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YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival., Competing Interests: Conflict of interest G. T. M. is the founder of Nexomics Biosciences Inc. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori is a common pathogen that is estimated to infect half of the human population, causing several diseases such as duodenal ulcer. Despite one of the first pathogens to be sequenced, its proteome remains poorly characterized as about one-third of its proteins have no functional annotation. Here, we integrate and analyze known protein interactions with proteomic and genomic data from different sources. We find that proteins with similar abundances tend to interact. Such an observation is accompanied by a trend of interactions to appear between proteins of similar functions, although some show marked cross-talk to others. Protein function prediction with protein interactions is significantly improved when interactions from other bacteria are included in our network, allowing us to obtain putative functions of more than 300 poorly or previously uncharacterized proteins. Proteins that are critical for the topological controllability of the underlying network are significantly enriched with genes that are up-regulated in the spiral compared with the coccoid form of H. pylori Determining their evolutionary conservation, we present evidence that 80 protein complexes are identical in composition with their counterparts in Escherichia coli , while 85 are partially conserved and 120 complexes are completely absent. Furthermore, we determine network clusters that coincide with related functions, gene essentiality, genetic context, cellular localization, and gene expression in different cellular states., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mycobacteriophage are viruses that infect mycobacteria. More than 1,400 mycobacteriophage genomes have been sequenced, coding for over one hundred thousand proteins of unknown functions. Here we investigate mycobacteriophage Giles-host protein-protein interactions (PPIs) using yeast two-hybrid screening (Y2H). A total of 25 reproducible PPIs were found for a selected set of 10 Giles proteins, including a putative virion assembly protein (gp17), the phage integrase (gp29), the endolysin (gp31), the phage repressor (gp47), and six proteins of unknown function (gp34, gp35, gp54, gp56, gp64, and gp65). We note that overexpression of the proteins is toxic to M. smegmatis, although whether this toxicity and the associated changes in cellular morphology are related to the putative interactions revealed in the Y2H screen is unclear.

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF K for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the half for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.K half for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Protein-protein interactions (PPIs) can offer compelling evidence for protein function, especially when viewed in the context of proteome-wide interactomes. Bacteria have been popular subjects of interactome studies: more than six different bacterial species have been the subjects of comprehensive interactome studies while several more have had substantial segments of their proteomes screened for interactions. The protein interactomes of several bacterial species have been completed, including several from prominent human pathogens. The availability of interactome data has brought challenges, as these large data sets are difficult to compare across species, limiting their usefulness for broad studies of microbial genetics and evolution., Results: In this study, we use more than 52,000 unique protein-protein interactions (PPIs) across 349 different bacterial species and strains to determine their conservation across data sets and taxonomic groups. When proteins are collapsed into orthologous groups (OGs) the resulting meta-interactome still includes more than 43,000 interactions, about 14,000 of which involve proteins of unknown function. While conserved interactions provide support for protein function in their respective species data, we found only 429 PPIs (~1% of the available data) conserved in two or more species, rendering any cross-species interactome comparison immediately useful. The meta-interactome serves as a model for predicting interactions, protein functions, and even full interactome sizes for species with limited to no experimentally observed PPI, including Bacillus subtilis and Salmonella enterica which are predicted to have up to 18,000 and 31,000 PPIs, respectively., Conclusions: In the course of this work, we have assembled cross-species interactome comparisons that will allow interactomics researchers to anticipate the structures of yet-unexplored microbial interactomes and to focus on well-conserved yet uncharacterized interactors for further study. Such conserved interactions should provide evidence for important but yet-uncharacterized aspects of bacterial physiology and may provide targets for anti-microbial therapies.

Helicobacter pylori (H. pylori) is a ε-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence. We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome. Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-α inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori., Biological Significance: Our study shows that SILAC can be employed to study protein abundance changes in H. pylori. We have chosen to establish SILAC for H. pylori because it facilitates fractionation on both, protein and peptide level and thus enables deep proteome coverage. Furthermore, SILAC allows robust and highly accurate protein quantification. The manuscript includes a detailed description of the applied method, suggestions for further improvement as well as a practical application. The investigation of differences between the coccoid and infectious spiral morphology of H. pylori with SILAC revealed the regulation of proteins that are involved in host colonization, motility, cell division as well as transcriptional and translational processes. The data will help molecular biologist to focus on relevant pathways that were found to be regulated in response to morphological changes. Furthermore, the application of SILAC offers new possibilities to study the biology of H. pylori. It enables to monitor protein abundance changes in response to certain stimuli such as oxygen stress or antibiotics. Moreover, SILAC raises the possibility to study co-cultures of host cells and H. pylori on protein level. Additionally, pulsed SILAC experiments enable the quantification of protein turnover., (Copyright © 2015. Published by Elsevier B.V.)

