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The dissimilatory sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

Author(s): Trotter, Valentine V; Shatsky, Maxim; Price, Morgan N; Juba, Thomas R; Zane, Grant M; De Leon, Kara B; Majumder, Erica L; Gui, Qin; Ali, Rida; Wetmore, Kelly M; Kuehl, Jennifer V; Arkin, Adam P; Wall, Judy D; Deutschbauer, Adam M; Chandonia, John-Marc; Butland, Gareth P | Abstract: ABSTRACTSulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources, and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

Sulfate-reducing microbes, such as Desulfovibrio vulgaris Hildenborough, cause “souring” of petroleum reservoirs through produced sulfide and precipitate heavy metals, either as sulfides or by alteration of the metal reduction state. Thus, inhibitors of these microbes, including nitrate and nitrite ions, are studied in order to limit their impact. Nitrite is a potent inhibitor of sulfate reducers, and it has been suggested that nitrate does not inhibit these microbes directly but by reduction to nitrite, which serves as the ultimate inhibitor. Here we provide evidence that nitrate inhibition of D. vulgaris can be independent of nitrite production. We also show that D. vulgaris can use nitrite as a nitrogen source or terminal electron acceptor for growth. Moreover, we report that use of nitrite as a terminal electron acceptor requires nitrite reductase (nrfA) as a D. vulgaris nrfA mutant cannot respire nitrite but remains capable of utilizing nitrite as a nitrogen source. These results illuminate previously uncharacterized metabolic abilities of D. vulgaris that may allow niche expansion in low-sulfate environments. Understanding these abilities may lead to better control of sulfate-reducing bacteria in industrial settings and more accurate prediction of their interactions in the environment.

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in product loss, a health hazard to workers, and ultimately abandonment of wells. Identification of the required genes is a critical step for determining the mechanism of biofilm formation by sulfate reducers. Here, the transporter by which putative biofilm structural proteins are exported from sulfate-reducing Desulfovibrio vulgaris Hildenborough cells was discovered, and a single nucleotide change within the gene coding for this transporter was found to be sufficient to completely stop formation of biofilm.

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-L-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Numerous affinity purification-mass spectrometry (APMS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homologue of E. coli BolA) in yeast and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G., et al. (2008) Nature Methods 5, 789-795]. These data connected the only E. coli mono-Grx, GrxD to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes to ultimately uncover if either of these complexes performs functions linked to FeS cluster biosynthesis.

The yjeE , yeaZ , and ygjD genes are highly conserved in the genomes of eubacteria, and ygjD orthologs are also found throughout the Archaea and eukaryotes. In this study, we have constructed conditional expression strains for each of these genes in the model organism Escherichia coli K12. We show that each gene is essential for the viability of E. coli under laboratory growth conditions. Growth of the conditional strains under nonpermissive conditions results in dramatic changes in cell ultrastructure. Deliberate repression of the expression of yeaZ results in cells with highly condensed nucleoids, while repression of yjeE and ygjD expression results in at least a proportion of very enlarged cells with an unusual peripheral distribution of DNA. Each of the three conditional expression strains can be complemented by multicopy clones harboring the rstA gene, which encodes a two-component-system response regulator, strongly suggesting that these proteins are involved in the same essential cellular pathway. The results of bacterial two-hybrid experiments show that YeaZ can interact with both YjeE and YgjD but that YgjD is the preferred interaction partner. The results of in vitro experiments indicate that YeaZ mediates the proteolysis of YgjD, suggesting that YeaZ and YjeE act as regulators to control the activity of this protein. Our results are consistent with these proteins forming a link between DNA metabolism and cell division.

High throughput methods are increasingly being used to examine the functions and interactions of gene products on a genome-scale. These include systematic large-scale proteomic studies of protein complexes and protein–protein interaction networks, functional genomic studies examining patterns of gene expression and comparative genomics studies examining patterns of conservation. Since these datasets offer different yet highly complementary perspectives on cell behavior it is expected that integration of these datasets will lead to conceptual advances in our understanding of the fundamental design and evolutionary principles that underlie the organization and function of proteins within biochemical pathways. Here we present Bacteriome.org, a resource that combines locally generated interaction and evolutionary datasets with a previously generated knowledgebase, to provide an integrated view of the Escherichia coli interactome. Tools are provided which allow the user to select and visualize functional, evolutionary and structural relationships between groups of interacting proteins and to focus on genes of interest. Currently the database contains three interaction datasets: a functional dataset consisting of 3989 interactions between 1927 proteins; a ‘core’ high quality experimental dataset of 4863 interactions between 1100 proteins and an ‘extended’ experimental dataset of 9860 interactions between 2131 proteins. Bacteriome.org is available online at http://www.bacteriome.org.

Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling., IMPORTANCE A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

A specific amperometric assay was developed for the membrane-bound NOR [NO (nitric oxide) reductase] from the model denitrifying bacterium Paracoccus denitrificans using its natural electron donor, pseudoazurin, as a co-substrate. The method allows the rapid and specific assay of NO reduction catalysed by recombinant NOR expressed in the cytoplasmic membranes of Escherichia coli. The effect on enzyme activity of substituting alanine, aspartate or glutamine for two highly conserved glutamate residues, which lie in a periplasmic facing loop between transmembrane helices III and IV in the catalytic subunit of NOR, was determined using this method. Three of the substitutions (E122A, E125A and E125D) lead to an almost complete loss of NOR activity. Some activity is retained when either Glu122 or Glu125 is substituted with a glutamine residue, but only replacement of Glu122 with an aspartate residue retains a high level of activity. These results are interpreted in terms of these residues forming the mouth of a channel that conducts substrate protons to the active site of NOR during turnover. This channel is also likely to be that responsible in the coupling of proton movement to electron transfer during the oxidation of fully reduced NOR with oxygen [U. Flock, N. J. Watmough and P. Ädelroth (2005) Biochemistry 44, 10711–10719].

