Genotypic and phenotypic diversity differences of presumptive commensal and avian pathogenic E. coli

ABSTRACT Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection. Prolonged stationary phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starving Escherichia coli populations in lysogeny broth. A wide range of mutations accumulates over the course of 1 month in stationary phase. Single nucleotide polymorphisms (SNPs) constitute 64% of all mutations. A majority of these mutations are nonsynonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency of mutations observed in our experimental populations cannot be explained by neutral drift. Moreover, there is frequent genetic parallelism across populations, suggesting that these mutations are under positive selection. Finally, functional analysis of mutations suggests that regulatory mutations are frequent targets of selection. IMPORTANCE Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection.

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Acetate and glycerol are not uniquely suited for the evolution of cross-feeding in E. coli

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008433 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1008433

Author(s):

Magdalena San Roman ◽

Andreas Wagner

Keyword(s):

Carbon Source ◽

Experimental Evolution ◽

Phenotypic Diversity ◽

Carbon Sources ◽

Metabolic Modeling ◽

Metabolic Changes ◽

E Coli ◽

Biochemical Pathways ◽

Genome Scale ◽

Feeding Interactions

The evolution of cross-feeding among individuals of the same species can help generate genetic and phenotypic diversity even in completely homogeneous environments. Cross-feeding Escherichia coli strains, where one strain feeds on a carbon source excreted by another strain, rapidly emerge during experimental evolution in a chemically minimal environment containing glucose as the sole carbon source. Genome-scale metabolic modeling predicts that cross-feeding of 58 carbon sources can emerge in the same environment, but only cross-feeding of acetate and glycerol has been experimentally observed. Here we use metabolic modeling to ask whether acetate and glycerol cross-feeding are especially likely to evolve, perhaps because they require less metabolic change, and thus perhaps also less genetic change than other cross-feeding interactions. However, this is not the case. The minimally required metabolic changes required for acetate and glycerol cross feeding affect dozens of chemical reactions, multiple biochemical pathways, as well as multiple operons or regulons. The complexity of these changes is consistent with experimental observations, where cross-feeding strains harbor multiple mutations. The required metabolic changes are also no less complex than those observed for multiple other of the 56 cross feeding interactions we study. We discuss possible reasons why only two cross-feeding interactions have been discovered during experimental evolution and argue that multiple new cross-feeding interactions may await discovery.

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Effect of inoculum size and antibiotics on bacterial traveling bands in a thin microchannel defined by optical adhesive

Microsystems & Nanoengineering ◽

10.1038/s41378-021-00309-3 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Yang Liu ◽

Thomas Lehnert ◽

Martin A. M. Gijs

Keyword(s):

Phenotypic Diversity ◽

Swimming Performance ◽

Effective Diffusion ◽

Bacterial Chemotaxis ◽

Microfluidic Channel ◽

Adhesive Layer ◽

Inoculum Size ◽

E Coli ◽

Optical Adhesive ◽

The Impact

AbstractPhenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns, appearing as traveling bands with transient locally enhanced cell densities. Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments. In this work, we studied different parameters of Escherichia coli (E. coli) collective migration, in particular the quantity of bacteria introduced initially in a microfluidic chip (inoculum size) and their exposure to antibiotics (ampicillin, ciprofloxacin, and gentamicin). We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel, enabling high-content imaging of the migration dynamics in a single bacterial layer, i.e., bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective. On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms. Quantifications of swimming speed, tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity, resulting in variations in transient cell density distributions and swimming performance. We found that incubation of isogeneic E. coli with different inoculum sizes eventually generated different swimming phenotype distributions. Interestingly, incubation with antimicrobials promoted bacterial chemotaxis in specific cases, despite growth inhibition. Moreover, E. coli filamentation in the presence of antibiotics was assessed, and the impact on motility was evaluated. We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.

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Mass allelic exchange: enabling sexual genetics in Escherichia coli

10.1101/2020.11.30.405282 ◽

2020 ◽

Author(s):

Varnica Khetrapal ◽

Liyana Ow Yong ◽

Swaine L. Chen

Keyword(s):

Linkage Analysis ◽

Allelic Variation ◽

Phenotypic Diversity ◽

F1 Hybrids ◽

Natural Phenomenon ◽

Loss Of Function ◽

E Coli ◽

Allelic Exchange ◽

Strong Negative Selection ◽

Single Operon

AbstractDespite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent Mass Allelic Exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of E. coli. MAE reimplements the natural phenomenon of random crossovers, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.One Sentence SummaryWe create F1 hybrids of E. coli using MAE, bringing the power of linkage analysis to bear on phenotypic diversity (including virulence)

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Diversity of Hemagglutination Phenotypes among P-Fimbriated Wild-Type Strains of Escherichia coli in Relation to papG Allele Repertoire

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.2.160-170.1998 ◽

1998 ◽

Vol 5 (2) ◽

pp. 160-170 ◽

Cited By ~ 9

Author(s):

James R. Johnson ◽

Jennifer J. Brown ◽

Parvia Ahmed

Keyword(s):

