MOLECULAR-CLONING AND CHARACTERIZATION OF THE NONTYPABLE HAEMOPHILUS-INFLUENZAE-2019 RFAE GENE REQUIRED FOR LIPOPOLYSACCHARIDE BIOSYNTHESIS

The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (N THi) is an important factor in pathogenesis and virulence. In an attem pt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimuriu m rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutan t synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE g ene is postulated to be involved in ADP-heptose synthesis. Retransform ation with the plasmid containing 4 kb of NTHi DNA isolated from a rec onstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sod ium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confir med the conversion of the rfaE mutant LPS to a wild-type LPS phenotype . Sequence analysis of a 2.4-kb Bg/II fragment revealed two open readi ng frames. One open reading frame encodes the RfaE protein with a mole cular weight of 37.6 kDa, which was confirmed by in vitro transcriptio n and translation, and the other encodes a polypeptide highly homologo us to the Escherichia coli HtrB protein. These two genes are transcrib ed from the same promoter region into opposite directions. Primer exte nsion analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start. site. The u pstream promoter region contained a sequence (TA AAAT) homologous to t he -10 region of the bacterial sigma(70)-dependent promoters at an app ropriate distance (7 bp), but no sequence resembling the consensus seq uence of the -35 region was found. These studies demonstrate the abili ty to use complementation of defined LPS defects in members of the fam ily Enterobacteriaceae to identify LOS synthesis genes in NTHi.