scholarly journals Genome wide functional screen for calcium transients in E. coli identifies decreased membrane potential adaptation to persistent DNA damage

2020 ◽  
Author(s):  
Rose Luder ◽  
Giancarlo N. Bruni ◽  
Joel M. Kralj
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Membrane Potential ◽  
Calcium Transients ◽  
Eukaryotic Cells ◽  
Calcium Dynamics ◽  
E Coli ◽  
Genome Wide ◽  
A Genome

1.AbstractCalcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Unlike eukaryotic cells, far less is known about calcium signaling in bacteria, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed calcium influx is induced by voltage depolarization, which were enhanced by mechanical stimulation, suggesting a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live cell screen to monitor calcium dynamics in single cells across a genome wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased calcium transients, and 32 genes knockouts that increased transients. Knockouts involved in energy production and regulation appeared, as expected, as well as knockouts of the voltage sink, the F1Fo-ATPase. Knockouts in exopolysaccharide and outer membrane synthesis showed reduced transients and refined our model of electrophysiology mediated mechanosensation in E. coli. Additionally, knockouts annotated in DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and suggested that only long term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long term DNA damage responses in bacteria, which has implications for mitochondrial DNA damage in eukaryotes.ImportanceAll eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli, we created a single cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.

scholarly journals Genome-Wide Functional Screen for Calcium Transients in Escherichia coli Identifies Increased Membrane Potential Adaptation to Persistent DNA Damage

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Rose Luder ◽  
Giancarlo N. Bruni ◽  
Joel M. Kralj
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Membrane Potential ◽  
Calcium Transients ◽  
Eukaryotic Cells ◽  
Calcium Dynamics ◽  
Content Type ◽  
Genome Wide ◽  
Gene Knockouts

ABSTRACT Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Far less is known about calcium signaling in bacteria than in eukaryotic cells, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed that calcium influx was induced by voltage depolarization, which was enhanced by mechanical stimulation, which suggested a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live-cell screen to monitor calcium dynamics in single cells across a genome-wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased levels of calcium transients and 32 gene knockouts that increased levels of transients. Knockouts of proteins involved in energy production and regulation appeared, as expected, as well as knockouts of proteins of a voltage sink, F1Fo-ATPase. Knockouts of exopolysaccharide and outer membrane synthesis proteins showed reduced transients which refined our model of electrophysiology-mediated mechanosensation. Additionally, knockouts of proteins associated with DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and the results suggested that only long-term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long-term DNA damage responses in bacteria, which also has implications for mitochondrial DNA damage in eukaryotes. IMPORTANCE All eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli, we created a single-cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage-mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long-term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.

e-Membranome: a Database for Genome-Wide Analysis of Escherichia coli Outer Membrane Proteins

2020 ◽  
Vol 21 ◽  
Author(s):  
Kang Mo Lee ◽  
Seung-Hak Cho ◽  
Cheorl-Ho Kim ◽  
Jong Hyun Kim ◽  
Sung Soon Kim
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
3D Structure ◽  
Glycan Array ◽  
Epitope Region ◽  
E Coli ◽  
Genome Wide ◽  
A Genome

Objectives: Lectin-like adhesins of enteric bacterial pathogens such as Escherichia coli are an attractive target for vaccine or drug development. Here, we have developed e-Membranome as a database of genome-wide putative adhesins in Escherichia coli (E. coli). Methods: The outer membrane adhesins were predicted from the annotated genes of Escherichia coli strains using the PSORTb program. Further analysis was performed using Interproscan and the String database. The candidate proteins can be investigated for homology modeling of the three-dimensional (3D) structure (I-TASSER version 5.1), epitope region (ABCpred), and the glycan array. Results: e-Membranome is implemented using the Django (version 2.2.5) framework. The Web Application Server Apache Tomcat 6.0 is integrated in the platform on Ubuntu Linux (version 16.04). MySQL database (version 5.7) is used as a database engine. The information of homology model of the 3D structure, epitope region, and affinity information from the glycan array will be stored in the e-Membranome database. As a case study, we performed a genome-wide screening of outer membrane-embedded proteins from the annotated genes of E. coli using the e-Membranome pipeline. Conclusion: This platform is expected to be a valuable resource for advancing research of outer membrane proteins for the construction of lectin-glycan interaction network of E. coli. In addition, the e-Membranome pipeline can be extended to other similar biological systems that need to address host-pathogen interactions.

scholarly journals Genome-wide analysis of Escherichia coli fitness determinants in a cross-feeding mutualism with Rhodopseudomonas palustris

2020 ◽  
Author(s):  
Breah LaSarre ◽  
Adam M. Deutschbauer ◽  
Crystal E. Love ◽  
James B. McKinlay
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Rhodopseudomonas Palustris ◽  
Microbial Interactions ◽  
Dependent Manner ◽  
Fermentation Products ◽  
E Coli ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome

ABSTRACTMicrobial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. Additionally, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori.IMPORTANCEMicrobial communities impact life on earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodospeudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

scholarly journals Horizontally acquired papGII-containing pathogenicity islands underlie the emergence of invasive uropathogenic Escherichia coli lineages

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael Biggel ◽  
Basil B. Xavier ◽  
James R. Johnson ◽  
Karen L. Nielsen ◽  
Niels Frimodt-Møller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Large Scale ◽  
Asymptomatic Bacteriuria ◽  
Pathogenicity Islands ◽  
Phylogenomic Analysis ◽  
E Coli ◽  
Non Invasive ◽  
Genome Wide ◽  
A Genome

