Many important pathogens such as Bacillus anthracis, Staphylococcus aureus, or Mycoplasma pneumoniae belong to the Gram-positive group of Firmicutes. In contrast to the first two pathogens, Mycoplasmas are characterized by their strongly reduced genome (M. pneumoniae encodes 688 protein) and the loss of the cell wall. Due to their small genomes, Mycoplasma have attracted much attention in systems and synthetic biology. Since M. pneumoniae is a human pathogen, we are interested in virulence factors of this organism. We focus on one major virulence determinant, hydrogen peroxide. It is unclear how Mycoplasma species can produce and at the same time tolerate high amounts of hydrogen peroxide. Furthermore, a huge number of genes have an unknown function and no homology to known proteins from other bacteria. We want to investigate these unknown genes with respect to their influence on virulence, growth and essentiality. Therefore, we collaborate within a consortium of research institutes in the MiniCell project to delete non-essential genes to create a much more reduced organism. For this we developed new techniques to manipulate M. pneumoniae very efficiently and to create targeted deletions. However, this near-minimal organism is also of great interest for basic research. Together with colleagues in Berlin we study the complete in vivo interactome of M. pneumoniae which leads also to the identification of new gene function, deduced from their interaction partners.