Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR

The biosynthesis pathway for riboflavin (vitamin B-2), the precursor of the essential cofactors flavin mononucleotide and flavin adenine dinucleotide, is present in bacteria and plants but is absent in vertebrates, Due to the ir conservation in bacterial species and their absence in humans, the ribof lavin synthesis genes should be well suited either for detection of bacteri al DNA in human specimens or for the differentiation of pathogenic bacteria by molecular techniques. A DNA fragment carrying the genes ribD, ribC, and ribE, which encode homologues of riboflavin deaminase (RibD) and subunits of riboflavin synthetase (RibC and RibE), respectively, was isolated from a plasmid-based DNA library of the human pathogen Bartonella henselae by com plementation of a ribC mutation in Escherichia coli, Sequence analysis of t he ribC gene region in strains of B. henselae, which were previously shown to be genetically different, revealed that the ribC gene is highly conserve d at the species level, PCR amplification with primers derived from the rib C locus of B, henselae was used to isolate the corresponding DNA regions in B. bacilliformis, B. clarridgeiae, and B. quintana, Sequence analysis indi cated that the riboflavin synthesis genes are conserved and show the same o peron-like genetic organization in all four Bartonella species, Primer olig onucleotides designed on the basis of localized differences within the ribC DNA region were successfully used to develop species-specific PCR assays f or the differentiation of B. henselae, B. clarridgeiae, B, quintana, and B, bacilliformis, The results obtained indicate that the riboflavin synthesis genes are excellent targets for PCR-directed differentiation of these emer ging pathogens, The PCR assays developed should increase our diagnostic pot ential to differentiate Bartonella species, especially B. henselae and the newly recognized species B, clarridgeiae.