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 Marie-Anne Barny, 1 Eric Schoonejans, 2 Anastassios Economou, 3 Andrew W. B. Johnston 4 and J. Allan Downie* John Innes Centre, Colney, Norwich NR4 7UH, UK. Summary The nod C genes from rhizobia encode an N-acetyl- glucosaminyl transferase (chitin synthase) involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate root nodule morphogenesis in legume plants. NodC proteins have two hydrophobic domains, one of about 21 residues at the N-terminus and a longer one, which could consist of two or three transmembrane spans, near the C-terminus. These two hydrophobic domains flank a large hydro- philic region that shows extensive homology with other -glycosyl transferases. The topology of NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed using a series of gene fusions encoding proteins in which NodC was fused to alkaline phosphatase (PhoA) lacking an N-terminal transit sequence or to -galactosidase (LacZ). Our data support a model in which the N-terminal hydro- phobic domain spans the membrane in a N out –C in orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm. This orienta- tion appears to depend upon the presence of the hydrophobic region near the C-terminus. We propose that this hydrophobic region contains three trans- membrane spans, such that the C-terminus of NodC is located in the periplasm. A short region of about 40 amino acids, encompassing the last transmem- brane span, is essential for the function of NodC. Our model for NodC topology suggests that most of NodC, including the region showing most similarity to other -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 inner and outer membranes. Introduction Recognition between leguminous plants and the rhizobia that nodulate them is mediated via specific signalling mole- cules exchanged between the two symbiotic partners. The plant roots secrete flavonoid or isoflavonoid molecules that induce the bacterial nodulation (nod ) genes (Carlson et al., 1994; Downie, 1994). Several of the nod gene pro- ducts are involved in the synthesis of lipo-chito-oligosac- charide Nod factors that induce root nodule morphogenesis (Truchet et al., 1991; Spaink et al., 1991). The most crucial nod gene products required for the synthesis of these lipo-chito-oligosaccharides are NodA, NodB and NodC and the nodA nodB and nodC genes are highly conserved in all rhizobia studied. In the absence of the other nod gene products, they can form a ‘core’ sig- nal consisting of oligomers of four or five N-acetyl glucos- amine residues carrying an N-linked acyl group (Spaink et al., 1991). The function of each of the three proteins is known: NodC is an N-acetylglucosaminyl transferase (chitin synthase) which makes the chito-oligosaccharide chain (Geremia et al., 1994; Spaink et al., 1994); the N- acetyl group from the non-reducing N-acetyl glucosamine residue of the chito-oligosaccharide chain is removed by NodB, which acts as a chitin oligosaccharide deacetylase (John et al., 1993; Spaink et al., 1994); NodA is involved in the attachment of the acyl chain to the free amino group generated by the action of NodB (Atkinson et al., 1994; Ro ¨hrig et al., 1994). These reactions probably occur in the cytoplasm. It is evident from their predicted sequences that NodA and NodB (To ¨ro ¨k et al., 1984; Rossen et al., 1984; Schofield and Watson, 1986; Scott, 1986; Goethals et al., 1989; Krishnan and Pueppke, 1991) are relatively hydrophilic, do not have an N-terminal transit sequence and are, there- fore, likely to be cytoplasmically located. NodA was found Molecular Microbiology (1996) 19(3), 443–453 1996 Blackwell Science Ltd Received 5 July, 1995; revised 5 September, 1995; accepted 7 Sep- tember 1995. Present addresses: 1 Laboratoire de Pathologie Ve ´ge ´- tale, INRA, 16 rue Claude Bernard, 75231 Paris Cedex 05, France; 2 Institut des Sciences Ve ´ge ´tales (CNRS), F91198 Gif-sur-Yvette, France; 3 Department of Biology, University of Crete, GR-71409 Heraclio, Greece; 4 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. *For correspondence. E-mail DOWNIE @BBSRC.AC.UK; Tel. 01603 452571; Fax 01603 456844.