Screening for synthetic lethal mutants in <i>Escherichia coli</i> and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity

Molecular Microbiology - Tập 52 Số 5 - Trang 1255-1269 - 2004
Thomas G. Bernhardt1, Piet A. J. de Boer1
1Case Western Reserve University, School of Medicine, W239, Department of Molecular Biology and Microbiology, 10900 Euclid Ave., Cleveland, OH 44106, USA.

Tóm tắt

SummaryBacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid‐cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min–) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with ΔminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin‐like, metallo‐endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min– EnvC– cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC–green fluorescent protein fusion, when directed to the periplasm via the twin‐arginine export system, is both functional and part of the septal ring apparatus. Using an in‐gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.

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Tài liệu tham khảo

10.1101/gad.1039902

10.1111/j.1365-2958.1992.tb01380.x

10.1093/nar/30.1.276

10.1128/JB.180.4.881-884.1998

10.1128/MCB.11.3.1295

10.1046/j.1365-2958.2003.03511.x

10.1038/354161a0

10.1016/0092-8674(89)90586-2

10.1002/j.1460-2075.1991.tb05015.x

10.1074/jbc.273.29.18003

10.1073/pnas.092128499

10.1016/0022-2836(76)90119-4

10.1046/j.1365-2958.2001.02640.x

10.1093/emboj/21.8.1998

10.1111/j.1349-7006.2000.tb00907.x

10.1073/pnas.120163297

10.1128/jb.178.20.5971-5976.1996

10.1093/nar/16.13.6127

10.1128/MMBR.67.1.52-65.2003

10.1128/JB.182.14.4068-4076.2000

10.1101/gad.1014102

10.1101/SQB.1981.045.01.022

10.1128/JB.181.1.167-176.1999

10.1128/JB.184.9.2552-2556.2002

10.1128/JB.182.18.5153-5166.2000

10.1093/emboj/20.7.1563

10.1002/j.1460-2075.1992.tb05497.x

10.1111/j.1574-6968.2002.tb11271.x

10.1046/j.1365-2958.2001.02499.x

10.1128/JB.184.22.6093-6099.2002

10.1128/MMBR.62.1.181-203.1998

10.1103/PhysRevLett.87.278102

10.1046/j.1365-2958.1999.01575.x

10.1016/S1097-2765(01)00273-8

10.1073/pnas.96.26.14819

10.1073/pnas.102059099

10.1128/JB.185.1.196-203.2003

10.1073/pnas.2135445100

10.1128/jb.176.6.1630-1638.1994

10.1128/JB.184.10.2595-2602.2002

10.1046/j.1365-2958.2003.03504.x

10.1128/jb.172.10.5783-5788.1990

10.1128/JB.184.11.2951-2962.2002

10.1128/JB.186.8.2418-2429.2004

10.1016/0378-1119(87)90051-5

10.1016/S0006-3495(02)75426-X

10.1128/JB.185.3.735-749.2003

10.1111/j.1365-2958.1994.tb00466.x

10.1073/pnas.89.20.9885

10.1046/j.1365-2958.1999.01322.x

10.1073/pnas.251216598

Miller J.H., 1972, Experiments in Molecular Genetics

10.1128/jb.171.8.4303-4314.1989

10.1046/j.1365-2958.1998.00974.x

10.1046/j.1365-2958.1998.00797.x

10.1111/j.1365-2958.2005.05010.x

10.1128/JB.183.22.6630-6635.2001

10.1093/emboj/21.4.685

10.1128/JB.181.20.6419-6424.1999

10.1073/pnas.96.9.4971

10.1073/pnas.84.5.1127

10.1099/00221287-75-2-409

10.1074/jbc.C000833200

10.1073/pnas.1232225100

10.1042/bj1670293

Spratt B.G., 1980, Defective and plaque‐forming lambda transducing bacteriophage carrying penicillin‐binding protein cell shape genes: genetic and physical mapping and identification of gene products from the lip‐dacA‐rodA‐pbpA‐leuS region of the Escherichia coli chromosome, J Bacteriol, 143, 569, 10.1128/jb.143.2.569-581.1980

10.1073/pnas.262671699

10.1046/j.1365-2958.1998.00942.x

10.1128/JB.118.2.407-413.1974

10.1046/j.1365-2958.2001.02253.x

10.1128/jb.175.20.6704-6710.1993

Vinella D., 2000, Selected amplification of the cell division genes ftsQ‐ftsA‐ftsZ. Escherichia coli, Genetics, 156, 1483, 10.1093/genetics/156.4.1483

Vischer N.O.E., 1994, Object‐Image: an interactive image analysis program using structured point collection, Binary, 6, 160

10.1046/j.1365-2958.1999.01351.x

10.1128/JB.182.21.6203-6213.2000

10.1073/pnas.100127597

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Molecular Microbiology

Tập 52 Số 5

1255-1269

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