Abstract
PilT is a hexameric ATPase required for type IV pili (Tfp) retraction in gram-negative bacterium. Retraction of Tfp mediates intimate attachment and motility on inorganic solid surfaces. We investigated the cloning and expression of pilT and pilU genes of Acidithiobacillus ferrooxidans strains ATCC 23270, and the results indicate that PilT and PilU contain the canonical conserved AIRNLIRE and GMQTXXXXLXXL motifs that are the characteristic motifs of the PilT protein family; PilT and PilU also contain the canonical nucleotide-binding motifs, named with Walker A box (GxxGxGKT/S) and Walker B box (hhhhDE), respectively. The pilT and pilU genes were expressed to produce 37.1- and 42.0-kDa proteins, respectively, and co-transcribed induced by 10 % mineral powder. However, ATPase activity of PilT was distinctly higher than those of PilU. These results indicated that the PilT protein was the real molecular motor of Tfp, while PilU could play a key role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. However, PilT and PilU were nonetheless interrelated in the forming and function of the molecular motor of Tfp.
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References
Aukema KG, Kron EM, Herdendorf TJ, Forest KT (2005) Functional dissection of a conserved motif within the pilus retraction protein PilT. J Bacteriol 187:611–618
Bertrand JJ, West JT, Enge JN (2010) Genetic analysis of the regulation of type IV pilus function by the Chp chemosensory system of Pseudomonas aeruginosa. J Bacteriol 192:994–1010
Chakraborty S, Monfett M, Maier TM, Benach JL, Frank DW, Thanassi DG (2008) Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence, and virulence. Infect Immun 76:2852–2861
Chiang P, Habash M, Burrows LL (2005) Disparate subcellular localization patterns of Pseudomonas aeruginosa type IV pilus ATPases involved in twitching motility. J Bacteriol 187:829–839
Chiang P, Sampaleanu M, Ayers M, Pahuta M, Howell PL, Burrows LL (2008) Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU. Microbiology 154:114–126
Forsberg A, Guina T (2007) Type II secretion and type IV pili of Francisella. Ann N Y Acad Sci 1105:187–201
Fuente LDL, Montanes E, Meng YZ, Li YX, Burr TJ, Hoch HC, Wu MM (2007) Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber. Appl Envir Microbiol 73:2690–2696
Han XY, Kennan RM, Davies JK, Reddacliff LA, Dhungye OP, Whittington RJ, Turnbull L, Whitchurch CB, Rood JI (2008) Twitching motility is essential for virulence in Dichelobacter nodosus. J Bacteriol 190:3323–3335
Herdendorf TJ, McCaslin DR, Forest KT (2002) Aquifex aeolicus PilT, homologue of a surface motility protein, is a thermostable oligomeric NTPase. J Bacteriol 184:6465–6471
Li YQ, Wan DS, Huang SS, Leng FF, Yan L, Ni YQ, Li HY (2010) Type IV pili of Acidithiobacillus ferrooxidans are necessary for sliding, twitching motility, and adherence. Curr Microbiol 60:17–24
Maier B, Potter L, So M, Seifert H, Sheetz M (2002) The destruction of one pilus by PilT releases enough energy to rein in the bacterium. PNAS 99:16012–16017
Nakasugi K, Alexova R, Svenson CJ, Neilan BA (2007) Functional analysis of pilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806. J Bacteriol 189:1689–1697
Park HSM, Wolfgang M, Koomey M (2002) Modification of type IV pilus-associated epithelial cell adherence and multicellular behavior by the PilU protein of Neisseria gonorrhoeae. Infect Immun 70:3891–3903
Planet PJ, Kachlany SC, DeSalle R, Figurski DH (2001) Phylogeny of genes for secretion NTPases: identification of the wide spread tadA subfamily and development of a diagnostic key for gene classification. PNAS 98:2503–2508
Robien MA, Krumm BE, Sandkvist M, Hol WG (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333:657–674
Sexton JA, Pinkner JS, Roth R, Heuser JE, Hultgren SJ, Vogel JP (2004) The Legionella pneumophila pilT homologue dotB Exhibits ATPase activity that is critical for intracellular growth. J Bacteriol 186:1658–1666
Skerker JM, Berg HC (2001) Direct observation of extension and retraction of type IV pili. PNAS 98:6901–6904
Turgay K, Hamoen LW, Venema G, Dubnau D (1997) Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis. Genes Dev 11:119–128
Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951
Whitchurch CB, Mattick JS (2006) Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa. Mol Microbiol 13:1079–1091
Wolfgang M, Lauer P, Park HS, Brossay L, Hebert J, Koomey M (1998) PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 29:321–330
Yasukawa K, Martin P, Tinsley CR, Nassif X (2006) Pilus-mediated adhesion of Neisseria meningitidis is negatively controlled by the pilus-retraction machinery. Mol Microbiol 59:579–589
Yeo HJ, Savvides SN, Herr AB, Lanka E, Waksman G (2000) Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell 6:1461–1472
Acknowledgments
This work was supported by the Fundamental Research Funds for the Central Universities (Grant No. zyz 2011093) and the Science and Technique Program of Gansu Province (Grant No. 3YS061-A25-017). Dr. B. Johnson is gratefully acknowledged for providing A. ferrooxidans strain ATCC 19859, and Prof. Douglas E. Rawlings for A. ferrooxidans strain ATCC 23270.
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Li, Y., Huang, S., Zhang, X. et al. Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans . Appl Microbiol Biotechnol 97, 1251–1257 (2013). https://doi.org/10.1007/s00253-012-4271-1
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DOI: https://doi.org/10.1007/s00253-012-4271-1