ListiWiki: A database for the foodborne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram positive foodborne pathogen that regularly causes outbreaks of systemic infectious diseases. The bacterium maintains a facultative intracellular lifestyle; it thrives under a variety of environmental conditions and is able to infect human host cells. L. monocytogenes is genetically tractable and therefore has become an attractive model system to study the mechanisms employed by facultative intracellular bacteria to invade eukaryotic cells and to replicate in their cytoplasm. Besides its importance for basic research, L. monocytogenes also serves as a paradigmatic pathogen in genomic epidemiology, where the relative stability of its genome facilitates successful outbreak detection and elucidation of transmission chains in genomic pathogen surveillance systems. In both terms, it is necessary to keep the annotation of the L. monocytogenes genome up to date. Therefore, we have created the database ListiWiki (http://listiwiki.uni-goettingen.de/) which stores comprehensive information on the widely used L. monocytogenes reference strain EDG-e. ListiWiki is designed to collect information on genes, proteins and RNAs and their relevant functional characteristics, but also further information such as mutant phenotypes, available biological material, and publications. In its present form, ListiWiki combines the most recent annotation of the EDG-e genome with published data on gene essentiality, gene expression and subcellular protein localization. ListiWiki also predicts protein-protein interactions networks based on protein homology to Bacillus subtilis proteins, for which detailed interaction maps have been compiled in the sibling database SubtiWiki. Furthermore, crystallographic information of proteins is made accessible through integration of Protein Structure Database codes and AlphaFold structure predictions. ListiWiki is an easy-to-use web interface that has been developed with a focus on an intuitive access to all information. Use of ListiWiki is free of charge and its content can be edited by all members of the scientific community after registration. In our labs, ListiWiki has already become an important and easy to use tool to quickly access genome annotation details that we can keep updated with advancing knowledge. It also might be useful to promote the comprehensive understanding of the physiology and virulence of an important human pathogen.

PAE viewer: a webserver for the interactive visualization of the predicted aligned error for multimer structure predictions and crosslinks.

The development of AlphaFold for protein structure prediction has opened a new era in structural biology. This is even more the case for AlphaFold-Multimer for the prediction of protein complexes. The interpretation of these predictions has become more important than ever, but it is difficult for the non-specialist. While an evaluation of the prediction quality is provided for monomeric protein predictions by the AlphaFold Protein Structure Database, such a tool is missing for predicted complex structures. Here, we present the PAE Viewer webserver (http://www.subtiwiki.uni-goettingen.de/v4/paeViewerDemo), an online tool for the integrated visualization of predicted protein complexes using a 3D structure display combined with an interactive representation of the Predicted Aligned Error (PAE). This metric allows an estimation of the quality of the prediction. Importantly, our webserver also allows the integration of experimental cross-linking data which helps to interpret the reliability of the structure predictions. With the PAE Viewer, the user obtains a unique online tool which for the first time allows the intuitive evaluation of the PAE for protein complex structure predictions with integrated crosslinks.

Understudied proteins and understudied functions in the model bacterium Bacillus subtilis-A major challenge in current research.

Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for model organisms, no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Of poorly studied proteins, those that are strongly expressed are likely to be important to the cell and should therefore be considered high priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, for example, from global interaction, expression, or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport, and the control of metabolic homeostasis. The identification of the functions of the selected proteins not only will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.

Protein complexes in cells by AI-assisted structural proteomics.

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry and co-fractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

MycoWiki: Functional annotation of the minimal model organism Mycoplasma pneumoniae.

The human pathogen Mycoplasma pneumoniae is viable independently from host cells or organisms, despite its strongly reduced genome with only about 700 protein-coding genes. The investigation of M. pneumoniae can therefore help to obtain general insights concerning the basic requirements for cellular life. Accordingly, M. pneumoniae has become a model organism for systems biology in the past decade. To support the investigation of the components of this minimal bacterium, we have generated the database MycoWiki. (http://mycowiki.uni-goettingen.de) MycoWiki organizes data under a relational database and provides access to curated and state-of-the-art information on the genes and proteins of M. pneumoniae. Interestingly, M. pneumoniae has undergone an evolution that resulted in the limited similarity of many proteins to proteins of model organisms. To facilitate the analysis of the functions of M. pneumoniae proteins, we have integrated structure predictions from the AlphaFold Protein Structure Database for most proteins, structural information resulting from in vivo cross-linking, and protein-protein interactions based on a global in vivo study. MycoWiki is an important tool for the systems and synthetic biology community that will support the comprehensive understanding of a minimal organism and the functional annotation of so far uncharacterized proteins.

The current state of SubtiWiki, the database for the model organism Bacillus subtilis.

Bacillus subtilis is a Gram-positive model bacterium with extensive documented annotation. However, with the rise of high-throughput techniques, the amount of complex data being generated every year has been increasing at a fast pace. Thus, having platforms ready to integrate and give a representation to these data becomes a priority. To address it, SubtiWiki (http://subtiwiki.uni-goettingen.de/) was created in 2008 and has been growing in data and viewership ever since. With millions of requests every year, it is the most visited B. subtilis database, providing scientists all over the world with curated information about its genes and proteins, as well as intricate protein-protein interactions, regulatory elements, expression data and metabolic pathways. However, there is still a large portion of annotation to be unveiled for some biological elements. Thus, to facilitate the development of new hypotheses for research, we have added a Homology section covering potential protein homologs in other organisms. Here, we present the recent developments of SubtiWiki and give a guided tour of our database and the current state of the data for this organism.

SynWiki: Functional annotation of the first artificial organism Mycoplasma mycoides JCVI-syn3A.

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.