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The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs. [ABSTRACT FROM AUTHOR]

ABSTRACTBacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often overlooked in biofilm investigations include the examination of metabolic pathways as the assumption might be that interference with central pathways such as glycolysis would only reduce growth and thus not be meaningful. Using the Keio collection of Escherichia coli mutants, we investigated the influence of biofilm formation and planktonic growth in full-strength and diluted Luria–Bertani (LB) broths using strains with a disruption of glycolysis (Δpgi), the Entner–Doudoroff pathway (Δedd), or the pentose phosphate pathway (Δgnd). Unexpectedly, in contrast to the E. coli Keio parent strain (BW25113), planktonic growth was enhanced in full strength and diluted LB broths in the metabolic mutants. Using a microtiter biofilm assay, the E. coli parent strain showed the highest crystal violet staining. However, when analyzed by culture assays, there was an increase in biofilm populations in the mutants in comparison to the parent strain. Fluorescence microscopy showed differences in colonization patterns in the strains. Given the availability of mutant collections in many model organisms, similar metabolic studies are warranted for biofilms, given their importance in nature. [ABSTRACT FROM AUTHOR]

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology.

ABSTRACT Single-gene deletions can affect the expression levels of other genes in the same operon in bacterial genomes. Here, we used proteomics for 133 Escherichia coli gene deletion mutants and transcriptome sequencing (RNA-seq) data from 71 mutants to probe the extent of transcriptional and post-transcriptional effects of gene deletions in operons. Transcriptional effects were common on genes located downstream of the deletion and were consistent across all operon members, with nearly 40% of operons showing greater than 2-fold up- or downregulation. Surprisingly, we observed an additional post-transcriptional effect that leads to the downregulation of the gene located directly downstream of the targeted gene. This effect was correlated with their intergenic distance, despite the ribosome binding site of the gene downstream remaining intact during library construction. Overall, the data presented can guide future library construction and highlight the importance of follow-up experiments for assessing direct effects of single-gene deletions in operons. IMPORTANCE Single-gene deletion libraries have allowed genome-wide characterization of gene function and interactions. While each mutant intends to disrupt the function of a single gene, it can unintentionally target other genes, such as those located in the same operon as the deletion. The extent to which such polar effects occur in deletion libraries has not been assessed. In this work, we use proteomics and transcriptomics data to show that transcript level changes lead to nearly 40% of deletions in operons affecting the protein levels of genes located downstream by at least 2-fold. Furthermore, we observed a post-transcriptional effect on the gene located directly downstream of the deletion. These results can guide the design of future gene deletion libraries and emphasizes the importance of follow-up work when linking genotypes to phenotypes.

• A highly efficient biotechnological platform for cis -1,2-dihydrocatechol production was established. • A strongly regulated toluene dioxygenase plasmid for the dearomatizing cis -1,2-dihydroxylation of benzene was developed. • An unforeseen downstream reaction consuming cis -1,2-dihydrocatechol, yielding catechol, was detected. • The native glycerol dehydrogenase in E. coli was identified as the main responsible enzyme for catechol generation. • A yield of 1.4 g L−1 cis -1,2-dihydrocatechol was achieved at semi-preparative scale. The compound cis -1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 Δ gldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41 g L−1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis. [ABSTRACT FROM AUTHOR]

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus . We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆ fimE ) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus , which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks., Competing Interests: The authors declare no conflict of interest.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal–drug compounds.

Expression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains of Escherichia coli possess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in the E. coli single-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motile E. coli strains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of the flhDC operon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring. [ABSTRACT FROM AUTHOR]

Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is one of the most emergency issues in recent years. The latest research reported that in 2019 up to 4.95 million deaths worldwide were associated with bacteria resistant to antibiotics [1]. Therefore, new therapeutic strategies are constantly being searched for. A novel alternative to traditional ways of treatment can be antimicrobial Blue Light (aBL) which leads to effective bacteria eradication of both planktonic and biofilm cultures. aBL is thought to have a multitargeted mode of action that cause rapid bacteria eradication due to the generation of reactive oxygen species which are toxic to bacteria cells [2]. However, the detailed mechanism responsible for aBL effectiveness is still not fully understood [3]. In consequence, this promising antimicrobial approach is rarely used in clinical practice. The aim of the present studies was to identify aBL- protective genes that can contribute to better understanding of mechanisms of aBL antimicrobial action. The research was conducted with Escherichia coli K-12 strain, and well-described tool for genome-wide analysis - KEIO collection which consists of 3985 single-gene mutants of E. coli K-12 strain [4]. During the studies screening of all mutants was performed with a sublethal dose of aBL. Each strain was irradiated in triplicate. Mutants for which no growth was observed in at least two of three replicates were identified as aBL-hypersensitive in comparison to the wild-type strain. About 70 single-gene mutants were recognized, suggesting their significance in cellular protection to aBL. The identified genes are involved in DNA repair, SOS response, cell envelope stress response, response to DNA damage, and cellular transport. This work was supported by the National Science Centre, grant no. 2020/36/C/NZ7/00061 (A.R.-Z.). [ABSTRACT FROM AUTHOR]

Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli.

Background Precise quantitative growth measurements and detection of small growth changes in highthroughput manner is essential for fundamental studies of bacterial cell. However, an inherent tradeoff for measurement quality in high-throughput methods sacrifices some measurement quality. A key challenge has been how to enhance measurement quality without sacrificing throughput. Results We developed a new high-throughput measurement system, termed Colony-live. Here we show that Colony-live provides accurate measurement of three growth values (lag time of growth (LTG), maximum growth rate (MGR), and saturation point growth (SPG)) by visualizing colony growth over time. By using a new normalization method for colony growth, Colony-live gives more precise and accurate growth values than the conventional method. We demonstrated the utility of Colony-live by measuring growth values for the entire Keio collection of Escherichia coli single-gene knockout mutants. By using Colonylive, we were able to identify subtle growth defects of single-gene knockout mutants that were undetectable by the conventional method quantified by fixed time-point camera imaging. Further, Colony-live can reveal genes that influence the length of the lag-phase and the saturation point of growth. Conclusions Measurement quality is critical to achieving the resolution required to identify unique phenotypes among a diverse range of phenotypes. Sharing high-quality genome-wide datasets should benefit many researchers who are interested in specific gene functions or the architecture of cellular systems. Our Colony-live system provides a new powerful tool to accelerate accumulation of knowledge of microbial growth phenotypes. [ABSTRACT FROM AUTHOR]

This article presents a study on a temperature-sensitive mutation in the ribE gene of an Escherichia coli Keio collection strain. It discusses the classification of Escherichia coli genes, generation of temperature-sensitive mutants, the growth defect of the Keio deletion strain, and growth analysis on riboflavin-complemented media.

Background Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions. This led to the choice of a gene for knockout that only connects two essential arms of an essential metabolic pathway - the glycolysis. Results Triosephosphate isomerase was chosen because its knockout will have a tremendous effect on growth on glucose as well as on glycerol. On glycerol the effect is almost absolute whereas on glucose growth is still possible, but with considerably lower rate than usual. This feature is essential because it may render cloning easier. This enzymatic activity was successfully tested as an alternative to antibiotic-based plasmid selection. Expression of a model recombinant β-glucanase in continuous cultivation was possible with stable maintenance of the plasmid. In addition, the complementation of tpiA knockout strains by the corresponding plasmids and their growth characteristics were tested on a series of complex and synthetic media. The accumulation of methylglyoxal during the growth of tpiA-deficient strains was shown to be a possible cause for the growth disadvantage of these strains in comparison to the parent strain for the Keio Collection strain or the complemented knock-out strain. Conclusion Through the use of this new auxotrophic complementation system, antibiotic-free cloning and selection of recombinant plasmid were possible. Continuous cultivation and recombinant protein expression with high segregational stability over an extended time period was also demonstrated. [ABSTRACT FROM AUTHOR]

Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Koch, Baumann, Nickling, Dziegielewski and Budisa.)

We analyzed high hydrostatic pressure (HP)-induced inactivation ofEscherichia coliin the presence of sucrose, NaCl, KCl, or LiCl under 250–400 MPa. Salt stress response (osmotic or cationic) mechanism was demonstrated to be evaluated by HP-induced inactivation kinetics. We selected nine mutant strains from the Keio collection; five strains each lacked one osmoregulatory gene (ΔmzrA, ΔenvZ, ΔompR, ΔompCor ΔompF), and four strains each lacked one cationic stress-response gene (ΔmscS, ΔmscL, ΔchaAor ΔnhaA). The nine mutants exhibited three different patterns of HP-induced inactivation. Of the nine genes, four (mzrA, mscS, nhaAandmscL) were involved in piezotolerance at NaCl concentration of 0.0–0.35 M. [ABSTRACT FROM PUBLISHER]

