title: Genetic analysis of MraY inhibition by the phiX174 protein E.
abstract: Protein E, the lysis protein of bacteriophage phiX174, is a specific inhibitor of MraY, the phospho-MurNAc-pentapeptide translocase that catalyzes the synthesis of lipid I in the conserved pathway for peptidoglycan biosynthesis. The original evidence for this inhibition was the isolation of two spontaneous E-resistance mraY mutants. Here we report further genetic studies aimed at dissecting the interaction between E and MraY, using a genetic strategy that is facile, rapid, and does not depend on the availability of purified E, purified MraY, or its substrates. This system relies on the ability of mraY or its enzymatically inactive D267N allele to protect cells from lysis after induction of a chimeric lambda :: E prophage. Using this approach, the MraY protein from Bacillus subtilis, which shares 43% sequence identity with the Escherichia coli enzyme, was found to interact weakly, if at all, with E. A potential E binding site defined by transmembrane domains 5 and 9 has been identified by isolating more mraY mutants resistant to E inhibition. Genetic analysis indicates that these E-resistant alleles fall into three classes on the basis of the affinity of the encoded proteins for MraY.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-2128, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.forename: Douglas K
authors.initials: DK
authors.lastname: Struck
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Bernhardt
authors.forename: Ry
authors.initials: R
authors.lastname: Young
mesh_terms: D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D001433:Bacteriolysis; D002478:Cells, Cultured; D004926:Escherichia coli / Q000502:physiology*; D010457:Peptidoglycan / Q000378:metabolism; D010957:Plasmids; D014166:Transferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups); D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C029555:E protein, bacteriophage X174; D010457:Peptidoglycan; D014764:Viral Proteins; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria
references.citation: J Biol Chem. 2001 Mar 2;276(9):6093-7
references.pmid: 11078734
references.citation: Mol Gen Genet. 1986 Jul;204(1):120-5
references.pmid: 3018438
references.citation: Cell. 1983 Jul;33(3):877-85
references.pmid: 6871998
references.citation: Glycobiology. 1994 Dec;4(6):768-71
references.pmid: 7734839
references.citation: J Biol Chem. 1994 Jan 28;269(4):2902-10
references.pmid: 8300625
references.citation: Microbiology (Reading). 2006 Oct;152(Pt 10):2959-2967
references.pmid: 17005977
references.citation: Mol Microbiol. 2002 Jul;45(1):99-108
references.pmid: 12100551
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: EMBO J. 1983;2(9):1521-6
references.pmid: 11892805
references.citation: Gene. 1985;40(1):39-46
references.pmid: 2936651
references.citation: FEMS Microbiol Rev. 2008 Mar;32(2):208-33
references.pmid: 18081839
references.citation: PLoS One. 2008 Jun 11;3(6):e2399
references.pmid: 18545655
references.citation: Mol Gen Genet. 1986 Nov;205(2):285-90
references.pmid: 3100913
references.citation: Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):4297-302
references.pmid: 10760296
references.citation: Biochemistry. 2008 Aug 26;47(34):8919-28
references.pmid: 18672909
references.citation: Mol Gen Genet. 1982;185(3):493-7
references.pmid: 6285147
references.citation: Microbiology (Reading). 2008 Jun;154(Pt 6):1710-1718
references.pmid: 18524925
references.citation: J Bacteriol. 2005 Oct;187(19):6631-40
references.pmid: 16166524
references.citation: Mol Microbiol. 1999 Nov;34(3):576-85
references.pmid: 10564498
references.citation: Infect Disord Drug Targets. 2006 Jun;6(2):85-106
references.pmid: 16789873
references.citation: Nature. 1977 Feb 24;265(5596):687-95
references.pmid: 870828
references.citation: Res Microbiol. 2002 Oct;153(8):493-501
references.pmid: 12437210
references.citation: Proc Natl Acad Sci U S A. 1976 Oct;73(10):3519-23
references.pmid: 1068463
references.citation: Genome Res. 2002 Apr;12(4):640-7
references.pmid: 11932248
references.citation: J Bacteriol. 2004 Mar;186(6):1747-57
references.pmid: 14996806
references.citation: Mol Genet Genomics. 2001 Apr;265(2):345-53
references.pmid: 11361346
references.citation: Science. 2001 Jun 22;292(5525):2326-9
references.pmid: 11423662
references.citation: J Bacteriol. 1983 Sep;155(3):1130-7
references.pmid: 6224771
references.citation: J Virol. 1982 Dec;44(3):993-1002
references.pmid: 6294347
references.citation: J Bacteriol. 1997 Oct;179(20):6228-37
references.pmid: 9335267
references.citation: FEMS Microbiol Lett. 1993 Apr 1;108(2):139-43
references.pmid: 8486239
references.citation: Trends Biochem Sci. 1997 Sep;22(9):334-9
references.pmid: 9301333
references.citation: J Bacteriol. 1983 Feb;153(2):1098-100
references.pmid: 6337118
references.citation: J Biol Chem. 1978 Sep 25;253(18):6551-60
references.pmid: 681366