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

Abstract

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 their conservation in bacterial species and their absence in humans, the riboflavin synthesis genes should be well suited either for detection of bacterial 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 complementation of a ribC mutation in Escherichia coli. Sequence analysis of the ribC gene region in strains of B. henselae, which were previously shown to be genetically different, revealed that the ribC gene is highly conserved at the species level. PCR amplification with primers derived from the ribC locus of B. henselae was used to isolate the corresponding DNA regions in B. bacilliformis, B. clarridgeiae, and B. quintana. Sequence analysis indicated that the riboflavin synthesis genes are conserved and show the same operon-like genetic organization in all four Bartonella species. Primer oligonucleotides designed on the basis of localized differences within the ribC DNA region were successfully used to develop species-specific PCR assays for 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 emerging pathogens. The PCR assays developed should increase our diagnostic potential to differentiate Bartonella species, especially B. henselae and the newly recognized species B. clarridgeiae.

FIG. 1

Schematic representations of the DNA regions comprising the genes ribC, ribD, and ribE in B. henselae and other Bartonella species. The upper line represents the DNA region of B. henselae Houston-1 cloned into plasmid pBH-RIBC1. The binding sites of the oligonucleotide primers used for the differentiation of Bartonella species by PCR are marked by the arrowheads. The bars indicate the sequenced DNA regions amplified with primers PBH3 and PBH4 from B. quintana, B. bacilliformis, and B. clarridgeiae. Sequences of primer oligonucleotides are given in Table 2.

FIG. 2

Alignments of the RibD, RibC, and RibE proteins from B. henselae and E. coli. The amino acid sequences of RibD (A), RibC (B), and RibE (C) from B. henselae were deduced from the DNA sequence cloned into pBH-RIBC1 and aligned with the sequences of the same proteins from E. coli. For the RibD protein, only the N-terminal region is shown because the homology of the middle and C-terminal regions of the proteins is very low (//). Amino acids that were found to be identical or conservatively exchanged are marked by double or single dots, respectively. Stretches of more than two amino acids conserved between proteins from both species are overlined. These regions are also conserved in the corresponding proteins from other bacterial species (listed in the introduction).

FIG. 3

PCR analysis for the detection of the ribC DNA region in B. henselae and in other Bartonella species. PCR with primers PBH3 and PBH4 (Fig. 1) were used to amplify a 1.7-kb DNA fragment which carries the ribC gene (arrow). PCR analysis was performed with 100 ng (lane 1), 10 ng (lane 2), and 1 ng (lane 3) of isolated total DNA from B. henselae FR96/K4 as the target. Total DNA from B. henselae Houston-1 (100 ng) served as a positive control (lane 4). PCR analysis with primers PBH3 and PBH4 generated products with sizes similar to those of DNA of other B. henselae isolates, B. quintana, B. bacilliformis, and B. clarridgeiae, listed in Table 1 (not shown). Lanes M, marker DNA fragments. The PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide.

FIG. 4

Alignments of the ribC genes and of the RibC proteins from Bartonella species. (A) The sequences of the oligonucleotide primers used for the differentiation of Bartonella species are underlined and marked by arrows. Asterisks indicate ribC DNA regions variable among Bartonella species. (B) Stretches of amino acids conserved in RibC homologues from unrelated microorganisms are overlined. Amino acids highly variable in the RibC proteins from different Bartonella species are marked with asterisks.

FIG. 4

Alignments of the ribC genes and of the RibC proteins from Bartonella species. (A) The sequences of the oligonucleotide primers used for the differentiation of Bartonella species are underlined and marked by arrows. Asterisks indicate ribC DNA regions variable among Bartonella species. (B) Stretches of amino acids conserved in RibC homologues from unrelated microorganisms are overlined. Amino acids highly variable in the RibC proteins from different Bartonella species are marked with asterisks.

FIG. 5

Species-specific differentiation of Bartonella species by PCR analysis with primers designed from the ribC DNA region. Primers designed from the ribC DNA regions of B. bacilliformis (PBH-L1 and PBB-R1), B. clarridgeiae (PBH5 and PBH15), B. henselae (PBH-L1 and PBH-R1), and B. quintana (PBH-L1 and PBQ-R1) (as indicated at the top) were used for PCR analysis of Bartonella species under stringent species-specific conditions (Table 2). DNA isolated (100 ng) from B. bacilliformis (lanes 1), B. clarridgeiae (lanes 2), B. henselae (lanes 3), and B. quintana (lanes 4) was analyzed. Sizes of PCR products are listed in Table 2. Lanes M, marker DNA fragments. The PCR products were separated on a 1.6% agarose gel and stained with ethidium bromide.