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-acetylglucosaminyl 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 hydrophilic region that shows extensive homology with other beta -glycosyl transferases. The topology 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 beta-galactosidase (LacZ). Our data support a model in which the N-terminal hydrophobic domain spans the membrane in a Nout-Cin orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm. This orientation appears to depend upon the presence of the hydrophobic region near the C-terminus. We propose that this hydrophobic region contains three transmembrane spans, such that the C-terminus of NodC is located in the periplasm. A short region of about 40 amino acids, encompassing the last transmembrane 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 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 inner and other membranes.

Chromosomal mapping of the pel and cel genes in Erwinia chrysanthemi strain B374.

Using the RP4::mini-Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co-transfer mapping of MudI1734 insertion mutants and refined the map by co-transposition analysis. This analysis has enabled us to identify another cel gene.

Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.

Amber suppressors of Erwinia chrysanthemi.

Mutations trp1 and thyA1, both of a polyauxotrophic derivative of the Erwinia chrysanthemi strain B374, were characterized as amber mutations with an Escherichia coli suppressor, supA1P2, which inserts a glutamine in response to UAG. Simultaneous reversion of both mutations allowed us to isolate amber suppressor mutants of E. chrysanthemi. These suppressors were tested with a set of amber mutants of bacteriophage Mu which had been previously characterized on E. coli. The two independently isolated suppressors behaved as supD and supE mutants, respectively, of E. coli.

In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi.

Using an RP4 plasmid which carries a mini-Mu prophage which allows it to integrate spontaneously random pieces of its host chromosome, we cloned in vivo at least some of the pectate lyase and cellulase genes of the Erwinia chrysanthemi strain B374. The RP4-prime plasmids were used to localize the cloned genes on the B374 chromosome by co-transposition mapping and to subclone most of the genes in a classic high copy number plasmid vector.

phiEC2, a new generalized transducing phage of Erwinia chrysanthemi.

A collection of Erwinia strains (chrysanthemi and carotovora) was screened for temperate phages. One of them, phiEC2, turned out to be a generalized transducing phage. The structure of its DNA was found to be 62 kb long, terminally redundant, and circularly permuted. The transducing properties of the phage are also briefly described.

Utilization of plasmid pULB113 (RP4::mini-Mu) to construct a linkage map of Erwinia carotovora subsp. chrysanthemi.

We report experimental evidence that pULB113, an RP4::mini-Mu plasmid, mediates chromosome transfer in a strain of Erwinia carotovora subsp. chrysanthemi which does not accept the F episome. This allowed us to construct a genetic map of that strain by measuring the frequencies of cotransfer of different markers (thy, leu, pro, [his, trp], thyA, rpsL, ile).