scholarly journals Stochastic transcriptional pulses orchestrate flagellar biosynthesis in Escherichia coli

2020 ◽  
Vol 6 (6) ◽  
pp. eaax0947 ◽  
Author(s):  
J. Mark Kim ◽  
Mayra Garcia-Alcala ◽  
Enrique Balleza ◽  
Philippe Cluzel
Keyword(s):  
Escherichia Coli ◽  
Translational Regulation ◽  
Temporal Dynamics ◽  
Transcriptional Networks ◽  
Cell Level ◽  
E Coli ◽  
Flagellar Biosynthesis ◽  
Flagellar Assembly ◽  
Regulatory Dynamics ◽  
Flagellum Biosynthesis

The classic picture of flagellum biosynthesis in Escherichia coli, inferred from population measurements, depicts a deterministic program where promoters are sequentially up-regulated and are maintained steadily active throughout exponential growth. However, complex regulatory dynamics at the single-cell level can be masked by bulk measurements. Here, we discover that in individual E. coli cells, flagellar promoters are stochastically activated in pulses. These pulses are coordinated within specific classes of promoters and comprise “on” and “off” states, each of which can span multiple generations. We demonstrate that in this pulsing program, the regulatory logic of flagellar assembly dictates which promoters skip pulses. Surprisingly, pulses do not require specific transcriptional or translational regulation of the flagellar master regulator, FlhDC, but instead appears to be essentially governed by an autonomous posttranslational circuit. Our results suggest that even topologically simple transcriptional networks can generate unexpectedly rich temporal dynamics and phenotypic heterogeneities.

scholarly journals Stochastic transcriptional pulses orchestrate flagellum biosynthesis in E. coli

2018 ◽  
Author(s):  
J. Mark Kim ◽  
Mayra Garcia-Alcala ◽  
Enrique Balleza ◽  
Philippe Cluzel
Keyword(s):  
Promoter Activity ◽  
Temporal Dynamics ◽  
Transcriptional Network ◽  
Transcriptional Networks ◽  
Transcriptional Program ◽  
E Coli ◽  
Multiple Generations ◽  
Flagellar Assembly ◽  
Flagellum Biosynthesis

AbstractThe classic picture of flagellum biosynthesis in E. coli, inferred from population measurements, describes a tightly controlled, deterministic transcriptional program. In individual E. coli cells, we discover that flagellar promoters are in fact stochastically activated in pulses. Such pulses comprise coordinated ‘on’ and ‘off’ states of promoter activity, each of which can span multiple generations. We demonstrate that this pulsing program obeys the regulatory logic of flagellar assembly, which dictates whether some promoters skip pulses. Remarkably, pulses in this transcriptional network appear to be actually governed by a post-translational circuit. Our results suggest that even topologically simple transcriptional networks can generate unexpectedly rich temporal dynamics and phenotypic heterogeneities.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Heather J. Beck ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Translational Regulation ◽  
Current Knowledge ◽  
Upstream Open Reading Frame ◽  
Model Organisms ◽  
Regulation Mechanism ◽  
Rna Regulation ◽  
Content Type ◽  
E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

scholarly journals Emergence of antibiotic resistance from multinucleated bacterial filaments

2014 ◽  
Vol 112 (1) ◽  
pp. 178-183 ◽  
Author(s):  
Julia Bos ◽  
Qiucen Zhang ◽  
Saurabh Vyawahare ◽  
Elizabeth Rogers ◽  
Susan M. Rosenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Antibiotic Resistance ◽  
Sos Response ◽  
Single Cell Level ◽  
Environmental Niche ◽  
Cell Level ◽  
E Coli ◽  
Enhanced Resistance ◽  
New Alleles

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacteriumEscherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes theE. colirod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

scholarly journals Heterogeneous Absorption of Antimicrobial Peptide LL37 inEscherichia coliCells Enhances Population Survivability

2018 ◽  
Author(s):  
Mehdi Snoussi ◽  
John Paul Talledo ◽  
Nathan-Alexander Del Rosario ◽  
Bae-Yeun Ha ◽  
Andrej Košmrlj ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mathematical Model ◽  
Minimum Inhibitory Concentration ◽  
Cell Density ◽  
Inhibitory Concentration ◽  
Single Cell Level ◽  
Cell Level ◽  
E Coli ◽  
Rapid Absorption ◽  
Growing Population

AbstractAntimicrobial peptides (AMPs) are broad spectrum antibiotics that selectively target bacteria. Here we investigate the activity of human AMP LL37 againstEscherichia coliby integrating quantitative, population and single-cell level experiments with theoretical modeling. Our data indicate an unexpected, rapid absorption and retention of a large number of LL37 byE. colicells upon the inhibition of their growth, which increases the chance of survival for the rest of population. Cultures with high-enough cell density exhibit two distinct subpopulations: a non-growing population that absorb peptides and a growing population that survive owing to the sequestration of the AMPs by others. A mathematical model based on this binary picture reproduces the rather surprising behaviors ofE. colicultures in the presence of LL37, including the increase of the minimum inhibitory concentration with cell density (even in dilute cultures) and the extensive lag in growth introduced by sub-lethal dosages of LL37.

scholarly journals Temporal Dynamics and Impact of Manure Storage on Antibiotic Resistance Patterns and Population Structure of Escherichia coli Isolates from a Commercial Swine Farm

2007 ◽  
Vol 73 (17) ◽  
pp. 5486-5493 ◽  
Author(s):  
Patrick Duriez ◽  
Edward Topp
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Population Structure ◽  
Temporal Dynamics ◽  
Swine Farm ◽  
Bacterial Populations ◽  
E Coli ◽  
Resistance Patterns ◽  
Fresh Feces ◽  
Antibiotic Resistance Patterns

ABSTRACT Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.

