Cloning, Expression, And Functional Analysis Of Type IV Pili Gene And The Molecular Motor PilT Gene In Acidithiobacillus Ferrooxidans

The composition, motility, and adherence about the type IV pili has a lot of reports in many Gram-negative bacterium strains but no report about the type IV pili of A. ferrooxidans. It is important that the motility and adherence of the type IV pili of A. ferrooxidans effects their survive in mine because they have to move and adhere to the mineral surface, and obtain energy by oxidizing mineral. In our work, four genes pilV, pilW, pilT, and pilU for the motility and adherence of Tfp were isolaetd by PCR from A. ferrooxidans. Sequence analysis and function predietion were carried out with different biological program. Gene experssion and transcription by 10% mineral powder induced were investigated. These cofirmed that the pilV and pilW genes were genes of Tfp proteins composed, and pilT gene was the molecular motor proteins gene of Tfp, which correlated with the motility and adherence of Tfp.The sequence analysis of PilV protein indicated that PilV protein contains the conserved motifs Ile7, Ala8, Gly13 and Ala48. The sequence analysis of PilW protein indicated that PilW protein contains the conserved FLELVAVIGL (signal peptides) and GRXAL (functional motif) motifs that are the characteristic motifs of pili family.The protein sequence of PilT contains the canonical conserved AIRNLIRE (from 286 to 293 amino acid residue) and GMQTXXXXLXXL (from 310 to 321 amino acid residue) motifs that are the characteristic motifs of pilT protein family. The protein sequence of PilT also contains the canonical nucleotide binding motifs the Walker A box (from 126 to 135 amino acid residue) and Walker B box (from 187 to 201 amino acid residue) that are the standard signature motif used to predict nucleotide-binding proteins.The protein sequence of PilU contains the canonical conserved AIRNLIRE (from 285 to 292 amino acid residues) and GMQTXXXXLXXL (from 309 to 320 amino acid residue) motifs that are the characteristic motifs of pilT protein family. However to compare the canonical conserved AIRNLIRE in other Gram-negative bacterium strains and AIADLIYK (from 285 to 292 amino acid residue) in A. ferrooxidans strains ATCC23270 indicates them difference signally, and some amino acid residue are substituted in A. ferrooxidans strains ATCC23270. The protein sequence of PilU also contains the canonical nucleotide binding motifs the Walker A box (from 126 to 135 amino acid residue) and Walker B box (from 190 to 201 amino acid residue) that are the standard signature motif used to predict nucleotide-binding proteins.RT-PCR analysis of the putative pilV, pilW, pilT, and pilU genes of A. ferrooxidans demonstrated that pilV and pilW genes of not all A. ferrooxidans strains were transcribed, and pilT and pilU genes were transcribed at same time by 10% mineral powder induced. However there were possible the time different in pilV and pilW transcribed. E. coli BL21 cells strains with pET-28a pilT and pilU plasmid were induced by IPTG to produce 37.10 and 42.0 kDa proteins.We used conventional methods to investigate the mechanism by which A. ferrooxidans colonizes a solid surface by assessing pili-mediated sliding, twitching motility, and adherence. A. ferrooxidans slided to form circular oxidised zones around each colony. This suggested that chemotaxis occurs through Tfp or flagella, though A. ferrooxidans strains ATCC19859 and ATCC23270 lack flagella. The results of reverse transcription-PCR demonstrated that the putative major Tfp gene of A. ferrooxidans strains ATCC 19859, ATCC23270, and BY3 genes were transcribed. Culture of A. ferrooxidans between silicone gel and glass led to the abundant production of Tfp and the formation of rough twitching motility zones. When the bacteria were grown on lean ore cubes, pyrite was colonised readily by A. ferrooxidans and the cells attached strongly to the mineral surface through Tfp. However, non-pili bacteria attached minimally to the mineral surface. We conclude that Tfp of A. ferrooxidans are necessary for sliding, twitching motility, and adherence to a solid surface.The Tfp conduction of A. ferrooxidans was analysed with an atomic force microscope (AFM) equipped with a conductive tip indicated that Tfp of A. ferrooxidans were highly conductive. This also indicated that A. ferrooxidans requires Tfp in order to oxidize Fe(II) oxides because Tfp were the electrical connection between the cell and the surface of the Fe(II) oxides. The likely function of the Tfp is to complete the circuit between these various intermediary electron carriers and the Fe(II) oxides.ATPase activity of PilT protein was high by ATPase assay but PilU lower. This indicated PilT protein was real the molecular motor of Tfp, and PilU could play a role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. PilT and PilU were related in forming and function for the molecular motor of Tfp.