Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation

Abstract

Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides.

Fig 1

(A) Genomic organization of the pilus biosynthesis genes and gene cluster downstream of pilA (GSU1496). The black bars indicate DNA cloned into the plasmids constructed for complementation experiments. (B) Sequences of the pilA gene and PilA protein, including the mutations introduced for each strain used in this work. In the isogenic wild-type and all mutant strains, a kanamycin cassette was inserted upstream of the P1 and P2 promoter regions. The arrows indicate the two transcription initiation sites. The two ribosome-binding sites are underlined. The translation start codons (TTG and ATG) and the peptidase cleavage site (GGT) are in boldface. The base changes leading to amino acid replacements are depicted in italics for each mutant strain. The deleted sequence of the pilA gene in the ΔpilA2 strain is highlighted in gray. (C) DNA gel of semiquantitative reverse transcriptase PCRs. (1) proC control; (2) wild-type pilA in DL1; (3) ΔpilA2; (4) short-isoform (pilA4) strain; and (5) long-isoform (pilA5) strain.

Fig 2

Localization of wild-type and engineered PilA. (A) Protein bands were detected with the PilA-specific antibody. The PilA fractions are fraction 1 (secreted), fraction 2 (nonsecreted soluble), and fraction 3 (membrane associated), as defined in Materials and Methods. Control (Cntrl) samples (the secreted protein fraction of DL1) were included in null strains to verify the possibility of detecting PilA if it was present. All PilA protein fractions migrated at 7 kDa. (B) Western blot of PilA isolated from the prepilin strain (pilA6) using the PilA-specific antibody. Shown are PilA fraction 1 (secreted), fraction 2 (nonsecreted soluble), and fraction 3 (membrane associated) containing PilA protein of the wild-type DL1 and the prepilin (pilA6) strains. Each lane contains 10 μg total cell protein (A and B).

Fig 3

Heme-stained SDS-PAGE of loosely bound outer surface c-type cytochromes prepared from the wild-type and pilA mutant strains. Shown are wild-type DL1 (Wt), short-isoform (pilA4), long-isoform (pilA5), null (pilA7), and deletion (ΔpilA2) strains. The Omc distribution in samples from the ΔpilA2 and ΔpilA1 pilA deletion strains (48) were found to be comparable (unpublished observation). The protein concentrations in the loosely bound outer membrane preparations were determined with a bicinchoninic acid (BCA) assay, and 3 μg of protein was loaded into each lane.

Fig 4

Current production by the DL100 (Wt) and the short-isoform (pilA4) strains in microbial fuel cells. (A) Plot representative of data obtained with three biological replicates. The ΔpilA strain produces a maximum current of 1.2 mA under the same experimental conditions (45). (B) Confocal scanning micrographs of biofilms formed with the isogenic wild-type DL100 (Wt) and short-isoform (pilA4) strains on graphite anodes producing maximum current for 3 days in microbial fuel cells. Cross-sectional (top) and top-down (bottom) views are shown. Scale bars, 250 μm.

Fig 5

Average biomass of the mature biofilms formed on glass and graphite surfaces with the isogenic wild-type DL100 (Wt), short-isoform (pilA4), and ΔpilA2 strains. The results are the averages and standard errors of the mean from six biological replicates in two independent experiments.

Fig 6

Confocal microscope images of biofilms formed with wild-type DL100 (A and D) and the short-isoform (pilA4) (B and E) and ΔpilA2 (C and F) strains on Fe(III) oxide-coated glass. The images were taken after 24 h in medium with 40 mM fumarate present (A, B, and C) and 4 days later after the fumarate was removed (D, E, and F). The scale bars represent 75 μm. (G) Average numbers of cells in biofilms of the isogenic DL100 (Wt) and short-isoform (pilA4) and ΔpilA2 strains formed on Fe(III) oxide-coated glass. Cell densities were measured after 24 h of incubation in the presence of 40 mM fumarate and 4 days later after the fumarate was removed. The results are the averages of six biological replicates from two independent experiments. The error bars are standard errors of the mean.

Fig 7

Two models of pilus assembly and functions of the PilA isoforms. The short and long isoforms are depicted in green and blue, respectively. Both isoforms are shown cleaved after glycine −1 once they reach the inner membrane (I.M.), generating identical mature proteins. Model I (right) suggests that the mature PilA derived from the short PilA isoform remains intracellular and forms the base of the pilus fiber anchoring the long-isoform-derived PilA in the cell membrane. Model II (left) suggests that secreted PilA is derived from both preprotein isoforms, and long- and short-isoform-derived PilA proteins are assembled into the intra- and extracellular regions of the pilus fiber. O.M., outer membrane.