MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

Abstract

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to L lysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJ P330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJ P330Q mutant was recessive and could be suppressed by L overcomes dnaJ (L odj ) alleles selected for restoration of lysis. All three L odj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ϳ20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the L odj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of X174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Phage single-gene lysis: Finding the weak spot in the bacterial cell wall

“…Two genetics-based approaches were mounted, the first aimed at identifying host factors, using, as before, inducible plasmid-based clones of L (81,82). To avoid mutations that reduced the copy number or L transcription, a blue/white reporter plasmid was constructed, and from hundreds of colonies surviving L induction from this construct, two blue colonies were identified and designated as ill (insensitive to L lysis) mutants (82). Surprisingly, the ill mutations mapped to dnaJ, which encodes a widely conserved chaperone involved in the heat shock response (83).…”

RNA Phage Biology in a Metagenomic Era

“…Nonetheless, the molecular information and functioning schema of such an autolytic pathway have yet to be identified [ 82 ]. Recent findings have indicated that the lysis of host cells by MS2 lysin is dependent on a range of host factors, including host chaperone DnaJ [ 106 ]. This post-translational regulator allows for another level of control of both quantity and activity of the lysis protein of MS2.…”

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

“…Dual functions for PPH in peptidoglycan hydrolysis and blocking cell division are consistent with descriptions of single lysis proteins in phages with smaller genomes. For example, coliphages X174, MS2, and Q␤ encode lysis proteins E, L, and A 2 , respectively (65)(66)(67)(68). Protein E causes host cell lysis by inhibiting the activity of a host protein involved in peptidoglycan biosynthesis (66).…”