Increased Episomal Replication Accounts for the High Rate of Adaptive Mutation in recD Mutants of Escherichia coli

Abstract Adaptive mutation has been studied extensively in FC40, a strain of Escherichia coli that cannot metabolize lactose (Lac-) because of a frameshift mutation affecting the lacZ gene on its episome. recD mutants of FC40, in which the exonuclease activity of RecBCD (ExoV) is abolished but its helicase activity is retained, have an increased rate of adaptive mutation. The results presented here show that, in several respects, adaptive mutation to Lac+ involves different mechanisms in recD mutant cells than in wild-type cells. About half of the apparent increase in the adaptive mutation rate of recD mutant cells is due to a RecA-dependent increase in episomal copy number and to growth of the Lac- cells on the lactose plates. The remaining increase appears to be due to continued replication of the episome, with the extra copies being degraded or passed to recD+ recipients. In addition, the increase in adaptive mutation rate in recD mutant cells is (i) dependent on activities of the single-stranded exonucleases, RecJ and ExoI, which are not required for (in fact, slightly inhibit) adaptive mutation in wild-type cells, and (ii) enhanced by RecG, which opposes adaptive mutation in wild-type cells.

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Evidence That Selected Amplification of a Bacterial lac Frameshift Allele Stimulates Lac+ Reversion (Adaptive Mutation) With or Without General Hypermutability

Genetics ◽

10.1093/genetics/161.3.945 ◽

2002 ◽

Vol 161 (3) ◽

pp. 945-956 ◽

Cited By ~ 3

Author(s):

E Susan Slechta ◽

Jing Liu ◽

Dan I Andersson ◽

John R Roth

Keyword(s):

Model Selection ◽

Direct Effect ◽

Mutation Rate ◽

Side Effect ◽

Copy Number ◽

Fold Increase ◽

Genetic System ◽

Adaptive Mutation ◽

E Coli ◽

Frameshift Mutant

Abstract In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac+ revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification—induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.

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Roasted coffee wastes as a substrate for Escherichia coli to grow and produce hydrogen

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa088 ◽

2020 ◽

Vol 367 (11) ◽

Cited By ~ 1

Author(s):

Hripsime Petrosyan ◽

Liana Vanyan ◽

Satenik Mirzoyan ◽

Armen Trchounian ◽

Karen Trchounian

Keyword(s):

Escherichia Coli ◽

H2 Production ◽

High Rate ◽

Main Role ◽

Wild Type ◽

Roasted Coffee ◽

Energy Needs ◽

Genetic Modifications ◽

Coffee Grounds ◽

And Control

ABSTRACT After brewing roasted coffee, spent coffee grounds (SCGs) are generated being one of the daily wastes emerging in dominant countries with high rate and big quantity. Escherichia coli BW25113 wild-type strain, mutants with defects in hydrogen (H2)-producing/oxidizing four hydrogenases (Hyd) (ΔhyaB ΔhybC, ΔhycE, ΔhyfG) and septuple mutant (ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE) were investigated by measuring change of external pH, bacterial growth and H2 production during the utilization of SCG hydrolysate. In wild type, H2 was produced with rate of 1.28 mL H2 (g sugar)−1 h−1 yielding 30.7 mL H2 (g sugar)−1 or 2.75 L (kg SCG)−1 during 24 h. In septuple mutant, H2 production yield was 72 mL H2 (g sugar)−1 with rate of 3 mL H2 (g sugar)−1 h−1. H2 generation was absent in hycE single mutant showing the main role of Hyd-3 in H2 production. During utilization of SCG wild type, specific growth rate was 0.72 ± 0.01 h−1 with biomass yield of 0.3 g L−1. Genetic modifications and control of external parameters during growth could lead to prolonged and enhanced microbiological H2 production by organic wastes, which will aid more efficiently global sustainable energy needs resulting in diversification of mobile and fixed energy sources.

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TolC-Dependent Exclusion of Porphyrins in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00595-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6228-6233 ◽

Cited By ~ 36

Author(s):

Ryoko Tatsumi ◽

Masaaki Wachi

Keyword(s):

Escherichia Coli ◽

Uv Irradiation ◽

High Performance ◽

Aminolevulinic Acid ◽

Wild Type ◽

E Coli ◽

Chromatography Analysis ◽

Increased Sensitivity ◽

Mutant Cells ◽

Reddish Brown Color

ABSTRACT We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli.

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Escherichia coli wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities

International Journal of Hydrogen Energy ◽

10.1016/j.ijhydene.2018.07.045 ◽

2018 ◽

Vol 43 (33) ◽

pp. 15870-15879 ◽

Cited By ~ 7

Author(s):

Anna Poladyan ◽

Lusine Baghdasaryan ◽

Armen Trchounian

Keyword(s):

Escherichia Coli ◽

Hydrogen Production ◽

Wild Type ◽

Buffer Capacities ◽

Mutant Cells

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Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/w00-019 ◽

2000 ◽

Vol 46 (6) ◽

pp. 577-583 ◽

Cited By ~ 1

Author(s):

Takashi Kubo ◽

Toshiko Aiso ◽

Reiko Ohki

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Temperature Sensitive ◽

Lacz Gene ◽

Permissive Temperature ◽

Wild Type ◽

Double Mutation ◽

Mutant Genes ◽

Beta Galactosidase ◽

Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

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Restoration of wild-type motility to flagellin-knockout Escherichia coli

10.1101/700492 ◽

2019 ◽

Author(s):

Nicholas M. Thomson ◽

Mark J. Pallen

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Plasmid Copy Number ◽

Major Constituent ◽

Strongly Correlated ◽

Wild Type ◽

Plasmid Copy ◽

Inducible Promoters ◽

E Coli ◽

Flagellar Filament

AbstractFlagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

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96 Validation of the candidate mutation responsible for embryonic lethality in Holstein haplotype 2 carriers

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab96 ◽

2021 ◽

Vol 33 (2) ◽

pp. 155

Author(s):

K. Clark ◽

J. Cole ◽

D. Bickhart ◽

J. Hutchison ◽

D. Null ◽

...

