title: Characterization of a DNA region containing 5'-(CAAT)(n)-3' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus.
abstract: Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identified at the 5' end of an open reading frame (ORF) named lob1 from Haemophilus somnus strain 738. Based on sequence analysis, lob1 has 59% DNA homology to lex2B, which is involved in lipooligosaccharide (LOS) biosynthesis in H. influenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 varied from 31-35, but that 94% of colonies contained 33 repeats of 5'-CAAT-3' downstream of two potential start codons, as determined by DNA sequence analysis of the 5'-CAAT-3' region from individual colonies. If transcription began with the start codon closest to the 5'-CAAT-3' repeats, a protein of 34.5 kDa would be encoded when 33 repeats were present. However, we could not establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS monoclonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob1 containing 33 5 '-CAAT-3' repeats in shuttle vector pLS88 resulted in transformants 129Pt(pLSlob1-33A) and 129Pt(pLSlob1-33B), both of which demonstrated the same altered LOS electrophoretic profile. Unlike strain 129Pt, both transformants underwent limited LOS phase variation, which correlated with variation in the number of 5'-CAAT-3' repeats in pLSlob1-33. Nanoelectrospray-mass spectrometry of O-deacylated LOS indicated that transformant 129Pt(pLSlob1-33A) LOS was composed of a different distribution of glycoforms than LOS of the parent strain. The ratio of glucose to galactose changed from 1:2 in strain 129Pt LOS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chromatography-mass spectrometry. Nuclear magnetic resonance spectroscopy confirmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was constitutively more reactive in colony immunoblotting to polyclonal antiserum made to purified strain 738 LOS, and was more susceptible to complement-mediated killing in the presence of anti-738 LOS serum than parent strain 129Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus.
authors.affiliation: Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: McQuiston
authors.forename: J R
authors.initials: JR
authors.lastname: McQuiston
authors.forename: A D
authors.initials: AD
authors.lastname: Cox
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.forename: S M
authors.initials: SM
authors.lastname: Boyle
authors.forename: T J
authors.initials: TJ
authors.lastname: Inzana
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000940:Antigenic Variation; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000276:immunology; D015139:Blotting, Southern; D002417:Cattle; D015169:Colony Count, Microbial; D018274:Electroporation; D005690:Galactose / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D006190:Haemophilus / Q000235:genetics* / Q000378:metabolism; D007106:Immune Sera; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D007106:Immune Sera; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D005947:Glucose; D005690:Galactose