A pilin chaperone required for the expression of electrically conductive Geobacter sulfurreducens pili

Abstract

Summary Mechanisms controlling the expression of the electrically conductive pili (e‐pili) of Geobacter species are of interest because of the important role of e‐pili in diverse biogeochemical processes, anaerobic digestion and electromicrobiological applications. We investigated the function of the protein, designated Spc (short pilin chaperone), encoded by the gene immediately downstream from the gene for PilA, the monomer that assembles into e‐pili. Multiple lines of evidence suggest that Spc forms an oligomer that is associated with the inner membrane. Mutating the start codon of spc to prevent translation increased the transcript abundance of pilA but greatly diminished the abundance of PilA, and e‐pili could no longer be detected. Cross‐linking, protein capture and two‐hybrid studies demonstrated that Spc and PilA interacted. Two sites in PilA for electrostatic interaction with Spc were identified. The results demonstrate that Spc is required for PilA stability prior to incorporation into e‐pili, suggesting that Spc has a chaperone function that may be specific to the relatively short PilA monomers that assemble into e‐pili. These results are important for identifying microorganisms likely to express e‐pili from (meta)genomic data and for the construction of microbial strains expressing e‐pili.

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments

“…Gene GSU1497 ( spc ) is located downstream of pilA (GenBank locus GSU1496) (Figure a), and these two genes are predicted to be in the same operon (Juárez et al, ). A recent study demonstrated that Spc directly interacted with PilA, and acted as the chaperone to stabilize the PilA and facilitated the assembly of PilA, suggesting an intimate relationship between Spc and PilA (Liu, Zhan, et al, ). Furthermore, performing a structure prediction of full‐length PilA indicated that its structure is similar to that of P. aeruginosa strain PAK PilA (Figure b).…”

“…The predicted molecular weights for PilA and Spc are 6.57 kDa and 10.97 kDa respectively. However, the Spc will form an oligomer even under denaturing conditions (Liu, Zhan, et al, ). Similarly, the fused PilA also formed an oligomer (Figure a).…”

“…The GSU1497 protein (protein Spc) antiserum was generated against a polypeptide sequence that corresponded to the full‐length GSU1497 protein without its signal peptide (Liu, Zhan, et al, ). This gene fragment was amplified using the primer pair PilA‐CndeI/PilA‐CxhoI, and the amplified fragment was digested with NdeI (NEB) and XhoI (NEB) before being ligated into the NdeI and XhoI sites of the expression vector pET‐29a(+) (T4 DNA Ligase, NEB).…”

“…Compared to other type IV pilins, Gs‐PilA is short (61 amino acids) and lacks the large C‐terminal globular structure typical of these proteins (Malvankar et al, ; Reguera et al, ). However, the C‐terminal naked Gs‐PilA is not stable (Liu, Zhan, Jing, Zhou, & Lovley, ). The Gs‐PilA is encoded by gene GSU1496 and the gene GSU1497 locates immediately after it sharing the same promoter (Figure ).…”

“…The Gs‐PilA is encoded by gene GSU1496 and the gene GSU1497 locates immediately after it sharing the same promoter (Figure ). A recent study further indicated that the gene GSU1497 encoded a protein chaperone Spc which is necessary for the stability of Gs‐PilA and can facilitate the assembly of Gs‐PilA, suggesting the functional evolution of truncated Gs‐PilA (Liu, Zhan, et al, ). Actually, there are five aromatic amino acids at the C terminus of Gs‐PilA.…”