Wolfram R. Zückert

The PilZ domain‐containing protein, PlzA, is the only known cyclic di‐GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c‐di‐GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c‐di‐GMP were increased. A mutant PlzA that is incapable of binding c‐di‐GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G–C content play a role in DNA binding affinity. PlzA is a dual‐domain protein with a PilZ‐like N‐terminal domain linked to a canonical C‐terminal PilZ domain. Dissection of the domains demonstrated that the separated N‐terminal domain bound nucleic acids independently of c‐di‐GMP. The C‐terminal domain, which includes the c‐di‐GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c‐di‐GMP binding at the C‐terminal domain stabilizes the overall protein structure and facilitates PlzA‐DNA interactions via residues in the N‐terminal domain. Based on our data, we propose that levels of c‐di‐GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface‐exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane‐associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease‐accessible BB0838 peptide that mapped to a predicted β‐barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB 2 FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptD Bb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.