Assembly of the active site of the [NiFe]-hydrogenase enzymes involves a multi-step pathway and the coordinated activity of many accessory proteins. To analyze complex formation between these factors in Escherichia coli, they were genomically tagged and native multi-protein complexes were isolated. This method validated multiple interactions reported in separate studies from several organisms and defined a new complex containing the putative chaperone HybG and the large subunit of hydrogenase 1 or 2. The complex also includes HypE and HypD, which interact with each other before joining the larger complex.

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approximately 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in DeltaslyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.

Iron sulfur (FeS) cluster containing proteins are involved in processes targeted by environmentally relevant stressors. These stress conditions, for sulfate reducing bacteria (SRBs) such as Desulfo...

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi-protein systems such as Isc and Suf in E. coli. Additional accessory factors including the monothiol glutaredoxin, GrxD, are located outside of these defined systems. GrxD has been proposed to function as a shuttle protein acting to transfer FeS clusters between scaffold and carrier proteins, although specific functional role remain unknown. We have previously linked GrxD in FeS client protein interactions with the radical SAM enzyme MiaB. To address these different hypotheses, we conducted an extensive survey for physical interaction of GrxD with a comprehensive set of FeS cluster biosynthesis factors including cysteine desulfurase components (IscS, SufSE), FeS scaffold (IscU, SufBC2D) , carrier proteins (IscA SufA, ErpA, NfuA), HscA, HscB and various FeS apoproteins. We have employed various methods, including affinity co-purification, analytical gel filtration and surface plasmon resonance (SPR) to obtain in...

The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe B –heme-Fe dinuclear catalytic center rather than a Cu B –heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in Paracoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex in Escherichia coli . Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli . Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.

A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.

Molecular interactions define the functional organization of the cell. Epistatic (genetic, or gene-gene) interactions, one of the most informative and commonly encountered forms of functional relationships, are increasingly being used to map process architecture in model eukaryotic organisms. In particular, 'systems-level' screens in yeast and worm aimed at elucidating genetic interaction networks have led to the generation of models describing the global modular organization of gene products and protein complexes within a cell. However, comparable data for prokaryotic organisms have not been available. Given its ease of growth and genetic manipulation, the Gram-negative bacterium Escherichia coli appears to be an ideal model system for performing comprehensive genome-scale examinations of genetic redundancy in bacteria. In this review, we highlight emerging experimental and computational techniques that have been developed recently to examine functional relationships and redundancy in E. coli at a systems-level, and their potential application to prokaryotes in general. Additionally, we have scanned PubMed abstracts and full-text published articles to manually curate a list of approximately 200 previously reported synthetic sick or lethal genetic interactions in E. coli derived from small-scale experimental studies.

Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins., Author Summary One goal of modern biology is to chart groups of proteins that act together to perform biological processes via direct and indirect interactions. Such groupings are sometimes called functional modules. The types of protein interactions within modules include physical interactions that generate protein complexes and biochemical associations that make up metabolic pathways. We have combined proteomic and bioinformatic tools, and used them to decipher a large number of protein interactions, complexes, and functional modules with high confidence. In addition, exploring the topology of the resulting interaction networks, we successfully predicted specific biological roles for a number of proteins with previously unknown functions, and identified some potential drug targets. Although our work is focused on E. coli, our phylogenetic projections suggest that a considerable fraction of our observations and predictions can be extrapolated to many other bacterial taxa. As all the data derived from this study are publicly available, others may build on our work for further hypothesis-driven studies of gene function discovery., A novel resource integrating proteomic and genome context-based tools provides a "systems-wide" functional blueprint ofE. coli, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO(2) and H(2). The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

International audience; AAA+ ATPases are ubiquitous proteins that employ the energy obtained from ATP hydrolysis to remodel proteins, DNA, or RNA. The MoxR family of AAA+ proteins is widespread throughout bacteria and archaea but is largely uncharacterized. Limited work with specific members has suggested a potential role as molecular chaperones involved in the assembly of protein complexes. As part of an effort aimed at determining the function of novel AAA+ chaperones in Escherichia coli, we report the characterization of a representative member of the MoxR family, YieN, which we have renamed RavA (regulatory ATPase variant A). We show that the ravA gene exists on an operon with another gene encoding a protein, YieM, of unknown function containing a Von Willebrand Factor Type A domain. RavA expression is under the control of the sigma(S) transcription factor, and its levels increase toward late log/early stationary phase, consistent with its possible role as a general stress-response protein. RavA functions as an ATPase and forms hexameric oligomers. Importantly, we demonstrate that RavA interacts strongly with inducible lysine decarboxylase (LdcI or CadA) forming a large cage-like structure consisting of two LdcI decamers linked by a maximum of five RavA oligomers. Surprisingly, the activity of LdcI does not appear to be affected by binding to RavA in a number of in vitro and in vivo assays, however, complex formation results in the stimulation of RavA ATPase activity. Data obtained suggest that the RavA-LdcI interaction may be important for the regulation of RavA activity against its targets.

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.