Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex.
Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system.
*Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Grant
G
English
†College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8QQ, U.K.
Olwyn
O
Byron
*Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Francesca R
FR
Cianfanelli
‡Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Alan R
AR
Prescott
*Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Sarah J
SJ
Coulthurst
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000235:genetics*; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012706:Serratia marcescens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D008565:Membrane Proteins; D011994:Recombinant Proteins