title: Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.
abstract: The Gram-positive pathogenClostridium perfringenspossesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present inC. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of thepilB2andpilC2genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detectedin vivoorin vitroeven in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase fromC. perfringens(CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levelsin vivobut that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously fromin vivostudies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium.IMPORTANCEType IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable speciesClostridium perfringenswas used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.
authors.affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Hendrick
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
authors.forename: Mona W
authors.initials: MW
authors.lastname: Orr
authors.affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA.
authors.forename: Samantha R
authors.initials: SR
authors.lastname: Murray
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA melville@vt.edu.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Melville
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003016:Clostridium perfringens / Q000201:enzymology* / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D036781:Fimbriae Proteins / Q000378:metabolism*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D036781:Fimbriae Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C068255:pilB protein, Bacteria; D000251:Adenosine Triphosphatases; D006152:Cyclic GMP; D015032:Zinc
keywords: ATPase; bacterial pathogenesis; cyclic di-GMP; molecular motor; protein-protein interaction; type IV pili