Supplementary Materialsmbo30001-0071-SD1. of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK. TssB (VipA) and TssC (VipB) proteins have been shown to form tubular structures resembling the bacteriophage T4 sheath FOS (B?nemann et al. 2009; Cascales and Cambillau 2012), which can be disassembled by ClpV, an AAA+ ATPase (B?nemann et al. 2009, 2010; Pietrosiuk et al. 2011). Aside from bacteriophage-derived components, a number of membrane-associated proteins are associated with T6SS. At least three proteinsTssJ, TssL, and TssMform a trans-envelope complex, that may be augmented by TagL, an additional protein containing a peptidoglycan-binding domain (Aschtgen et al. 2010a). TssJ is an outer membrane lipoprotein (Aschtgen et al. 2008) whose structure has been reported recently (Felisberto-Rodrigues et al. 2011). TssJ interacts with the inner membrane TssM subunit, an IcmF-like protein (Zheng and Leung 2007; Felisberto-Rodrigues et al. 2011). TssM interacts with TssL, an IcmH-like protein (Zheng Tedizolid biological activity and Leung 2007; Ma et al. 2009). IcmF and IcmH are two components required for the optimal function of Type IVb secretion systems in and (Segal et al. 2005; Nagai and Kubori 2011). Tedizolid biological activity The T6SS TssL and T4bSS IcmH proteins are closely related (DUF2077 family). Although the enteroaggregative (EAEC) TssL and the IcmH proteins share 45% of similarity, they endow conserved secondary structure predictions (Fig. S1). TagL is a polytopic inner membrane protein shown Tedizolid biological activity to interact directly with TssL (Aschtgen et al. 2010a). The periplasmic domain of TagL carries a peptidoglycan-binding motif of the OmpA/Pal/MotB family (pfam PF05691; Aschtgen et al. 2010a, 2010b) that presumably anchors the T6SS to the cell wall. Herein, we further characterized the TssJLMCTagL complex of the EAEC Sci-1 T6SS focusing on the TssL subunit (GenBank accession number “type”:”entrez-protein”,”attrs”:”text”:”CBG37349″,”term_id”:”284924248″,”term_text”:”CBG37349″CBG37349; locus tag EC042_4527). We report that TssL is an essential protein for the function of the Sci-1 apparatus and demonstrate that TssL is an inner membrane protein. Further topology experiments using cysteine and protease accessibility assays showed that TssL is anchored to the inner membrane through a transmembrane domain located at the extreme C-terminus of the protein. This topology being unusual we tested the genetic requirements for TssL insertion in the inner membrane. Our Tedizolid biological activity data suggest that proper insertion of TssL is Tedizolid biological activity Tat independent, Sec independent and signal recognition particle (SRP) independent but requires the YidC protein and is modulated by the DnaK chaperone. Experimental Procedures Bacterial strains, media, growth conditions, and chemicals Bacteria strains are listed in Table S1. K12 DH5 was used for cloning procedures. The enteroaggregative strain 17-2 (kindly provided by Arlette Darfeuille-Michaud, University of Clermont-Ferrand, France) and its (K12 MC4100 conditional mutant strain (FTL10, an MC4100 gene under the control of the arabinose-inducible Ppromoter; Hatzixanthis et al. 2003) were kindly provided by Pierre Genevaux (LMGM, Toulouse, France). The FtsY-depletion strain (IY28 [Erez et al. 2010]) was kindly provided by Hans-Georg Koch (Freiburg Universit?t, Germany). The (DADE, Wexler et al. 2000) and the conditional temperature-sensitive (MM52, Oliver and Beckwith 1981) strains were kindly provided by Long-Fei Wu (LCB, Marseille, France). Except the conditional and mutants, strains were routinely grown in LB (Luria Broth) broth at 37C, with aeration. The YidC- and FtsY-depletion strains were grown overnight in LB medium supplemented with L-arabinose 0.5% and then diluted in fresh LB medium without arabinose and cultured for 3 h before induction of MM52 strain was grown at 28C for 3 h before induction. Expression of (from plasmid pIBA-TssL) was obtained by addition of anhydrotetracyclin (AHT). For the Hcp release, fractionation, selective solubilization, and cysteine and protease accessibility assays, the gene cluster was induced by addition of the iron chelator 2,2-dipyridyl (125 M final concentration) 30 min prior harvesting the cells (Brunet et al. 2011). Plasmids and mutant alleles were maintained by the addition of ampicillin (100 g mL?1 for K12, 200 g mL?1 for EAEC), kanamycin (50 g mL?1 for K12, 50 g mL?1 for chromosomal insertion on EAEC, 100 g mL?1 for plasmid-bearing EAEC), chloramphenicol (40 g mL?1), or tetracycline (12 g mL?1). Sodium lauroyl sarcosinate (SLS), L-arabinose, for 5.