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Anaplasma phagocytophilum , the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a singlep44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in theA. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved inp44 gene conversion. The recombination intermediate structure between a donorp44 and thep44 expression locus ofA. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donorp44 locus connected to the 3′ conserved region of the recipientp44 in thep44 expression locus in direct orientation. To determine whether thep44 recombination intermediate structure can be generated in a RecF-activeEscherichia coli strain, we constructed a double-origin plasmid carrying thep44 expression locus and a donorp44 locus and introduced the plasmid into variousE. coli strains. The recombination intermediate was recovered in anE. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that thep44 gene conversion inA. phagocytophilum occurs through the RecF pathway.

infection and immunity

Analysis of Involvement of the RecF Pathway in p44 Recombination in Anaplasma phagocytophilum and in Escherichia coli by Using a Plasmid Carrying the p44 Expression and p44 Donor Loci