Keyword(s):

Dna Sequences ◽

Frameshift Mutation ◽

Embryonic Lethality ◽

High Rate ◽

Reference Sequence ◽

Future Research ◽

Wild Type ◽

Protein Amino Acid ◽

Cell Stage ◽

Target Sequences

Holstein haplotype 2 (HH2) is embryonic lethal and carried by 1.21% of the US Holstein population. Using next-generation sequencing, we identified a high-impact frameshift mutation in intraflagellar protein 80 (IFT80) as the putative causal mutation. In bovine embryos, IFT80 expression begins at the 8-cell stage and decreases by the blastocyst stage. We hypothesised that the loss of function of IFT80 early in development causes the lethal phenotype. The aim of this study was to mimic the mutation observed invivo using a CRISPR-Cas9 approach to determine its effect on embryo development. Two guide RNAs (gRNAs) were designed to disrupt exon 11 (Ex11), one before and one after the known IFT80 mutation site, creating a 317-nucleotide (nt) cut to facilitate genotyping. Then, gRNAs annealed to a tracr-Cas9mRNA complex were delivered to 1-cell embryos by microinjection. Each replicate contained control embryos injected with only Cas9mRNA and treated embryos injected with gRNAs targeting IFT80. Embryos from each group were collected at the 8-cell stage for genotyping and gene expression analysis (n=47), or on Day 8 to validate genotypes of embryos left to develop (n=50). DNA sequences containing gRNA target sequences were amplified and visualised on an agarose gel. IFT80 expression was determined in biallelic embryos (n=13) using quantitative PCR and normalized to GAPDH. Primers were designed for the transcript regions before and after gRNAs target sequences, exons 9 and 12, respectively. Expression data were analysed using SAS software (v. 9.4; SAS Institute Inc.) using PROC GLM and LSMEANS to determine expression differences. Biallelic samples (n=9) were Sanger-sequenced (SS) and aligned with the reference sequence to determine exact cut sites. Protein amino acid (AA) sequences were predicted using SS data. Protein models were constructed using the I-Tasser platform, and then aligned and visualised using PyMol 2.4. Biallelic edits showed a significant decrease in exon 12 expression (P<0.05), and no difference in exon 9 compared with controls (P>0.05), indicating that the transcript was severely affected downstream of the edited sites. The reference protein model contained 777 AA, whereas the biallelic sample with the most accurate cut sites yielded a 385-AA protein, indicating that the mutation severely altered protein conformation and possible function. Embryos injected with CRISPR-Cas9 targeting Ex11 arrested at the 8-cell stage and failed to form blastocysts. Day 8 embryos were genotyped (n=24) and 58% were biallelic, 21% were monoallelic, and 21% appeared wild-type. Given the high rate of edits, the observed embryonic arrest is likely due to disruption of IFT80, and wild-type embryos may contain small edits not visible by gel. In conclusion, generation of CRISPR-Cas9 IFT80 knockouts demonstrated that the frameshift mutation in Ex11 results in a seemingly nonfunctional protein that is responsible for the embryonic lethality seen in HH2 carriers. Future research is needed to determine how IFT80 regulates embryonic development. This research was supported by USDA-NIFA National Needs Fellowship, USDA-NIFA AFRI Grant No. 2019-67015-28998.

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Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in Escherichia coli O157:H7

Journal of Bacteriology ◽

10.1128/jb.00001-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4253-4263 ◽

Cited By ~ 75

Author(s):

Amy J. Vogler ◽

Christine Keys ◽

Yoshimi Nemoto ◽

Rebecca E. Colman ◽

Zack Jay ◽

...

Keyword(s):

Escherichia Coli ◽

Mismatch Repair ◽

Mutation Rate ◽

Tandem Repeat ◽

Copy Number ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Mutation Rates ◽

Repeat Copy Number ◽

Repeat Copy

ABSTRACT Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 × 10−4 mutations/generation and a combined 28-locus rate of 6.4 × 10−4 mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r 2 = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r 2 = 0.833, P < 0.0001) or excluded (r 2 = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

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Unusual properties of a new division mutant of Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m83-113 ◽

1983 ◽

Vol 29 (6) ◽

pp. 694-699 ◽

Cited By ~ 3

Author(s):

G. R. Drapeau ◽

J. P. Chausseau ◽

F. Gariépy

Keyword(s):

Escherichia Coli ◽

Uv Irradiation ◽

Pleiotropic Effect ◽

Single Mutation ◽

Permissive Temperature ◽

Wild Type ◽

Genetic Lesion ◽

Host Cell Reactivation ◽

Mutant Forms ◽

Mutant Cells

The properties of a division mutant of Escherichia coli were investigated. At 42 °C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation. Temperature and UV sensitivities are probably due to a single mutation. The mutant phenotype is dominant to wild type. The mutant cells make DNA nearly as effectively as control cells at 42 °C or following UV irradiation. They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation. The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.

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