Molecular cloning and mutagenesis of a DNA locus involved in lipooligosaccharide biosynthesis in Haemophilus somnus

Haemophilus somnus undergoes antigenic and structural phase variation in it s lipooligosaccharide (LOS). A gene (lab-1) containing repetitive 5'-CAAT-3 ' sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675-4681, 1997). We hav e now identified another putative gene (lob-2A) immediately upstream from l ob-1, Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Nei sseria meningitidis and N. gonorrhoeae. Unlike lob-1, lob-2A contained 18 t o 20 5'-GA-3' repeats 141 bp upstream of the termination codon as determine d by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5'-GA-3' repeats were present a stop codon would occur 1 bp after the last 5'-GA-3' repeat. A 630-bp SalI-BsgI fragment with in lob-2A was deleted, and a kanamycin resistance (Km(r)) gene was inserted into this site to create pCAAT Delta lob2A. Following electroporation of p CAAT Delta lob2A into H. somnus 738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1: :Km, was altered, and the phase variation rate was reduced but phase variat ion was not eliminated. A variant with 19 5'-GA-3' repeats in lob-2A had an LOS profile similar to that of 738-lob2A1::Km, suggesting that lob-2A was turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and n uclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was defic ient in the terminal beta Gal(1-3)beta GlcNAc residue present in parent str ain 738, Mutant 738-lob2A1::Km was significantly more sensitive to the bact ericidal action of normal bovine serum and was less virulent in mice than w as parent strain 738, When H. somnus 129Pt was electrotransformed with shut tle vector pLS88 containing lob-2A, its LOS electrophoretic profile was mod ified and additional N-acetylhexosamine residues were present, as determine d by nES-MS analysis. These results indicated that lob-2A may be an N-acety lglucosamine transferase involved in LOS biosynthesis and phase variation a nd that LOS structure is important to H. somnus virulence.