scholarly journals The Effects of Sequence Length and Composition of Random Sequence Peptides on the Growth of E. coli Cells

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1913
Author(s):  
Johana Fajardo ◽  
Diethard Tautz
Keyword(s):  
Cell Growth ◽  
De Novo ◽  
Random Sequence ◽  
Large Fraction ◽  
Gc Content ◽  
Sequence Length ◽  
E Coli ◽  
Aggregation Propensity ◽  
Evolutionary Innovation ◽  
Frequency Changes

We study the potential for the de novo evolution of genes from random nucleotide sequences using libraries of E. coli expressing random sequence peptides. We assess the effects of such peptides on cell growth by monitoring frequency changes in individual clones in a complex library through four serial passages. Using a new analysis pipeline that allows the tracing of peptides of all lengths, we find that over half of the peptides have consistent effects on cell growth. Across nine different experiments, around 16% of clones increase in frequency and 36% decrease, with some variation between individual experiments. Shorter peptides (8–20 residues), are more likely to increase in frequency, longer ones are more likely to decrease. GC content, amino acid composition, intrinsic disorder, and aggregation propensity show slightly different patterns between peptide groups. Sequences that increase in frequency tend to be more disordered with lower aggregation propensity. This coincides with the observation that young genes with more disordered structures are better tolerated in genomes. Our data indicate that random sequences can be a source of evolutionary innovation, since a large fraction of them are well tolerated by the cells or can provide a growth advantage.

scholarly journals The effects of sequence length and composition of random sequence peptides on the growth of E. coli cells

2021 ◽  
Author(s):  
Johana R. C. Fajardo ◽  
Diethard Tautz
Keyword(s):  
Cell Growth ◽  
De Novo ◽  
Random Sequence ◽  
Large Fraction ◽  
Gc Content ◽  
Sequence Length ◽  
E Coli ◽  
Aggregation Propensity ◽  
Evolutionary Innovation ◽  
Frequency Changes

We study the potential for the de novo evolution of genes from random nucleotide sequences using libraries of E. coli expressing random sequence peptides. We assess the effects of such peptides on cell growth by monitoring frequency changes of individual clones in a complex library through four serial passages. Using a new analysis pipeline that allows to trace peptides of all lengths, we find that over half of the peptides have consistent effects on cell growth. Across nine different experiments, around 16 % of clones increase in frequency and 36 % decrease, with some variation between individual experiments. Shorter peptides (8 - 20 residues), are more likely to increase in frequency, longer ones are more likely to decrease. GC content, amino acid composition, intrinsic dis-order and aggregation propensity show slightly different patterns between peptide groups. Sequences that increase in frequency tend to be more disordered with lower aggregation propensity. This coincides with the observation that young genes with more disordered structures are better tolerated in genomes. Our data indicate that random sequences can be a source of evolutionary innovation, since a large fraction of them are well tolerated by the cells or can provide a growth advantage.

scholarly journals Random Sequences Rapidly Evolve into de novo Promoters

2017 ◽  
Author(s):  
Avihu H. Yona ◽  
Eric J. Alm ◽  
Jeff Gore
Keyword(s):  
Protein Interactions ◽  
De Novo ◽  
Random Sequence ◽  
Lac Operon ◽  
Protein Protein Interactions ◽  
Random Sequences ◽  
E Coli ◽  
Promoter Recognition ◽  
Motif Recognition ◽  
Active Promoter

AbstractHow do new promoters evolve? To follow evolution of de novo promoters, we put various random sequences upstream to the lac operon in Escherichia coli and evolved the cells in the presence of lactose. We found that a typical random sequence of ~100 bases requires only one mutation in order to enable growth on lactose by increasing resemblance to the canonical promoter motifs. We further found that ~10% of random sequences could serve as active promoters even without any period of evolutionary adaptation. Such a short mutational distance from a random sequence to an active promoter may improve evolvability yet may also lead to undesirable accidental expression. We found that across the E. coli genome accidental expression is minimized by avoiding codon combinations that resemble promoter motifs. Our results suggest that the promoter recognition machinery has been tuned to allow high accessibility to new promoters, and similar findings might also be observed in higher organisms or in other motif recognition machineries, like transcription factor binding sites or protein-protein interactions.

scholarly journals Suppressors of dGTP Starvation in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Mark Itsko ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Dna Replication ◽  
De Novo ◽  
Large Fraction ◽  
Cell Mass ◽  
Purine Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Major Mode

ABSTRACT dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving an E. coli gpt optA1 strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included the purEK operon as well as the acrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions. IMPORTANCE Concentrations of the four precursors for DNA synthesis (2′-deoxynucleoside-5′-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition, E. coli cells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels.

scholarly journals Differences between the de novo proteome and its non-functional precursor can result from neutral constraints on its birth process, not necessarily from natural selection alone

