Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors

AbstractThe evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

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Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

eLife ◽

10.7554/elife.53535 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Andrew B Morgenthaler ◽

Wallis R Kinney ◽

Christopher C Ebmeier ◽

Corinne M Walsh ◽

Daniel J Snyder ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Pathway ◽

Weak Link ◽

Experimental Studies ◽

Wild Type ◽

Gene Encoding ◽

Evolutionary Trajectory ◽

Adaptive Mutations ◽

E Coli ◽

Essential Function

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the ‘weak-link’ enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

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Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.5158-5162.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 5158-5162 ◽

Cited By ~ 39

Author(s):

Hiroaki Iwaki ◽

Yoshie Hasegawa ◽

Masahiro Teraoka ◽

Tai Tokuyama ◽

Hélène Bergeron ◽

...

Keyword(s):

Gene Sequence ◽

Expression System ◽

Transcriptional Activator ◽

Fold Increase ◽

Gene Arrangement ◽

Catabolic Pathway ◽

Gene Encoding ◽

Cyclohexanone Monooxygenase ◽

E Coli ◽

Molecular Masses

ABSTRACT We identified chnR, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression ofchnB, the structural gene for cyclohexanone monooxygenase (CHMO) in Acinetobacter sp. strain NCIMB 9871. The gene sequence of chnE, which encodes an NADP+-linked 6-oxohexanoate dehydrogenase, the enzyme catalyzing the fifth step of cyclohexanol degradation, was also determined. The gene arrangement ischnB-chnE-chnR. The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system. Inducible expression of cloned chnB inEscherichia coli depended upon the presence ofchnR. A transcriptionalchnB::lacZ fusion experiment revealed that cyclohexanone induces chnB expression in E. coli, in which a 22-fold increase in activity was observed.

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Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽

10.3390/biom11060843 ◽

2021 ◽

Vol 11 (6) ◽

pp. 843

Author(s):

Balagra Kasim Sumabe ◽

Synnøve Brandt Ræder ◽

Lisa Marie Røst ◽

Animesh Sharma ◽

Eric S. Donkor ◽

...

Keyword(s):

Mammalian Cells ◽

Mutation Frequency ◽

Nucleoside Analogues ◽

Cancer Therapies ◽

E Coli ◽

Replicative Stress ◽

Pol V ◽

Dna And Rna ◽

Sos Induction ◽

Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

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Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli

Nature Communications ◽

10.1038/s41467-021-25142-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chun-Ying Wang ◽

Martin Lempp ◽

Niklas Farke ◽

Stefano Donati ◽

Timo Glatter ◽

...

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Metabolic Pathways ◽

Glycerol Production ◽

Inducible Promoters ◽

E Coli ◽

Inhibition Of Glycolysis ◽

Underlying Mechanisms ◽

Rate Limiting ◽

Engineered Bacteria

AbstractSynthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

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Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gkaa1221 ◽

2020 ◽

Author(s):

Seungwoo Cha ◽

Chang Pyo Hong ◽

Hyun Ah Kang ◽

Ji-Sook Hahn

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Target Genes ◽

Gene Encoding ◽

Metabolic Switch ◽

Differential Activation ◽

N Terminus ◽

Activation Mechanisms ◽

In Trans ◽

Separate Activation

Abstract Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

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Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

Applied and Environmental Microbiology ◽

10.1128/aem.02920-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 130-138 ◽

Cited By ~ 30

Author(s):

James Kirby ◽

Minobu Nishimoto ◽

Ruthie W. N. Chow ◽

Edward E. K. Baidoo ◽

George Wang ◽

...

Keyword(s):

Bacterial Species ◽

Xylose Reductase ◽

Pentose Phosphate ◽

Terpene Biosynthesis ◽

Content Type ◽

E Coli ◽

Pathway Expression ◽

Terpene Synthesis ◽

Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

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