The location of the complex during the intracellular infection phase as well as whether this location also corresponds to the sites of active CG biosynthesis, transport, and modification remains to be determined. Supplementary Material Supplemental material: Click here to view. ACKNOWLEDGMENTS This work was supported by grants PICT 2011/1485 and PICT 2014/3359 from Agencia Nacional de Promocin Cientfica y Tecnolgica (ANPCyT), Argentina. with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the presence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in species synthesize cyclic OPGs consisting of a cyclic chain of 17 to 25 glucose residues linked in -1,2 glycosidic bonds and substituted with sn-1-phosphoglycerol, succinic acid, methylmalonic acid, or a combination of them (1,C3). Cyclic -1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of cyclic -1,2-glucans (CG), is present in a restricted number of symbiotic or pathogenic bacteria, most of them belonging to the group, in which CG are a symbiosis or virulence factor required for successful host conversation (4,C6). In to reach an endoplasmic reticulum-derived vacuole permissive for bacterial replication (4). Additionally, CG can be used to enhance cellular immunity by activation of human and mouse dendritic cells (10). Cgs is usually a 320-kDa (2,867-amino-acid-residue) polytopic integral inner membrane protein with six transmembrane segments (TMSs) and the amino and carboxy terminus located on the cytoplasm (11). Cgs itself acts as a protein intermediate and catalyzes the four enzymatic reactions required for the synthesis of CG. The first glucose is transferred from UDP-glucose to a not-yet-identified amino FGF23 acid residue of the protein (initiation reaction). Successive glucoses are then transferred from UDP-glucose to the protein-bound glucose, thus elongating a linear polyglucose chain (elongation reaction) linked to the protein. The initiation and elongation reactions are catalyzed by the Cgs glycosyltransferase domain name (amino acid residues 475 to 818) (12). The glucose-removing activity catalyzed by the -1,2-glucooligosaccharide phosphorylase domain name (Cgs residues 1545 to 2867) Trabectedin counteracts the Cgs elongation reaction, thus controlling the length of the -1,2-glucoologosaccharide protein-linked intermediate (13). The cyclization reaction catalyzed by the Cgs region from positions 991 to 1544 puts an end to the balance between the elongation and phosphorolysis reactions, the linear -1,2-glucooligosaccharide protein-linked intermediate is usually cyclized, and CG (with the appropriate ring size) are released from the protein to the cytoplasm (14). Once in the cytoplasm, CG are transported to the periplasm by the cyclic glucan transporter Cgt, an ABC transporter of 66 kDa with six predicted TMSs (9). During transport or once they have reached the periplasmic space, a fraction of the CG are substituted with CG metabolism. Using bacterial two-hybrid (BACTH) and coimmunoprecipitation (Co-IP) strategies, we obtained evidence of homotypic and heterotypic protein-protein interactions among Cgs, Cgt, and Cgm, creating a membrane-located protein complex dedicated to cyclic -1,2-glucan biosynthesis, transport, and succinylation. Single-cell fluorescence microscopy analysis in exhibited that these proteins are focally distributed in the membrane, particularly at the cell poles. MATERIALS AND METHODS Bacterial strains and growth conditions. Bacterial strains used in this work are listed in Table S1 in the Trabectedin supplemental material. strains were grown in Luria-Bertani (LB) broth (20) at 37C, unless otherwise indicated. When necessary, medium was supplemented with 50 to 100 g/ml ampicillin (Amp), 25 to Trabectedin 50 g/ml kanamycin (Km), 2 g/ml tetracycline (Tet), 20 g/ml chloramphenicol (Cm), and 0.5 to 1 1 mM isopropyl–d-1-thiogalactopyranoside (IPTG). strains were grown in tryptic soy broth (TSB) (Difco/Becton-Dickinson, Sparks, MD) at 37C. If Trabectedin necessary, TSB was supplemented with 50 g/ml Amp, 50 g/ml Km, 20 g/ml Cm, and/or 5 g/ml nalidixic acid (Nal). All work with live was performed.