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.