The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

The molecular architecture and function of the Gram-negative bacterial cell envelope are dictated by protein composition and localization. Proteins that localize to the inner membranes (IM) and outer membranes (OM) of Gram-negative bacteria play critical and distinct roles in cellular physiology; however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. Here, we employed multiplexed quantitative mass spectrometry using tandem mass tag (TMT) labeling to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing Escherichia coli K-12 cells on a sucrose density gradient. The migration patterns for >1,600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli For 559 proteins that are currently annotated as peripherally associated with the IM (G. Orfanoudaki and A. Economou, Mol Cell Proteomics 13:3674-3687, 2014, https://doi.org/10.1074/mcp.O114.041137) and that display potential for dual localization to either the IM or cytoplasm, we could allocate 110 proteins to the IM and 206 proteins to the soluble cell fraction based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of these cases, we were able to find supportive evidence for our localization findings in the literature. We anticipate that our systems-level analysis of the E. coli membrane proteome will serve as a useful reference data set to query membrane protein localization, as well as to provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species.IMPORTANCE Current knowledge of protein localization, particularly outer membrane proteins, is highly dependent on bioinformatic predictions. To date, no systematic experimental studies have directly compared protein localization spanning the inner and outer membranes of E. coli By combining sucrose density gradient fractionation of inner membrane (IM) and outer membrane (OM) proteins with multiplex quantitative proteomics, we systematically quantified localization patterns for >1,600 proteins, providing high-confidence localization annotations for 1,368 proteins. Of these proteins, we resolve the predominant localization of 316 proteins that currently have dual annotation (cytoplasmic and IM) in protein databases and identify new annotations for 42 additional proteins. Overall, we present a novel quantitative methodology to systematically map membrane proteins in Gram-negative bacteria and use it to unravel the biological complexity of the membrane proteome architecture in E. coli.

Escherichia colimazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme.

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1'β2, mβ1-β5, mβ1-β6 and mβ4-β5) by transplanting β1, β1'β2, β1-β5, β1-β6 and β4-β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1'β2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the β2 strand of mβ1'β2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1-β6, MtuSSB, mβ1'β2 and mβ1-β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1'β2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Antimicrobial resistance threatens our current standards of care for the treatment and prevention of infectious disease. Antibiotics that have multiple targets have a lower propensity for the development of antibiotic resistance than those that have single targets and therefore represent an important tool in the fight against antimicrobial resistance. In this work, groups of essential paralogous proteins were identified in the important Gram-negative pathogen Escherichia coli that could represent novel targets for multitargeting antibiotics. These groups include targets from a broad range of essential macromolecular and biosynthetic pathways, including cell wall synthesis, membrane biogenesis, transcription, translation, DNA replication, fatty acid biosynthesis, and riboflavin and isoprenoid biosynthesis. Importantly, three groups of clinically validated antibiotic multitargets were identified using this method: the two subunits of the essential topoisomerases, DNA gyrase and topoisomerase IV, and one pair of penicillin-binding proteins. An additional eighteen protein groups represent potentially novel multitargets that could be explored in drug discovery efforts aimed at developing compounds having multiple targets in E. coli and other bacterial pathogens. IMPORTANCE Many types of bacteria have gained resistance to existing antibiotics used in medicine today. Therefore, new antibiotics with novel mechanisms must continue to be developed. One tool to prevent the development of antibiotic resistance is for a single drug to target multiple processes in a bacterium so that more than one change must arise for resistance to develop. The work described here provides a comprehensive search for proteins in the bacterium Escherichia coli that could be targets for such multitargeting antibiotics. Several groups of proteins that are already targets of clinically used antibiotics were identified, indicating that this approach can uncover clinically relevant antibiotic targets. In addition, eighteen currently unexploited groups of proteins were identified, representing new multitargets that could be explored in antibiotic research and development.

Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product.

