IDENTIFICATION OF THE ADP-L-GLYCERO-D-MANNO-HEPTOSE-6-EPIMERASE (RFAD) AND HEPTOSYLTRANSFERASE-II (RFAF) BIOSYNTHESIS GENES FROM NONTYPABLEHAEMOPHILUS-INFLUENZAE-2019

Haemophilus influenzae is an important human pathogen, The lipooligosa ccharide (LOS) of H. influenzae has been implicated as a virulence det erminant, To better understand the assembly of LOS in nontypeable H. i nfluenzae (NtHi), we have cloned and characterized the rfaD and rfaF g enes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-ep imerase and heptosyltransferase II enzymes, respectively, This cloning was accomplished by the complementation of Salmonella typhimurium lip opolysaccharide (LPS) biosynthesis gene mutants, These deep rough muta nts are novobiocin susceptible until complemented with the appropriate gene, In this manner, we are able to use novobiocin resistance to sel ect for specific NtHi LOS inner core biosynthesis genes, Such a screen ing system yielded a plasmid with a 4,8-kb insert, This plasmid was ab le to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Sal monella LPS, The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli, The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated b y in vitro transcription-translation studies. Isogenic mutants which d emonstrated truncated LOS consistent with inner core biosynthesis muta nts were constructed in the NtHi strain 2019, Primer extension analysi s demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF, Complementation studies, however, suggest that the rfaF gene does have an independent promoter, Mass spectrometric analysis shows that the LOS molecules exp ressed by H. influenzae rfaD and rfaF mutant strains have identical mo lecular masses. Additional studies verified that in the rfaD mutant st rain, D-glycero-D-mannoheptose is added to the LOS molecule in place o f the usual L-glycero-D-manno-heptose. Finally, the genetic organizati ons of the inner core biosynthesis genes of S. typhimurium, E. coli, a nd several strains of H. influenzae were examined, and substantial dif ferences were uncovered.