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 "gold standard" protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 "gold standard" protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial "model" species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies.

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

Unlabelled: More than 20% of all protein domains are currently annotated as "domains of unknown function" (DUFs). About 2,700 DUFs are found in bacteria compared with just over 1,500 in eukaryotes. Over 800 DUFs are shared between bacteria and eukaryotes, and about 300 of these are also present in archaea. A total of 2,786 bacterial Pfam domains even occur in animals, including 320 DUFs. Evolutionary conservation suggests that many of these DUFs are important. Here we show that 355 essential proteins in 16 model bacterial species contain 238 DUFs, most of which represent single-domain proteins, clearly establishing the biological essentiality of DUFs. We suggest that experimental research should focus on conserved and essential DUFs (eDUFs) for functional analysis given their important function and wide taxonomic distribution, including bacterial pathogens., Importance: The functional units of proteins are domains. Typically, each domain has a distinct structure and function. Genomes encode thousands of domains, and many of the domains have no known function (domains of unknown function [DUFs]). They are often ignored as of little relevance, given that many of them are found in only a few genomes. Here we show that many DUFs are essential DUFs (eDUFs) based on their presence in essential proteins. We also show that eDUFs are often essential even if they are found in relatively few genomes. However, in general, more common DUFs are more often essential than rare DUFs.

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens.

Yeast two-hybrid screens often produce vastly non-overlapping interaction data when the screens are conducted in different laboratories, or use different vectors, strains, or reporter genes. Here we investigate the underlying reasons for such inconsistencies and compare the effect of seven different vectors and their yeast two-hybrid interactions. Genome-wide array screens with 49 motility-related baits from Treponema pallidum yielded 77 and 165 interactions with bait vectors pLP-GBKT7 and pAS1-LP, respectively, including 21 overlapping interactions. In addition, 90 motility-related proteins from Escherichia coli were tested in all pairwise combinations and yielded 140 interactions when tested with pGBKT7g/pGADT7g vectors but only 47 when tested with pDEST32/pDEST22. We discuss the factors that determine these effects, including copy number, the nature of the fusion protein, and species-specific differences that explain non-conserved interactions among species. The pDEST22/pDEST32 vectors produce a higher fraction of interactions that are conserved and that are biologically relevant when compared with the pGBKT7/pGADT7-related vectors, but the latter appear to be more sensitive and thus detect more interactions overall.

Unlabelled: Prokaryotic protein-protein interactions are underrepresented in currently available databases. Here, we describe a 'gold standard' dataset (MPI-LIT) focusing on microbial binary protein-protein interactions and associated experimental evidence that we have manually curated from 813 abstracts and full texts that were selected from an initial set of 36 852 abstracts. The MPI-LIT dataset comprises 1237 experimental descriptions that describe a non-redundant set of 746 interactions of which 659 (88%) are not reported in public databases. To estimate the curation quality, we compared our dataset with a union of microbial interaction data from IntAct, DIP, BIND and MINT. Among common abstracts, we achieve a sensitivity of up to 66% for interactions and 75% for experimental methods. Compared with these other datasets, MPI-LIT has the lowest fraction of interaction experiments per abstract (0.9) and the highest coverage of strains (92) and scientific articles (813). We compared methods that evaluate functional interactions among proteins (such as genomic context or co-expression) which are implemented in the STRING database. Most of these methods discriminate well between functionally relevant protein interactions (MPI-LIT) and high-throughput data., Availability: http://www.jcvi.org/mpidb/interaction.php?dbsource=MPI-LIT., Supplementary Information: Supplementary data are available at Bioinformatics online.

Summary: The microbial protein interaction database (MPIDB) aims to collect and provide all known physical microbial interactions. Currently, 22,530 experimentally determined interactions among proteins of 191 bacterial species/strains can be browsed and downloaded. These microbial interactions have been manually curated from the literature or imported from other databases (IntAct, DIP, BIND, MINT) and are linked to 24,060 experimental evidences (PubMed ID, PSI-MI methods). In contrast to these databases, interactions in MPIDB are further supported by 8150 additional evidences based on interaction conservation, co-purification and 3D domain contacts (iPfam, 3did)., Availability: http://www.jcvi.org/mpidb/

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this "minimal" bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions ("interologs") for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed.

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.

TP0658 (FliW) and its orthologs, conserved proteins of unknown function in Treponema pallidum and other species, interact with a C-terminal region of flagellin (FlaB1-3 in T. pallidum; FliC in most other species). Mutants of orthologs in Bacillus subtilis and Campylobacter jejuni (yviF, CJ1075) showed strongly reduced motility. TP0658 stabilizes flagellin in a way similar to FliS, suggesting that TP0658 is a conserved assembly factor for the bacterial flagellum.

The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.