The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174.
Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn. All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.
Bernhardt
Thomas G
TG
Department of Biochemistry and Biophysics, Texas A and M University, 77843-2128, USA.
Roof
William D
WD
Young
Ry
R
D000595:Amino Acid Sequence; D001433:Bacteriolysis; D010584:Bacteriophage phi X 174 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000821:virology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D022021:Tacrolimus Binding Proteins / Q000145:classification; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D002352:Carrier Proteins; C029555:E protein, bacteriophage X174; D029968:Escherichia coli Proteins; C085606:SlyD protein, E coli; D014764:Viral Proteins; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase