The unphosphorylated form of the PilR two-component system regulates pilA gene expression in Geobacter sulfurreducens

Abstract

In Geobacter sulfurreducens, metal reduction and generation of bioelectricity require the participation of several elements, and among them, the type IV pili has an essential role. The pilus is composed of multiple PilA monomers. Expression of pilA gene depends mainly on the σ54 factor and the response regulator protein PilR. In this work, we characterized the role of the PilS-PilR two-component system in the regulation of the pilA gene expression. Experimental evidence indicates that PilS is autophosphorylated at the His-334 residue, which in turn is transferred to the conserved Asp-53 in PilR. Contrary to other PilS-PilR systems, substitution D53N in PilR resulted in higher activation of the pilA gene. By using a pilA::luxCDABE fusion with different promoter fragments and in vitro DNA-binding assays, we demonstrated the existence of multiple functional PilR binding sites. A regulatory model in which the non-phosphorylated PilR protein directs activation of pilA expression by binding to two sites in the promoter region of this gene is presented.

Type IV‐pili dependent motility is co‐regulated by PilSR and PilS2R2 two‐component systems via distinct pathways in Myxococcus xanthus

“…This result is in contrast to a recent report that phosphorylation of PilR in G. sulfurreducens, another d-proteobacteria with similar pil locus organisation to that of M. xanthus (Supporting Information Fig. S1), is not necessary for expression of pilA in that species (Hernandez-Eligio et al, 2016). This may reflect underlying differences of T4P regulation between these related bacteria, or may be due to experimental design; we chose to mutate the critical aspartic acid residue to alanine while the G. sulfurreducens pilR aspartic acid residue was mutated to asparagine.…”

“…The Δ pilR and Δ pilR2 mutants were complemented with vectors expressing the corresponding wild‐type RR, or constructs in which the putative phosphorylated aspartic acid (D) residue was changed to alanine (A) or glutamic acid (E), to mimic an unphosphorylated or phosphorylated state respectively. The G. sulfurreducens PilR sequence was used as a guide for mutation design (Hernandez‐Eligio et al ., ) (A). T4P‐dependent motility was assessed on 0.5% CYE after 48 h (B and C) and the gross rate of movement determined by measuring the diameter of the colonies over 5 days.…”

“…First, that phosphorylation of PilR and/or PilR2 is not essential to their activity. This was especially important to address as it has recently pil locus been demonstrated that Geobacter sulfurreducens retains the ability to express pilA in a strain producing PilR in which the critical aspartic acid residue for phosphorylation was changed to asparagine (Hernandez-Eligio et al, 2016). The alternative explanation for the motility phenotypes of these strains is that PilR and/or PilR2 are alternatively phosphorylated in the absence of their cognate HK.…”

“…The corresponding region of G. sulfurreducens PilR was used to predict the phosphorylated aspartic acid residues ( Fig. 3A) (Hernandez-Eligio et al, 2016). The DpilR strain was complemented for motility with wild-type PilR, although not completely.…”

A review on sediment microbial fuel cells as a new source of sustainable energy and heavy metal remediation: mechanisms and future prospective

“…The configurable protein type IV pili are encoded by gene PilA . PilA is 10–20 µm long, is 3–5 nm wide, and have 7‐ to 20‐kDa molecular weight . The pili have metal‐like conductivity, including the following characteristics: (i) decreased temperature increases conductivity, as observed with metals ; (ii) the N‐terminal of the PilA protein of G. sulfurreducens contains an α‐helix, and the C‐terminal has a conserved chain of amino acids (Trp ,Met, Phe, His, and Tyr), which are aligned to provide π–π stacking as in metals ; and (iii) the addition of protons increases the pilus conduction as occurs with polyaniline .…”