NDLI: Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnus

Content Provider	PubMed Central
Author	Wu, Yanping Mcquiston, Jennifer H. Cox, Andrew Pack, Todd D. Inzana, Thomas J.
Editor	Orndorff, P. E.
Copyright Year	2000
Abstract	Haemophilus somnus undergoes 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 from lob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Neisseria meningitidis and N. gonorrhoeae. Unlike lob-1, lob-2A contained 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-bp SalI-BsgI fragment within lob-2A was deleted, and a kanamycin resistance (Kmr) gene was inserted into this site to create pCAATΔlob2A. Following electroporation of pCAATΔlob2A 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 variation 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 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. When H. somnus 129Pt was electrotransformed with shuttle vector pLS88 containing lob-2A, its LOS electrophoretic profile was modified and additional N-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated that lob-2A may be an N-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important to H. somnus virulence.
Starting Page	310
File Format	PDF
ISSN	10985522
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	1
Volume Number	68
Language	English
Publisher	American Society for Microbiology
Publisher Date	2000-01-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

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