Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis inHaemophilus somnus

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Abstract
Haemophilus somnusundergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-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 have now identified another putative gene (lob-2A) immediately upstream fromlob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the familyPasteurellaceaeand the LgtB and LgtE galactosyltransferases ofNeisseria meningitidisandN. gonorrhoeae. Unlikelob-1,lob-2Acontained 18 to 20 5′-GA-3′ repeats 141 bp upstream of the termination codon as determined 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-bpSalI-BsgI fragment withinlob-2Awas deleted, and a kanamycin resistance (Kmr) gene was inserted into this site to create pCAATΔlob2A. Following electroporation of pCAATΔlob2A intoH. somnus738, 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 variation was not eliminated. A variant with 19 5′-GA-3′ repeats inlob-2Ahad an LOS profile similar to that of 738-lob2A1::Km, suggesting thatlob-2Awas turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal βGal(1-3)βGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. WhenH. somnus129Pt was electrotransformed with shuttle vector pLS88 containinglob-2A, its LOS electrophoretic profile was modified and additionalN-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated thatlob-2Amay be anN-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important toH. somnusvirulence.