Escherichia Coli ◽

Phenotypic Diversity ◽

Class Ii ◽

Class I ◽

Class Iii ◽

Wild Type ◽

Sheep Erythrocytes ◽

E Coli ◽

Naturally Occurring ◽

Type Strains

ABSTRACT Data regarding the hemagglutination (HA) patterns of the three variants (classes I, II, and III) of the Escherichia coliadhesin PapG are conflicting. These HA patterns usually have been assessed for each papG allele separately with recombinant strains in slide HA assays. We rigorously evaluated an alternative microtiter tray HA assay and then used it to assess the HA of four erythrocyte types (human A1P1 and OP1, rabbit, and sheep erythrocytes) by multiple wild-typeE. coli strains representing the four naturally occurring combinations of the papG alleles, i.e., class I plus III, class III only, class II plus III, and class II only. The microtiter tray HA assay displayed significantly better reproducibility of intraobserver (83%) and interobserver (86%) results than did slide HA assays (39 and 73%, respectively). Novel findings from the study of 32 wild-type P-fimbriated strains included reproducible determinations of phenotypic diversity among different papG categories, among strains within each papG category, and from day to day for individual strains. There was also substantial overlap of phenotypes between papG categories I plus III and III only and between II plus III and II only. A class III papG recombinant strain’s HA pattern differed significantly from that of the wild-type class III strains. These data demonstrate that HA phenotypes of wild-type P-fimbriated E. coli strains can be reproducibly assessed by a microtiter HA assay and that they correspond broadly topapG genotype but in a more complex and varied fashion than previously recognized.

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Optimizing complex phenotypes through model-guided multiplex genome engineering

10.1101/086595 ◽

2016 ◽

Author(s):

Gleb Kuznetsov ◽

Daniel B. Goodman ◽

Gabriel T. Filsinger ◽

Matthieu Landon ◽

Nadin Rohland ◽

...

Keyword(s):

Genome Engineering ◽

De Novo ◽

Phenotypic Diversity ◽

Genotypic Diversity ◽

Unintended Effects ◽

Laboratory Evolution ◽

E Coli ◽

Recombination Efficiency ◽

Complex Phenotypes ◽

Regression Techniques

AbstractOptimization of complex phenotypes in engineered microbial strains has traditionally been accomplished by laboratory evolution. However, only a subset of the resulting mutations may affect the phenotype of interest and many others may have unintended effects. Multiplexed genome editing can complement evolutionary approaches by creating diverse combinations of targeted changes, but in both cases it remains challenging to identify which alleles influence the desired phenotype. We present a method for identifying a minimal set of genomic modifications that optimizes a complex phenotype by combining iterative cycles of multiplex genome engineering and predictive modeling. We applied our method to the 63-codon E. coli strain C321.ΔA, which has 676 mutations relative to its wild-type ancestor, and identified six single nucleotide mutations that together recover 59% of the fitness defect exhibited by the strain. The resulting optimized strain, C321.DA.opt, is an improved chassis for production of proteins containing non-standard amino acids. Our data reveal how multiple cycles of multiplex automated genome engineering (MAGE) and inexpensive sequencing can generate rich genotypic and phenotypic diversity that can be combined with linear regression techniques to quantify individual allelic effects. While laboratory evolution relies on enrichment as a proxy for allelic effect, our model-guided approach is less susceptible than enrichment to bias from population dynamics and recombination efficiency. We also show that the method can identify beneficial de novo mutations that arise adventitiously. Beyond improving the fitness of C321, ΔA, our work provides a proof-of-principle for high-throughput quantification of individual allelic effects which can be used with any method for generating targeted genotypic diversity.

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Phenotypic Diversity Caused by Differential RpoS Activity among Environmental Escherichia coli Isolates

Applied and Environmental Microbiology ◽

10.1128/aem.05274-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 7915-7923 ◽

Cited By ~ 47

Author(s):

Sarah M. Chiang ◽

Tao Dong ◽

Thomas A. Edge ◽

Herb E. Schellhorn

Keyword(s):

Escherichia Coli ◽

Phenotypic Diversity ◽

Carbon Sources ◽

Enteric Bacteria ◽

Environmental Isolates ◽

Bacterial Populations ◽

Content Type ◽

Phenotypic Differences ◽

Adaptive Mutations ◽

E Coli

ABSTRACTEnteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmentalEscherichia coliisolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. TherpoSsequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after platingrpoS+isolates on succinate, RpoS mutants could be readily selected from environmentalE. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than theirrpoS+isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects forrpoSmutations in environmentalE. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival ofE. coliin the environment.

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Nongenetic individuality, changeability, and inheritance in bacterial behavior

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023322118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2023322118

Author(s):

Maroš Pleška ◽

David Jordan ◽

Zak Frentz ◽

BingKan Xue ◽

Stanislas Leibler

Keyword(s):

Phenotypic Diversity ◽

E Coli ◽

Behavioral Traits ◽

Behavioral Diversity ◽

Two Generations ◽

Model Independent ◽

Positive Correlations ◽

Nongenetic Inheritance ◽

Low Dimensional ◽

General Studies

Isogenic populations often display remarkable levels of phenotypic diversity even in constant, homogeneous environments. Such diversity results from differences between individuals (“nongenetic individuality”) as well as changes during individuals’ lifetimes (“changeability”). Yet, studies that capture and quantify both sources of diversity are scarce. Here we measure the swimming behavior of hundreds of Escherichia coli bacteria continuously over two generations and use a model-independent method for quantifying behavior to show that the behavioral space of E. coli is low-dimensional, with variations occurring mainly along two independent and interpretable behavioral traits. By statistically decomposing the diversity in these two traits, we find that individuality is the main source of diversity, while changeability makes a smaller but significant contribution. Finally, we show that even though traits of closely related individuals can be remarkably different, they exhibit positive correlations across generations that imply nongenetic inheritance. The model-independent experimental and theoretical framework developed here paves the way for more general studies of microbial behavioral diversity.

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Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050755 ◽

1974 ◽

Vol 32 ◽

pp. 148-149

Author(s):

D. E. Philpott ◽

A. Takahashi

Keyword(s):

Skeletal Muscle ◽

Endothelial Cells ◽

Salivary Gland ◽

Carotid Artery ◽

Basement Membrane ◽

Coronary Vessels ◽

Ruthenium Red ◽

E Coli ◽

Old Rats ◽

The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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