The protein network of bacterial motility
2007; Springer Nature; Volume: 3; Issue: 1 Linguagem: Inglês
10.1038/msb4100166
ISSN1744-4292
AutoresSeesandra V. Rajagopala, Bjoern Titz, Johannes B. Goll, Jodi R. Parrish, Katrin Wohlbold, Matthew McKevitt, Timothy Palzkill, Hirotada Mori, Russell L. Finley, Peter Uetz,
Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle31 July 2007Open Access The protein network of bacterial motility Seesandra V Rajagopala Seesandra V Rajagopala Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany The Institute for Genomic Research, Rockville, MD, USA Search for more papers by this author Björn Titz Björn Titz Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Johannes Goll Johannes Goll Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Jodi R Parrish Jodi R Parrish Center for Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Katrin Wohlbold Katrin Wohlbold Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Matthew T McKevitt Matthew T McKevitt Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Timothy Palzkill Timothy Palzkill Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Hirotada Mori Hirotada Mori Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Russell L Finley Jr Russell L Finley Jr Center for Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Peter Uetz Corresponding Author Peter Uetz Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany The Institute for Genomic Research, Rockville, MD, USA Search for more papers by this author Seesandra V Rajagopala Seesandra V Rajagopala Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany The Institute for Genomic Research, Rockville, MD, USA Search for more papers by this author Björn Titz Björn Titz Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Johannes Goll Johannes Goll Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Jodi R Parrish Jodi R Parrish Center for Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Katrin Wohlbold Katrin Wohlbold Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany Search for more papers by this author Matthew T McKevitt Matthew T McKevitt Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Timothy Palzkill Timothy Palzkill Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Hirotada Mori Hirotada Mori Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Russell L Finley Jr Russell L Finley Jr Center for Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Peter Uetz Corresponding Author Peter Uetz Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany The Institute for Genomic Research, Rockville, MD, USA Search for more papers by this author Author Information Seesandra V Rajagopala1,2,‡, Björn Titz1,‡, Johannes Goll1,‡, Jodi R Parrish3, Katrin Wohlbold1, Matthew T McKevitt4, Timothy Palzkill4, Hirotada Mori5,6, Russell L Finley3 and Peter Uetz 1,2 1Institute of Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany 2The Institute for Genomic Research, Rockville, MD, USA 3Center for Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA 4Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA 5Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan 6Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan ‡These authors contributed equally to this work *Corresponding author. Intitute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, D-76021 Karlsruhe, Germany. Tel.: +49 7247 826103; Fax: +49 7247 823354 or J Craig Venter Institute (JCVI), 9712 Medical Center Drive, Rockville, MD 20850, USA. Tel.: +1 301 795 7589; Fax: +1 301 294 3142; E-mail: [email protected] Molecular Systems Biology (2007)3:128https://doi.org/10.1038/msb4100166 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Motility is achieved in most bacterial species by the flagellar apparatus. It consists of dozens of different proteins with thousands of individual subunits. The published literature about bacterial chemotaxis and flagella documented 51 protein–protein interactions (PPIs) so far. We have screened whole genome two-hybrid arrays of Treponema pallidum and Campylobacter jejuni for PPIs involving known flagellar proteins and recovered 176 and 140 high-confidence interactions involving 110 and 133 proteins, respectively. To explore the biological relevance of these interactions, we tested an Escherichia coli gene deletion array for motility defects (using swarming assays) and found 159 gene deletion strains to have reduced or no motility. Comparing our interaction data with motility phenotypes from E. coli, Bacillus subtilis, and Helicobacter pylori, we found 23 hitherto uncharacterized proteins involved in motility. Integration of phylogenetic information with our interaction and phenotyping data reveals a conserved core of motility proteins, which appear to have recruited many additional species-specific components over time. Our interaction data also predict 18 110 interactions for 64 flagellated bacteria. Synopsis Motility is achieved in most bacterial species by a complex machine called the flagellar apparatus. This mechanical nanomachine consists of dozens of different proteins, most of which are present in multiple, sometimes thousands of copies (as in the case of the filament protein FliC). The bacterial flagellum rotates at a rotation frequency of 300 Hz, has an energy conversion rate of nearly 100%, and is able to self assemble (Berg, 2003; Macnab, 1999, 2003; Kojima and Blair, 2004). Systematic analysis of hundreds of completely sequenced bacterial genomes has predicted many additional motility genes. Most of these predicted motility genes lie in known flagellar operons or gene clusters, although often their actual roles in motility remain unknown. A major goal of this study was to find novel flagellar components among the many proteins of still unknown function. In addition, we attempted an integrative systems biology approach to assemble a comprehensive picture of the flagellar protein complex in different bacterial species. In this study, we first identified genes essential for bacterial motility by systematically testing the swarming capability of 3985 gene deletion strains of Escherichia coli (Baba et al, 2006) and identified 159 mutants showing a reduced or nonmotile phenotype. Out of these genes, 116 are "new" motility genes, that is, they were not known previously to play a role in motility. Second, we screened all previously known motility proteins for protein–protein interactions (PPIs) in two distantly related bacteria, Treponema pallidum, the causative agent of syphilis, and Campylobacter jejuni, a common cause of gastroenteritis. We reasoned that unknown motility proteins can be discovered by interactions with known flagellar and chemotaxis components. The motility protein interactions were identified using comprehensive array-based yeast two-hybrid screens (Uetz et al, 2000, Parrish et al, submitted). Indeed, 28 and 33% of the 176 and 140 high-confidence interactions found in T. pallidum and