The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli We applied DRaCALA (differential radial capillary action of ligand assay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets in E. coli that, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp in E. coli are identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved between E. coli and Firmicutes In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets in E. coliIMPORTANCE Antibiotic resistance and tolerance exhibited by pathogenic bacteria have resulted in a global public health crisis. Remarkably, almost all bacterial pathogens require the alarmone (p)ppGpp to be virulent. Thus, (p)ppGpp not only induces tolerance of nutritional limitations and chemical insults, including antibiotics, but is also often required for induction of virulence genes. However, understanding of the molecular targets of (p)ppGpp and the mechanisms by which (p)ppGpp influences bacterial physiology is incomplete. In this study, a systematic approach was used to uncover novel targets of (p)ppGpp in E. coli, the best-studied model bacterium. Comprehensive comparative studies of the targets revealed conserved target pathways of (p)ppGpp in both Gram-positive and -negative bacteria and novel targets of (p)ppGpp, including an alternative degradative pathway of (p)ppGpp. Thus, our discoveries may help in understanding of how (p)ppGpp increases the stress resilience and multidrug tolerance not only of the model organism E. coli but also of the pathogenic organisms in which these targets are conserved.

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.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Anopheles gambiae is a major vector of human malaria and its immune system in part determines the fate of ingested parasites. Proteins, hemocytes and fat body in hemolymph are critical components of this system, mediating both humoral and cellular defenses. Here we assessed differences in the hemolymph proteomes of water- and E. coli-pricked mosquito larvae by a gel-LC-MS approach. Among the 1756 proteins identified, 603 contained a signal peptide but accounted for two-third of the total protein amount on the quantitative basis. The sequence homology search indicated that 233 of the 1756 may be related to defense. In general, we did not detect substantial differences between the control and induced plasma samples in terms of protein numbers or levels. Protein distributions in the gel slices suggested post-translational modifications (e.g. proteolysis) and formation of serpin-protease complexes and high Mr immune complexes. Based on the twenty-five most abundant proteins, we further suggest that major functions of the larval hemolymph are storage, transport, and immunity. In summary, this study provided first data on constitution, levels, and possible functions of hemolymph proteins in the mosquito larvae, reflecting complex changes occurring in the fight against E. coli infection.

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.

Protein mobility affects most cellular processes, such as the rates of enzymatic reactions, signal transduction, and assembly of macromolecular complexes. Despite such importance, little systematic information is available about protein diffusion inside bacterial cells. Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the bacterial cytoplasm, the plasma membrane, and in the nucleoid. Estimated diffusion coefficients of cytoplasmic and membrane proteins show steep dependence on the size and on the number of transmembrane helices, respectively. Protein diffusion in both compartments is thus apparently obstructed by a network of obstacles, creating the so-called molecular sieving effect. These obstructing networks themselves, however, appear to be dynamic and allow a slow and nearly size-independent movement of large proteins and complexes. The obtained dependencies of protein mobility on the molecular mass and the number of transmembrane helices can be used as a reference to predict diffusion rates of proteins in Escherichia coli. Mobility of DNA-binding proteins apparently mainly depends on their binding specificity, with FRAP recovery kinetics being slower for the highly specific TetR repressor than for the relatively nonspecific H-NS regulator.

RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Mechanical properties such as physical constraint and pushing of chromosomes are thought to be important for chromosome segregation in Escherichia coli and it could be mediated by a hypothetical molecular "tether." However, the actual tether that mediates these features is not known. We previously described that SecA (Secretory A) and Secretory Y (SecY), components of the membrane protein translocation machinery, and AcpP (Acyl carrier protein P) were involved in chromosome segregation and homeostasis of DNA topology. In the present work, we performed three-dimensional deconvolution of microscopic images and time-lapse experiments of these proteins together with MukB and DNA topoisomerases, and found that these proteins embraced the structures of tortuous nucleoids with condensed regions. Notably, SecA, SecY, and AcpP dynamically localized in cells, which was interdependent on each other requiring the ATPase activity of SecA. Our findings imply that the membrane protein translocation machinery plays a role in the maintenance of proper chromosome partitioning, possibly through "tethering" of MukB [a functional homolog of structural maintenance of chromosomes (SMC) proteins], DNA gyrase, DNA topoisomerase IV, and SeqA (Sequestration A).

Proline residues are unique in the extent to which they constrain the conformational space available to the protein backbone. Because the conformational preferences of proline cannot be recapitulated by any of the other proteinogenic amino acids, standard mutagenesis approaches that seek to introduce new chemical functionality at proline positions unavoidably perturb backbone flexibility. Here, we detail the incorporation of proline analogs into recombinant proteins in Escherichia coli via a residue-specific mutagenesis strategy. This approach results in global replacement of proline residues with high yields of the recombinant protein of interest, minimal genetic manipulation, and maintenance of backbone conformational constraints.

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P(1)' position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P(1)' position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.