Abstract: Acquiring new genetic traits by lateral gene transfer is a bacterial strategy for environment adaptation. We previously showed that Escherichia coli could laterally transmit non-conjugative plasmids in co-cultures containing strains with and without the plasmid. In this study, using the Keio collection, a comprehensive library of E. coli knock-out mutants for non-essential genes, we screened for genes responsible for repressing cell-to-cell plasmid transfer in recipient cells. By stepwise screening, we identified 55 ‘transfer-up’ mutants that exhibited approximately 2- to 30-fold increased activities. We confirmed plasmid acquisition by these ‘up’ mutants and revealed that there were no significant changes in antibiotic resistance in the original Keio strains. The presumed functions of these gene products covered a wide range of activities, including metabolism and synthesis, transport, transcription or translation and others. Two competence-gene homologues (ybaV and yhiR) were identified from among these genes. The presumed localizations of these 55 gene products were estimated to be 34 cytoplasmic proteins, 20 in or around the cell surface and 1 unknown location. Our results suggest that these 55 genes may be involved in repressing plasmid uptake during cell-to-cell plasmid transfer. [Copyright &y& Elsevier]

Abstract: Acquisition of new genetic traits by horizontal gene transfer is a bacterial strategy for adaptation to the environment. We previously showed that Escherichia coli can transmit non-conjugative plasmids laterally in a co-culture containing strains with and without the plasmid. In this study, using the Keio collection, a comprehensive library of E. coli knock-out mutants for non-essential genes, we screened for genes responsible for the execution and promotion of cell-to-cell plasmid transfer in recipient cells. By stepwise screening of ‘transfer-down’ mutants, two essential genes and six promoting genes were obtained. One of the essential genes was priA, which is involved in DNA replication. This priA mutant was also unable to be transformed by artificial transformation methods, probably due to the deficiency of the plasmid maintenance function. The other essential gene was rodZ (yfgA), a gene involved in the regulation of rod-shaped structure of E. coli cells. This rodZ mutant was transformable by all three methods of artificial transformation tested, suggesting that this gene is essential for cell-to-cell plasmid transfer but not for artificial transformation. These are the first data that suggest that rodZ plays an essential role in DNA acquisition. [Copyright &y& Elsevier]

Synthetic biology depends on the ability to rapidly produce strains with improved phenotypes but is limited by the ability to rapidly produce strain collections with directed mutations. Here, we present a system capable of overcoming this limitation through automated P1-phage transductions of Escherichia coli. By combining the Keio collection of single-gene deletion E. coli mutants with P1-phage, it is possible to generate an engineered host-strain collection consisting of every possible gene deletion mutant. This strategy was tested by transducing 355 genetic markers from the Keio collection into five different host strains, and it achieved a 98% success rate. This method offers an improved mechanism for rapidly engineering collections of microbes and provides one method for rapidly deploying a broader synthetic biology effort. [Copyright &y& Elsevier]

Microorganisms have the capacity to utilize a variety of nutrients and adapt to continuously changing environmental conditions. Many microorganisms, including yeast and bacteria, accumulate carbon and energy reserves to cope with the starvation conditions temporarily present in the environment. Glycogen biosynthesis is a main strategy for such metabolic storage, and a variety of sensing and signaling mechanisms have evolved in evolutionarily distant species to ensure the production of this homopolysaccharide. At the most fundamental level, the processes of glycogen synthesis and degradation in yeast and bacteria share certain broad similarities. However, the regulation of these processes is sometimes quite distinct, indicating that they have evolved separately to respond optimally to the habitat conditions of each species. This review aims to highlight the mechanisms, both at the transcriptional and at the post-transcriptional level, that regulate glycogen metabolism in yeast and bacteria, focusing on selected areas where the greatest increase in knowledge has occurred during the last few years. In the yeast system, we focus particularly on the various signaling pathways that control the activity of the enzymes of glycogen storage. We also discuss our recent understanding of the important role played by the vacuole in glycogen metabolism. In the case of bacterial glycogen, special emphasis is placed on aspects related to the genetic regulation of glycogen metabolism and its connection with other biological processes. [ABSTRACT FROM AUTHOR]

