Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.

The new field of synthetic biology aims at the creation of artificially designed organisms. A major breakthrough in the field was the generation of the artificial synthetic organism Mycoplasma mycoides JCVI-syn3A. This bacterium possesses only 452 protein-coding genes, the smallest number for any organism that is viable independent of a host cell. However, about one third of the proteins have no known function indicating major gaps in our understanding of simple living cells. To facilitate the investigation of the components of this minimal bacterium, we have generated the database SynWiki (http://synwiki. uni-goettingen.de/). SynWiki is based on a relational database and gives access to published information about the genes and proteins of M. mycoides JCVI-syn3A. To gain a better understanding of the functions of the genes and proteins of the artificial bacteria, protein-protein interactions that may provide clues for the protein functions are included in an interactive manner. SynWiki is an important tool for the synthetic biology community that will support the comprehensive understanding of a minimal cell as well as the functional annotation of so far uncharacterized proteins.

Bacteria evolved many ways to invade, colonize and survive in the host tissue. Such complex infection strategies of other bacteria are not present in the cell-wall less Mycoplasmas. Due to their strongly reduced genomes, these bacteria have only a minimal metabolism. Mycoplasma pneumoniae is a pathogenic bacterium using its virulence repertoire very efficiently, infecting the human lung. M. pneumoniae can cause a variety of conditions including fever, inflammation, atypical pneumoniae, and even death. Due to its strongly reduced metabolism, M. pneumoniae is dependent on nutrients from the host and aims to persist as long as possible, resulting in chronic diseases. Mycoplasmas evolved strategies to subvert the host immune system which involve proteins fending off immunoglobulins (Igs). In this study, we investigated the role of MPN400 as the putative factor responsible for Ig-binding and host immune evasion. MPN400 is a cell-surface localized protein which binds strongly to human IgG, IgA, and IgM. We therefore named the protein MPN400 immunoglobulin binding protein of Mycoplasma (IbpM). A strain devoid of IbpM is slightly compromised in cytotoxicity. Taken together, our study indicates that M. pneumoniae uses a refined mechanism for immune evasion.

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