Identification of a plasmid-borne locus in Rhizobium etli KIM5s involved in lipopolysaccharide O-chain biosynthesis and nodulation of Phaseolus vulgaris

Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgus/A30 resulted in the identification of strain KIM-G1. Its rough co lony appearance, flocculation in liquid culture, and Ndv(-) Fix(-) phenotyp e mere indicative of a lipopolysaccharide (LPS) defect. Electrophoretic ana lysis of cell-associated polysaccharides showed that KIM-G1 produces only r ough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (alpha-D-GalA- 1-->4[alpha-D-GalA-1-->5]-Kdo) and tetrasaccharide (alpha-D-Gal-l-->6[alpha -D-GalA-1-->4]-alpha-D-Man-1-->5Kdo), strongly suggesting that the transpos on insertion disrupted a locus involved in O-antigen biosynthesis. Five mon osaccharides (Glc, Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identifie d as the components of the repeating O unit of the smooth parent strain, KI M5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s t hat, when provided in trans on a broad-host-range vector, restored the smoo th LPS and the full capacity of nodulation and fixation on its host Phaseol us vulgaris, The mTn5 insertion in KIM-G1 was located at the N terminus of a putative alpha-glycosyltransferase, which most likely had a polar effect on a putative beta-glycosyltransferase located downstream. A third open rea ding frame with strong homology to sugar epimerases and dehydratases was lo cated upstream of the insertion site. The two glycosyltransferases are stra in specific, as suggested by Southern hybridization analysis, and are invol ved in the synthesis of the variable portion of the LPS, i.e., the O antige n. This newly identified LPS locus was mapped to a 680-kb plasmid and is li nked to the Ips beta 2 gene recently reported for R erli CFN42.