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (Δ cpxA, Δ gcvR, Δ glnL, and an unknown-gene-deleted one (Δ uk)) were able to increase the CYP154A1 activity by approximately 1.4–1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (Δ cpxA, Δ gcvR, and Δ glnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Δ uk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of Δ cpxA, Δ gcvR, and Δ glnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst. [ABSTRACT FROM AUTHOR]

Abstract: Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with k cat and K m values of 260±45s−1 and 229±26μM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a ΔbioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15mM simvastatin acid in less than 12h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive. [Copyright &y& Elsevier]

Abstract: A systematic and comprehensive gene-disrupted mutant collection of E. coli K-12 was used to identify genes whose deletions affect glycogen accumulation. Of the 3985 non-essential gene mutants of the collection, 35 displayed a glycogen-excess phenotype, whereas 30 displayed either glycogen-less or glycogen-deficient phenotypes. The genes whose deletions affect glycogen accumulation were classified into various functional categories, including energy production, envelope composition and integrity, protein translation and stability, transport of inorganic ions and nucleotides, and metabolism of carbohydrates and amino acids. The overall data indicate that glycogen metabolism is highly interconnected with a wide variety of cellular processes in E. coli. [Copyright &y& Elsevier]

Use of bacteriophages as a therapy for bacterial infection has been attempted over the last century. Such an endeavor requires the elucidation of basic aspects of the host-virus interactions and the resistance mechanisms of the host. Two recently developed bacterial collections now enable a genomewide search of the genetic interactions between Escherichia coil and bacteriophages. We have screened >85% of the E. co/i genes for their ability to inhibit growth of 17 phage and >90% of the host genes for their ability to be used by the virus. In addition to identifying all of the known interactions, several other interactions have been identified. E. coli CMP kinase is essential for T7 growth, whereas overexpression of the E. coil uridine/cytidine kinase inhibits T7 growth. Mutations in any one of nine genes that encode enzymes for the synthesis of the E. coil lipopolysaccharide receptor for T7 adsorption leads to T7 resistance. Selection of T7 phage that can recognize these altered receptors has enabled the construction of phage to which the host is 100-fold less resistant. [ABSTRACT FROM AUTHOR]

Single-gene deletions can affect the expression levels of other genes in the same operon in bacterial genomes. Here, we used proteomics for 133 Escherichia coli gene deletion mutants and transcriptome sequencing (RNA-seq) data from 71 mutants to probe the extent of transcriptional and post-transcriptional effects of gene deletions in operons. Transcriptional effects were common on genes located downstream of the deletion and were consistent across all operon members, with nearly 40% of operons showing greater than 2-fold up- or downregulation. Surprisingly, we observed an additional post-transcriptional effect that leads to the downregulation of the gene located directly downstream of the targeted gene. This effect was correlated with their intergenic distance, despite the ribosome binding site of the gene downstream remaining intact during library construction. Overall, the data presented can guide future library construction and highlight the importance of follow-up experiments for assessing direct effects of single-gene deletions in operons. IMPORTANCE Single-gene deletion libraries have allowed genome-wide characterization of gene function and interactions. While each mutant intends to disrupt the function of a single gene, it can unintentionally target other genes, such as those located in the same operon as the deletion. The extent to which such polar effects occur in deletion libraries has not been assessed. In this work, we use proteomics and transcriptomics data to show that transcript level changes lead to nearly 40% of deletions in operons affecting the protein levels of genes located downstream by at least 2-fold. Furthermore, we observed a post-transcriptional effect on the gene located directly downstream of the deletion. These results can guide the design of future gene deletion libraries and emphasizes the importance of follow-up work when linking genotypes to phenotypes.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal-drug compounds.

cis -Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of cis -dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant E. coli , harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the E. coli catalyzed degradation of cis -dihydrodiendiols into catechols via dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant E. coli platforms pursuing the production of cis -dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in E. coli BW25113, catalyzing the degradation of cis -dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a cis -dihydrodiendiol of choice.•Novel screening strategy for E. coli BW25113 dehydrogenase knock-outs, incapable of degrading cis -dihydrodiendiols was validated for cis -1,2-dihydrocatechol as substrate•Corresponding knock-outs can be used for recombinant production of cis -dihydrodiendiols•Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD)., Competing Interests: The Authors confirm that there are no conflicts of interest., (© 2020 The Author(s). Published by Elsevier B.V.)

