title: Identification of pseudomurein cell wall binding domains.
abstract: Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.
authors.affiliation: Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands.
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Steenbakkers
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Geerts
authors.forename: Nilgün A
authors.initials: NA
authors.lastname: Ayman-Oz
authors.forename: Jan T
authors.initials: JT
authors.lastname: Keltjens
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002473:Cell Wall / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017013:Methanobacteriaceae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase