title: Experimental Methods for Studying the BAM Complex in Neisseria meningitidis.
abstract: Neisseria meningitidis is a human pathogen. It is intensively studied for host-pathogen interactions and vaccine development. However, its favorable growth properties, genetic accessibility, and small genome size also make it an excellent model organism for studying fundamental biological processes, such as outer membrane biogenesis. Indeed, the first component of the assembly machinery for outer-membrane proteins, the BAM complex, was identified in N. meningitidis. Here, we describe protocols to inactivate chromosomal genes and to express genes from a well-controlled promoter on a plasmid in N. meningitidis. Together, these protocols can be used, for example, to deplete cells from essential components of the BAM complex. We also describe a simple, gel-based assay to assess the proper functioning of the BAM complex in vivo.
authors.affiliation: Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands.|Department of Medical Microbiology and Infection Control, VU University Medical Center, van der Boechorststraat 7, Amsterdam, 1081 BT, The Netherlands.
authors.forename: Martine P
authors.initials: MP
authors.lastname: Bos
authors.affiliation: Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands.
authors.forename: Ria Tommassen-van
authors.initials: RT
authors.lastname: Boxtel
authors.affiliation: Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands. j.p.m.tommassen@uu.nl.
authors.forename: Jan
authors.initials: J
authors.lastname: Tommassen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002876:Chromosomes, Bacterial / Q000235:genetics; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D055786:Gene Knockout Techniques; D005821:Genetic Techniques; D009345:Neisseria meningitidis / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D014170:Transformation, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial
keywords: BAM complex; Expression vector; Gene cloning; Mutagenesis; Neisseria meningitidis; Outer membrane protein; Semi-native SDS-PAGE