Proline 21, a residue within the α‐helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

Abstract

Summary⌽X174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis-trans isomerization of proline residues within ␣-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded ␣-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli. Oligomerization of protein P21G-StrpA was not disturbed.

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

“…However, there is no observable change in the secondary structure of E pep upon addition of SlyD, as shown by CD spectroscopy; therefore, under these conditions, SlyD does not appear to catalyse a prolyl isomerization upon E pep , as proposed by Witte et al (1997). Bernhardt et al (2002) have proposed that the role of SlyD is to protect E from proteolysis, which is consistent with our data.…”

“…However, it was not certain from these earlier results whether the interaction of E with MraY was at the MraY active site, or through a site in the transmembrane region; nor was the precise role of prolyl isomerase SlyD certain, although it was possible that the enzyme catalysed prolyl isomerization of E (Witte et al, 1997).…”

“…The sequence of E contains a proline residue close to the centre of the transmembrane a-helix, Pro-21, which has been shown to be essential for the activity of E (Witte et al, 1997). Using the synthetic peptide E pep , it was possible to examine whether treatment with SlyD resulted in any change in conformation of E pep .…”

Online Monitoring of Escherichia coli Ghost Production

“…The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.The formation of bacterial ghosts of Escherichia coli is a well-characterized process (19,24). Initiated by the expression of cloned phage X174 lysis gene E, a transmembrane tunnel structure is formed in consequence of the oligomerization of protein E. Driven by the high osmotic pressure inside the cell, the cytoplasmic content is expelled into the surrounding medium, thus giving rise to empty bacterial cell envelopes.…”

Evolutionary dominance of holin lysis systems derives from superior genetic malleability

“…One notion was that the lytic lesion supported by the single-gene system was sufficient for the release of small phages like wX174 but not for the much larger, more complex dsDNA phages. This idea was based on a model for E-mediated lysis in which the E protein would oligomerize into a 'transmembrane tunnel' that spanned the entire envelope of the host cell (Witte et al, 1990b(Witte et al, , 1997. The tunnel would be adequate for release of the small wX174 virion but not for a large, complex dsDNA phage like l. However, the elucidation of the molecular mechanism of E, as an inhibitor of MraY, and the complete cell lysis that derives from it, leaves the transmembrane tunnel hypothesis untenable.…”