus RNase E. Though electron microscopy provides evidence that - likewise to the E. coli homolog - RNase E is associated with the membrane, until now it is unclear if Prochlorococcus RNase E is organized in a multi-protein-complex, comparable to the degradosome, or functions as a single protein unit.

• DFG Programme: Research Grants