Heterogeneity in Tandem Octanucleotides within Haemophilus influenzae Lipopolysaccharide Biosynthetic Gene losA Affects Serum Resistance

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Abstract
Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA . Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA . Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies ( losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA -on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA , a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.

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