Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27

Abstract

Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.

Fig. 1.

Classification of the identified phosphoproteins in T. thermophilus HB27. The phosphoproteins were grouped by (A) cellular location and (B) biological function based on Gene Ontology terms as assigned by Blast2GO. Hypothetical proteins were grouped into the “Unknown” category of the pie chart. C, this simplified diagram shows the central metabolic pathways that link glycolysis and the tricarboxylic acid cycle for glucose. Other carbohydrates can also be metabolized, via glucose-1-phosphate or glucosamine-1-phosphate. The shaded boxes show the phosphorylated enzymes identified in this study.

Fig. 2.

MS/MS spectra of the threonine and serine phosphorylated peptides belonging to ATP-binding-motif-containing protein PilF. Rich backbone fragmentation (RGGGRLEDpTLVQSGK and RGGGRLEDTLVQpSGK) was shown in the MS/MS spectra, in which (A) Thr-368 and (B) Ser-372 were identified as the sites of PilF phosphorylation.

Fig. 3.

Colony morphology and piliation of T. thermophilus HB27 strains. A, the effects of the overexpression of various PilF proteins in T. thermophilus HB27. Strains containing a plasmid expressing the indicated PilF allele were incubated on 1.6% Thermus medium plates for 3 days at 70 °C under humid conditions. Scale bar represents 5 mm. B, precipitation of the overexpressed PilF proteins in T. thermophilus HB27. After a 6-h incubation at 70 °C and an initial A₆₀₀ of 0.6, the cells were transferred to 2-ml tubes containing PBS buffer and subjected to gravity sedimentation at 4 °C for 1 day. C, images of T. thermophilus HB27 strains overexpressing the various PilF proteins. Cells from 2-day cultures on agar plates of the indicated strains were directly transferred to a grid and stained with 2% uranyl acetate for transmission electron microscopy. Approximately 20 cells of each strain were analyzed for piliation. Bar, 0.1 μm.

Fig. 4.

Biofilm formation and EPS production by T. thermophilus HB27 strains. A, biofilm formation by strains of T. thermophilus HB27 overexpressing various PilF proteins. Biofilms formed on 96-well microtiter plates after 24 h were stained with crystal violet. Surface-attached cells were quantified by solubilizing the dye in ethanol and determining the absorbance at 600 nm. Data are the means of three independent experiments, with each sample tested in triplicate. The asterisks (*) indicate statistical significance (p < 0.05). B, the total EPS content extracted from T. thermophilus HB27 strains expressing wild-type or mutant PilF proteins normalized to each of their bacterial concentrations, as determined in a phenol–sulfuric acid assay. Error bar represents the standard deviations of three independent measurements. Asterisks indicate statistical significance (p < 0.05).