Mapping of sequences required for the translation of the β subunit of Escherichia coli RNA polymerase

1997 ◽  
Vol 43 (9) ◽  
pp. 819-826
Author(s):  
Luciano Passador ◽  
Thomas Linn
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Translational Regulation ◽  
Rpob Gene ◽  
Β Subunit ◽  
E Coli ◽  
Lacz Fusions ◽  
Translational Start ◽  
Genetic Constructs ◽  
Expression Plasmids

Previous experiments using expression plasmids which overproduce the β and β′ subunits of Escherichia coli RNA polymerase suggested that regions considerably upstream of the start of the rpoB gene, which encodes the β subunit, are required for its efficient synthesis. To further delineate the required regions, a collection of genetic constructs that contained varying amounts of the region either upstream or downstream of the translational start of rpoB was assembled. Measurements of β and β′ synthesis and rpoB mRNA production from a series of rpoBC expression plasmids indicated that sequences extending more than 43 bp but less than 79 bp upstream of rpoB are required for the efficient translation of rpoB mRNA. This result was confirmed by β-galactosidase measurements from a series of rpoB-lacZ fusions that have the same set of end points upstream of rpoB as the expression plasmids. A second set of gene fusions containing differing amounts of the sequence distal to the start of rpoB fused in frame to lacZ revealed that more than 29 bp but less than 70 bp of rpoB was required for efficient translation.Key words: RNA polymerase, E. coli, translational regulation.

scholarly journals Requirements for Conversion of the Na+-Driven Flagellar Motor of Vibrio cholerae to the H+-Driven Motor of Escherichia coli

2000 ◽  
Vol 182 (15) ◽  
pp. 4234-4240 ◽  
Author(s):  
Khoosheh K. Gosink ◽  
Claudia C. Häse
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Proton Translocation ◽  
Specific Inhibitor ◽  
The Other ◽  
E Coli ◽  
Alkaline Conditions ◽  
Flagellar Assembly ◽  
And Function ◽  
Hybrid Motor

ABSTRACT Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H+ or Na+ into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions inpomA, pomB, motX, ormotY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complementedfliG-null strains of the parent species but notfliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB,motX, and motY became weakly motile when theE. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins ofV. cholerae and that the hybrid motors are driven by the proton motive force.

scholarly journals A 12-Base-Pair Deletion in the Flagellar Master Control Gene flhC Causes Nonmotility of the Pathogenic German Sorbitol-Fermenting Escherichia coli O157:H− Strains

2004 ◽  
Vol 186 (8) ◽  
pp. 2319-2327 ◽  
Author(s):  
Steven R. Monday ◽  
Scott A. Minnich ◽  
Peter C. H. Feng
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Antigen Expression ◽  
Pcr Analysis ◽  
E Coli ◽  
Detection And Identification ◽  
Base Pair Deletion ◽  
Uremic Syndrome ◽  
Flagellum Biosynthesis ◽  
Master Control

ABSTRACT An atypical, Stx2-producing, pathogenic Escherichia coli O157:H− strain has been isolated with increasing frequency from hemolytic uremic syndrome patients in Germany. The lack of the H7 antigen coupled with the strain's ability to ferment sorbitol and express β-glucuronidase have complicated its detection and identification. In this study, we have determined that the loss of motility in these German sorbitol-fermenting (SF) O157 strains is due to a 12-bp in-frame deletion in flhC that is required for transcriptional activation of genes involved in flagellum biosynthesis. Either complementation with a functional flhC or repair of this mutation restored H7 antigen expression and motility. PCR analysis of several nonmotile E. coli O157 strains from various geographical sources confirmed that the 12-bp flhC deletion is found only in the cluster of German SF O157 strains, providing a potentially useful marker by which these atypical strains can be identified. The loss of motility via mutations in the flhDC operon that we observed in the German SF O157 strains is consistent with a similar phenomenon currently observed in a significant subset of other important gram-negative pathogens.

scholarly journals Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

2019 ◽  
Author(s):  
Madeleine Huber ◽  
Jörg Soppa
Keyword(s):  
Escherichia Coli ◽  
Translational Regulation ◽  
Enzyme Assay ◽  
Microtiter Plate ◽  
E Coli ◽  
Reporter Enzyme ◽  
Keio Collection ◽  
Beta Galactosidase ◽  
Transcriptional Fusions ◽  
Microtiter Plate Format

Abstract The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

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