Topology and membrane interaction of Helicobacter pylori ComB proteins involved in natural transformation competence.
The human gastric pathogen Helicobacter pylori is naturally competent for genetic transformation. The H. pylori comB gene duster encodes the VirB4-homologous ATPase ComB4 and the structural proteins ComB7-ComB10, which share significant sequence identity to the Agrobacterium tumefaciens virB-encoded type IV secretion system. To study the topology of the ComB7-10 proteins, we applied TnMax transposon mutagenesis by generating fusions of ComB proteins with mature beta-lactamase (BlaM) or alkaline phosphatase (PhoA). Our data show that the putative lipoprotein ComB7 is secreted and is found membrane-attached, probably by its lipid anchor. According to our topology mapping ComB8 is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space. ComB9 was verified as a periplasmic protein, tightly attached to the membrane. The N-terminus of ComB10 is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm. Limited protease digestion and protein extraction under different salt and pH conditions confirmed the periplasmic localization and the tight membrane association of the ComB protein complex. A hypothetical model of the ComB DNA transformation pore in H. pylori is presented.
Hofreuter
Dirk
D
Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, LMU München, München, Germany. dirk.hofreuter@yale.edu
Karnholz
Arno
A
Haas
Rainer
R
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004251:DNA Transposable Elements; D010450:Endopeptidases; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D018272:Porins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014170:Transformation, Genetic; D037521:Virulence Factors; D001618:beta-Lactamases / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D018272:Porins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D001618:beta-Lactamases