Characterization of a DNA region containing 5 '-(CAAT)(n)-3 ' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus

Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identifie d 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. i nfluenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 vari ed 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 ana lysis of the 5'-CAAT-3' region from individual colonies. If transcription b egan with the start codon closest to the 5'-CAAT-3' repeats, a protein of 3 4.5 kDa would be encoded when 33 repeats were present. However, we could no t establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS mono clonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob-1containing 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 L OS electrophoretic profile. Unlike strain 129Pt, both transformants underwe nt limited LOS phase variation, which correlated with variation in the numb er 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 s train. The ratio of glucose to galactose changed from 1:2 in strain 129Pt L OS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chro matography-mass spectrometry. Nuclear magnetic resonance spectroscopy confi rmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was c onstitutively more reactive in colony immunoblotting to polyclonal antiseru m made to purified strain 738 LOS, and was more susceptible to complement-m ediated killing in the presence of anti-738 LOS serum than parent strain 12 9Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus. (C) 2000 Academic P ress.