Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export.
The pulmonary pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to replicate inside host cells. This apparatus translocates proteins into macrophages to alter their endocytic pathway and enable bacterial growth. Although the secretion ATPase DotB is critical for T4SS function, its specific role in type IV secretion remains undefined. Due to similarity to the VirB11 and PilT ATPases, DotB has been proposed to play a role in assembly of the T4SS, retraction of pili and/or export of substrates. With the goal of understanding the protein's function(s), we isolated and characterized 30 dotB alleles using a variety of phenotypic and biochemical assays. Twenty-four of these alleles possess several dot/icm mutant phenotypes, including a complete lack of intracellular replication, plasmid mobilization and contact-dependent cytotoxicity. These 24 non-functional alleles fall into three classes: those with a known biochemical defect, those with a predicted enzymatic defect and those with an unknown defect. Six other alleles display partial activity in dot/icm phenotypic assays, thus constituting a fourth class. Two mutants in this class are unable to export a subset of T4SS substrates, providing the first evidence for a DotB function in substrate export and suggesting a possible role in substrate selection.
Sexton
Jessica A
JA
Department of Molecular Microbiology, Washington University, Campus Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Yeo
Hye-Jeong
HJ
Vogel
Joseph P
JP
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured / Q000382:microbiology; D005260:Female; D005799:Genes, Dominant; D016952:Legionella pneumophila / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D021381:Protein Transport / Q000502:physiology
D001426:Bacterial Proteins; D000251:Adenosine Triphosphatases; C503088:DotB protein, Legionella pneumophila