2018 ◽  
Author(s):  
Lou Nielly-Thibault ◽  
Christian R Landry
Keyword(s):  
Natural Selection ◽  
De Novo ◽  
Random Sequence ◽  
Gc Content ◽  
Intrinsic Disorder ◽  
Raw Material ◽  
Published Data ◽  
Novel Proteins ◽  
Evolutionary Forces ◽  
The Mean

ABSTRACTProteins are among the most important constituents of biological systems. Because all proteins ultimately evolved from previously non-coding DNA, the properties of these non-coding sequences and how they shape the birth of novel proteins are also expected to influence the organization of biological networks. When trying to explain and predict the properties of novel proteins, it is of particular importance to distinguish the contributions of natural selection and other evolutionary forces. Studies in the field typically use non-coding DNA and GC-content-based random-sequence models to generate random expectations for the properties of novel functional proteins. Deviations from these expectations have been interpreted as the result of natural selection. However, interpreting such deviations requires a yet-unattained understanding of the raw material of de novo gene birth and its relation to novel functional proteins. We mathematically show how the importance of the “junk” polypeptides that make up this raw material goes beyond their average properties and their filtering by natural selection. We find that the mean of any property among novel functional proteins also depends on its variance among junk polypeptides and its correlation with their rate of evolutionary turnover. In order to exemplify the use of our general theoretical results, we combine them with a simple model that predicts the means and variances of the properties of junk polypeptides from the genomic GC content alone. Under this model, we predict the effect of GC content on the mean length and mean intrinsic disorder of novel functional proteins as a function of evolutionary parameters. We use these predictions to formulate new evolutionary interpretations of published data on the length and intrinsic disorder of novel functional proteins. This work provides a theoretical framework that can serve as a guide for the prediction and interpretation of past and future results in the study of novel proteins and their properties under various evolutionary models. Our results provide the foundation for a better understanding of the properties of cellular networks through the evolutionary origin of their components.

scholarly journals Characteristics of Glucose Oxidase Gene (GGOx) from Aspergillus niger IPBCC 08.610

2020 ◽  
Vol 6 (1) ◽  
pp. 10-19
Author(s):  
Popi Asri Kurniatin ◽  
Laksmi Ambarsari ◽  
Annisa Dhiya Athiyyah Khanza ◽  
Inda Setyawati ◽  
Djarot Sasongko Hami Seno ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
Gc Content ◽  
Sequence Length ◽  
Oxidase Gene ◽  
E Coli ◽  
Gene Recognition ◽  
Enzymatic Fuel Cell ◽  
Ecori Restriction ◽  
Glucose Oxidase Gene

Glucose oxidase is used in various industries for the development of enzymatic fuel cell. Based on prior studies, this compound is sourced from the local isolates of Aspergillus niger IPBCC 08.610, although investigations on the encoding gene have not been conducted. The purpose of this research, therefore, is to identify and characterized the gene responsible for encoding glucose oxidase, in the aspect of sequence, length, and restriction patterns. This experiment involved the amplification of genomic DNA using specific primers for gene recognition, which was followed by the restriction technique with EcoRI and PstI endonucleases. Furthermore, the gene is inserted into vector pGEM®T-Easy and transformed into competent E. coli DH5α cells, in an attempt to perform sequencing. The glucose oxidase gene from A. niger IPBCC 08.610 was confirmed to possess a size of 1848 bp, and a GC content of 57.8%, with a possibility of restriction into two fragments of size 908 bp and 980 bp, using the EcoRI restriction.

scholarly journals Epidemiological and phylogenetic analysis reveals Flavobacteriaceae as potential ancestral source of tigecycline resistance gene tet(X)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rong Zhang ◽  
Ning Dong ◽  
Zhangqi Shen ◽  
Yu Zeng ◽  
Jiauyue Lu ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Resistance Gene ◽  
Gc Content ◽  
Zhejiang Province ◽  
Bacterial Strains ◽  
Gram Negative ◽  
E Coli ◽  
Transmission Mechanisms ◽  
Low Prevalence ◽  
Potential Origin

Abstract Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994–2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X).

scholarly journals Effect of Cell Growth on Hepatitis C Virus (HCV) Replication and a Mechanism of Cell Confluence-Based Inhibition of HCV RNA and Protein Expression

2006 ◽  
Vol 80 (3) ◽  
pp. 1181-1190 ◽  
Author(s):  
Heather B. Nelson ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Growth ◽  
De Novo ◽  
Physiological State ◽  
Inhibitory Effect ◽  
Cell Number ◽  
Serum Starvation ◽  
Hcv Rna ◽  
Cell Growth Arrest

ABSTRACT An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

2012 ◽  
Vol 78 (20) ◽  
pp. 7407-7413 ◽  
Author(s):  
Qian Zhang ◽  
Tao Yan
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Potassium Acetate ◽  
Desiccation Stress ◽  
Desiccation Resistance ◽  
Content Type ◽  
Soil Desiccation ◽  
E Coli ◽  
Organic Solute ◽  
Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

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