Background: Alteration of commensal bacterial composition is associated with many inflammatory diseases. However, few studies have pinpointed the specific bacterial genes that may suppress host immune responses against microbes and maintain homeostasis in the host intestine., Methods: High-throughput screening was performed in Caenorhabditis elegans with a single gene knockout ut screening was performed in Caenorhabditis elegans with a single gene knockout Escherichia coli (E. coli) library and identified the immune suppression gene blc. The coding sequences of blc among different kinds of E. coli strains were aligned to identify the single nucleotide polymorphisms (SNPs). Physiological and biochemical experiments were performed in C. elegans and mice to explore the function of the blc variant., Findings: By screening 3983 E. coli mutants, we discovered that 9 bacterial genes, when deleted, activate innate immunity in the host C. elegans. Among these 9 genes, the gene encoding blc showed a distinctive SNP in many clinically pathogenic bacteria. We found that bacteria with this SNP, which converts Blc G84 to Blc E84 , are highly enriched in the faeces of patients with inflammatory bowel disease (IBD). Exposure to Blc E84 -encoding bacteria resulted in epithelial barrier disruption and immune activation in both worms and mice. Detailed analysis indicated that infection with Blc E84 -encoding bacteria causes a significant decrease in LPE levels in the intestine and subsequently disrupts gut epithelial integrity in mice. Consistently, the levels of LPE in patients with IBD are significantly lower than those in healthy people. Finally, supplementation with LPE, which activates LPA 1 /PLCβ/PKC signaling, reversed the defects induced by Blc E84 -encoding bacteria., Interpretation: Our results identified a novel bacterial gene, blc, in E. coli that regulates host gut integrity and immunity. FUND: The Ministry of Science and Technology of China; the National Natural Science Foundation of China; and the Natural Science Foundation of Jiangsu Province., Competing Interests: Declaration of competing interest We declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article, except a patent that we have (Number: 201911273194.1)., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

[NiFe]-hydrogenases have attracted attention as potential therapeutic targets or components of a hydrogen-based economy. [NiFe]-hydrogenase production is a complicated process that requires many associated accessory proteins that supply the requisite cofactors and substrates. Current methods for measuring hydrogenase activity have low throughput and often require specialized conditions and reagents. In this work, we developed a whole-cell high-throughput hydrogenase assay based on the colorimetric reduction of benzyl viologen to explore the biological networks of these enzymes in Escherichia coli We utilized this assay to screen the Keio collection, a set of nonlethal single-gene knockouts in E. coli BW25113. The results of this screen highlighted the assay's specificity and revealed known components of the intricate network of systems that underwrite [NiFe]-hydrogenase activity, including nickel homeostasis and formate dehydrogenase activities as well as molybdopterin and selenocysteine biosynthetic pathways. The screen also helped identify several new genetic components that modulate hydrogenase activity. We examined one E. coli strain with undetectable hydrogenase activity in more detail (Δ eutK ), finding that nickel delivery to the enzyme active site was completely abrogated, and tracked this effect to an ancillary and unannotated lack of the fumarate and nitrate reduction (FNR) anaerobic regulatory protein. Collectively, these results demonstrate that the whole-cell assay developed here can be used to uncover new information about bacterial [NiFe]-hydrogenase production and to probe the cellular components of microbial nickel homeostasis., (© 2019 Lacasse et al.)

It is essential to understand the mechanisms by which a toxicant is capable of poisoning the bacterial cell. The mechanism of action of many biocides and toxins, including numerous ubiquitous compounds, is not fully understood. For example, despite the widespread clinical and commercial use of silver (Ag), the mechanisms describing how this metal poisons bacterial cells remains incomplete. To advance our understanding surrounding the antimicrobial action of Ag, we performed a chemical genetic screen of a mutant library of Escherichia coli—the Keio collection, in order to identify Ag sensitive or resistant deletion strains. Indeed, our findings corroborate many previously established mechanisms that describe the antibacterial effects of Ag, such as the disruption of iron-sulfur clusters containing proteins and certain cellular redox enzymes. However, the data presented here demonstrates that the activity of Ag within the bacterial cell is more extensive, encompassing genes involved in cell wall maintenance, quinone metabolism and sulfur assimilation. Altogether, this study provides further insight into the antimicrobial mechanism of Ag and the physiological adaption of E. coli to this metal. [ABSTRACT FROM AUTHOR]

Defensin attack! Here we report the screening of human defensin 5 against the Keio Collection of E. coli strains. The results of this screen further our understanding of how this important hostdefense peptide kills bacteria and how bacteria protect themselves against the attack from the human host., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)