AbstractEscherichia coli is the leading cause of urinary tract infection, one of the most common bacterial infections in humans. Despite this, a genomic perspective is lacking regarding the phylogenetic distribution of isolates associated with different clinical syndromes. Here, we present a large-scale phylogenomic analysis of a spatiotemporally and clinically diverse set of 907 E. coli isolates, including 722 uropathogenic E. coli (UPEC) isolates. A genome-wide association approach identifies the (P-fimbriae-encoding) papGII locus as the key feature distinguishing invasive UPEC, defined as isolates associated with severe UTI, i.e., kidney infection (pyelonephritis) or urinary-source bacteremia, from non-invasive UPEC, defined as isolates associated with asymptomatic bacteriuria or bladder infection (cystitis). Within the E. coli population, distinct invasive UPEC lineages emerged through repeated horizontal acquisition of diverse papGII-containing pathogenicity islands. Our findings elucidate the molecular determinants of severe UTI and have implications for the early detection of this pathogen.

scholarly journals Experimental Determination and System Level Analysis of Essential Genes in Escherichia coli MG1655

2003 ◽  
Vol 185 (19) ◽  
pp. 5673-5684 ◽  
Author(s):  
S. Y. Gerdes ◽  
M. D. Scholle ◽  
J. W. Campbell ◽  
G. Balázsi ◽  
E. Ravasz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
System Level ◽  
Context Analysis ◽  
E Coli ◽  
Genome Wide ◽  
Rich Media ◽  
A Genome ◽  
Evolutionary Context ◽  
Functional Context ◽  
Level Analysis

ABSTRACT Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

scholarly journals Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Michael A. Olson ◽  
Timothy W. Siebach ◽  
Joel S. Griffitts ◽  
Eric Wilson ◽  
David L. Erickson
Keyword(s):  
Escherichia Coli ◽  
Mammary Glands ◽  
Data Set ◽  
Bacterial Gene ◽  
Content Type ◽  
E Coli ◽  
Global Comparison ◽  
Genome Wide ◽  
A Genome ◽  
Highly Virulent

ABSTRACTVirulence factors of mammary pathogenicEscherichia coli(MPEC) have not been identified, and it is not known how bacterial gene content influences the severity of mastitis. Here, we report a genome-wide identification of genes that contribute to fitness of MPEC under conditions relevant to the natural history of the disease. A highly virulent clinical isolate (M12) was identified that killedGalleria mellonellaat low infectious doses and that replicated to high numbers in mouse mammary glands and spread to spleens. Genome sequencing was combined with transposon insertion site sequencing to identify MPEC genes that contribute to growth in unpasteurized whole milk, as well as duringG. mellonellaand mouse mastitis infections. These analyses show that strain M12 possesses a unique genomic island encoding a group III polysaccharide capsule that greatly enhances virulence inG. mellonella. Several genes appear critical for MPEC survival in bothG. mellonellaand in mice, including those for nutrient-scavenging systems and resistance to cellular stress. Insertions in the ferric dicitrate receptor genefecAcaused significant fitness defects under all conditions (in milk,G. mellonella, and mice). This gene was highly expressed during growth in milk. Targeted deletion offecAfrom strain M12 caused attenuation inG. mellonellalarvae and reduced growth in unpasteurized cow's milk and lactating mouse mammary glands. Our results confirm that iron scavenging by the ferric dicitrate receptor, which is strongly associated with MPEC strains, is required for MPEC growth and may influence disease severity in mastitis infections.IMPORTANCEMastitis caused byE. coliinflicts substantial burdens on the health and productivity of dairy animals. Strains causing mastitis may express genes that distinguish them from otherE. colistrains and promote infection of mammary glands, but these have not been identified. Using a highly virulent strain, we employed genome-wide mutagenesis and sequencing to discover genes that contribute to mastitis. This extensive data set represents a screen for mastitis-associatedE. colifitness factors and provides the following contributions to the field: (i) global comparison of genes required for different aspects of mastitis infection, (ii) discovery of a unique capsule that contributes to virulence, and (iii) conclusive evidence for the crucial role of iron-scavenging systems in mastitis, particularly the ferric dicitrate transport system. Similar approaches applied to other mastitis-associated strains will uncover conserved targets for prevention or treatment and provide a better understanding of their relationship to otherE. colipathogens.

scholarly journals Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Massimiliano Rossi ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
De Bruijn Graph ◽  
Anabas Testudineus ◽  
E Coli ◽  
Genome Wide ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps ◽  
Definition Of ◽  
Numeric Representation

AbstractGenome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

scholarly journals A Genome-Wide Scan for Evidence of Selection in a Maize Population Under Long-Term Artificial Selection for Ear Number

Genetics ◽  
2013 ◽  
Vol 196 (3) ◽  
pp. 829-840 ◽  
Author(s):  
Timothy M. Beissinger ◽  
Candice N. Hirsch ◽  
Brieanne Vaillancourt ◽  
Shweta Deshpande ◽  
Kerrie Barry ◽  
...  
Keyword(s):  
Artificial Selection ◽  
Maize Population ◽  
Genome Wide ◽  
A Genome ◽  
Selection For ◽  
Ear Number ◽  
Genome Wide Scan

Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1599-1610 ◽  
Author(s):  
Bradley T Smith ◽  
Graham C Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Cellular Response ◽  
Sos Response ◽  
Posttranslational Regulation ◽  
E Coli ◽  
Sos Mutagenesis ◽  
Induced Mutagenesis ◽  
Mechanistic Basis ◽  
Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

scholarly journals A Genome-Wide Assay Specifies Only GreA as a Transcription Fidelity Factor in Escherichia coli

2018 ◽  
Vol 8 (7) ◽  
pp. 2257-2264 ◽  
Author(s):  
Charles C. Traverse ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Genome Wide ◽  
A Genome
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