The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of <i>Borrelia burgdorferi</i>

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with <i>Borrelia burgdorferi sensu lato</i> bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the <i>B. burgdorferi</i> RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (<i>bbi42</i>, <i>bbk53</i>, and <i>bbq03</i>) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in <i>B. burgdorferi</i>'s reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.