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Journal of Bacteriology, September 2009, p. 5603-5612, Vol. 191, No. 18
0021-9193/09/$08.00+0     doi:10.1128/JB.00627-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria{triangledown}

Irene K. Guttilla,1,{ddagger} Andrew H. Gaspar,1,{ddagger} Arlene Swierczynski,1,{ddagger} Anu Swaminathan,1,{ddagger} Prabhat Dwivedi,2,{ddagger} Asis Das,1 and Hung Ton-That1,2*

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030,1 Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, Texas 770302

Received 12 May 2009/ Accepted 6 July 2009

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, 6431 Fannin Street, Houston, TX 77030. Phone: (713) 500-5468. Fax: (713) 500-5499. E-mail: ton-that.hung{at}uth.tmc.edu

{triangledown} Published ahead of print on 10 July 2009.

{ddagger} I.K.G., A.H.G., A.S., A.S., and P.D. contributed equally to this work.

Journal of Bacteriology, September 2009, p. 5603-5612, Vol. 191, No. 18
0021-9193/09/$08.00+0     doi:10.1128/JB.00627-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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