C. jejuni, respectively, connect a known motility protein to a conserved hypothetical protein (Supplementary Table S4), suggesting that there are still unidentified proteins with a motility function. The diversity of information on different genomes, proteins, phenotypes, and so on makes it difficult to keep track of all details. Therefore, we combined PPI data sets of T. pallidum, Campylobacter pylori, H. pylor (Rain et al, 2001), and E. coli (Arifuzzaman et al, 2006), as well as interactions curated from the literature, from genome-wide motility phenotyping data sets of E. coli and Bacillus subtilis (Schumann et al, 2001) and from small-scale mutant screens of C. jejuni (Golden et al, 2000; Hendrixson et al, 2001), and Helicobacter pylori (Salama et al, 2004). The resulting network summarizes the current knowledge about functional and protein interaction data from multiple species (Figure 4). We assigned motility functions to 23 hitherto uncharacterized proteins based on their interaction with known motility proteins and/or their motility phenotype (Table I). For example, multiple members of the cluster of orthologous group COG1664, for example, TP0048 and HP1542 (Figure 4B), show interactions with the FliC–FliS cluster (note that FliC is called FlaA or FlaB in other species). Additional evidence for their role in motility comes from the double mutant of the B. subtilis orthologs, yhbE and yhbF, which also show reduced motility. To represent multiple species in the integrated motility network, homologous proteins are combined into "clusters of orthologous groups" (COGs), rather than individual proteins. This allowed us to reduce the overwhelming complexity of the network and improve the quality of links, which are supported by multiple evidence. The bacterial flagellum has attracted attention because of its amazing complexity, which appears to have evolved from a much simpler type III secretion channel. We believe that our interaction data and phenotypes support this model. First, a phylogenetic supertree of 30 species soley based on 35 flagellar protein families (Supplementary Figure 4) supports the phylogeny of bacteria as reported previously, for example an rRNA tree (Olsen et al, 1994) and a tree which was based on 31 highly conserved protein families (Ciccarelli et al, 2006). This shows that the flagellar system evolved together with other cellular systems and not independently. Evolution of the flagellum is also consistent with the fact that neither any flagellar protein nor any of its interactions is conserved. In fact, our Treponema data set predicted 173 interactions for C. jejuni, of which we found only 49 (Supplementary Table S4d). This indicates that protein interactions may be evolutionarily less conserved than generally believed. An evolutionary model also predicts that core proteins, which have been associated with the flagellum, should be tightly integrated, and thus have more interactions than peripheral proteins, which have been only recently recruited to the flagellar machinery. Indeed, we did find a weak, but statistically significant linear relationship between the number of interactions of an orthologous group and its conservation ratio among flagellated bacteria (r=0.43, P 0.9) to be strongly associated. These numbers indicate that this integrated network is more reliable and biologically relevant than individual networks. In addition, links among orthologous groups can usually be transferred to proteins of other species. However, because of the stringent filtering not all interactions are included in this network. Figure 4.Integrated motility network. Multispecies summary of interactions among motility proteins. (A) Homologous protein nodes of pairwise aligned networks were merged into orthologous groups (COGs). Nodes were labelled according to KEGG descriptions (Kanehisa et al, 2006); their shape corresponds to motility phenotypes in E. coli (squares), B. subtilis (octagons), or both (rounded squares). Edges represent either direct, indirect, or literature interactions among orthologous groups and are color-coded (see legend in Figure). The border shading of a COG nodes indicates its conservation ratio among 68 flagellated bacteria. (B–E) Aligned protein interactions showing COG interactions from (A) in more detail: each node represents two homologous proteins from either T. pallidum (TPA), C. jejuni (CJE), H. pylori (HPY), or E. coli (ECO). Edges represent either direct or indirect interactions and are color-coded (see legend in Figure). The border color of a protein node indicates the BLAST E-value of its homologous protein pair. (F) Interactions of paralogous FliG1 and FliG2 proteins taken from T. pallidum network. Download figure Download PowerPoint Several insights into the internal organization of the bacterial flagellum can be obtained. For example, the aligned network shows that the flagellum filament protein, FliC, and its homolog FlgL, a hook-associated protein, are members of the same COG. FlgL is connected to the second hook-associated protein FlgK and both are stabilized by their export chaperone, FlgN. The interaction of FliC with its chaperone FliS is conserved in all species. The basal body complex with FliN/FliY, FliG, FliM, and FliF, forms another cluster, which is connected to the motor proteins, MotA and MotB, and to rod proteins such as FlgC and FlgG. The chemotaxis protein cluster in the network is only connected to the flagellum switch complex. The interaction of CheY with FliM depends on CheY's phosphorylation, which is not detected in our yeast two-hybrid assays, because we do not coexpress the pertinent kinase CheA. Nevertheless, the integrated network reflects the fact that external signals are detected by homodimerizing methyl-accepting chemoreceptors (Mcps), which are linked by an adapter protein, CheW, to the kinase CheA, which transfers the phosphate group to CheY. By its interaction with the basal body complex, phosphorylated CheW controls the rotation state of the flagellum. In addition to these previously known interactions, we find conserved links between chemotaxis proteins and rod proteins such as FlgB and FlgG, which are difficult to explain by the standard model of the flagellum, but allow for interesting speculations about the organization of chemotaxis signalling in the cytoplasm. Another striking connection is the conserved MotB–FliL interaction in C. jejuni and Helicobacter. For Proteus mirabilis, FliL is thought to be involved in sensing of the actual flagellum status (Belas and Suvanasuthi, 2005). Here, we found evidence that this sensing is mediated by a direct interaction with the motor apparatus (Figure 4E). Discussion New bona fide motility proteins A major goal of this study was to find novel flagellar components among the many proteins of still unknown function. In addition, we suspected that there must b
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