THE C-TERMINAL DOMAIN OF THE RHIZOBIUM-LEGUMINOSARUM CHITIN SYNTHASE NODC IS IMPORTANT FOR FUNCTION AND DETERMINES THE ORIENTATION OF THE N-TERMINAL REGION IN THE INNER MEMBRANE

The nodC genes from rhizobia encode an N-acetyl-glucosaminyl transfera se (chitin synthase) involved in the formation of lipo-chito-oligosacc haride Nod factors that initiate root nodule morphogenesis in legume p lants, NodC proteins have two hydrophobic domains, one of about 21 res idues at the N-terminus and a longer one, which could consist of two o r three transmembrane spans, near the C-terminus, These two hydrophobi c domains flank a large hydrophilic region that shows extensive homolo gy with other beta-glycosyl transferases. The topology of NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed u sing a series of gene fusions encoding proteins in which NodC was fuse d to alkaline phosphatase (PhoA) lacking an N-terminal transit sequenc e or to beta-galactosidase (LacZ). Our data support a model in which t he N-terminal hydrophobic domain spans the membrane in a N-out-C-in or ientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm, This orientation appears to depend upon the presence o f the hydrophobic region near the C-terminus, We propose that this hyd rophobic region contains three transmembrane spans, such that the C-te rminus of NodC is located in the periplasm, A short region of about 40 amino acids, encompassing the last transmembrane span, is essential f or the function of NodC. Our model for NodC topology suggests that mos t of NodC, including the region showing most similarity to other beta- glycosyl transferases, is exposed to the cytoplasm, where it is likely that polymerization of N-acetyl glucoasamine occurs, Such a model is incompatible with previous reports suggesting that NodC spans both inn er and outer membranes.