The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA

Wegener, Marius; Vogtmann, Kristina; Huber, Madeleine; Laaß, Sebastian; Soppa, Jörg

doi:10.1016/j.mimet.2016.10.015

Cited by 10 publications

(7 citation statements)

References 31 publications

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“…To test whether AzuC affects GlpD activity, we employed a dehydrogenase activity assay in which glycerol-3-phosphate oxidation to dihydroxyacetone phosphate (DHAP) is coupled to the reduction of yellow 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2 H -tetrazolium bromide (MTT) to blue formazan, which is detected at OD 570 (Wegener et al ., 2016). Dehydrogenase activity based on this assay was found to be almost 2-fold lower in the absence of AzuC when extracts were made from a WT or a Δ azuC strain grown in M63 glucose medium and shifted to glycerol (pH 5.5) for 3 h. In contrast, overexpression of wild type AzuC, but not AzuC L3STOP , led to an increase in the dehydrogenase activity for the extracts (Fig 4A, middle panel).…”

Section: Resultsmentioning

confidence: 99%

AzuCR RNA modulates carbon metabolism as a dual-function RNA inEscherichia coli

Raina

Aoyama

Bhatt

et al. 2021

Preprint

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Bacteria have evolved small RNAs (sRNAs) to regulate numerous biological processes and stress responses. While sRNAs generally are considered to be "noncoding", a few have been found to also encode a small protein. Here we describe one such dual-function RNA that modulates carbon utilization in Escherichia coli. The 164 nucleotide RNA was previously shown to encode a 28 amino acid protein (denoted AzuC). We discovered the membrane-associated AzuC protein interacts with GlpD, the aerobic glycerol-3-phosphate dehydrogenase, leading to increased GlpD activity. Overexpression of the RNA encoding AzuC results in a growth defect in glycerol and galactose medium. The defect in galactose medium was still observed for a stop codon mutant derivative, suggesting a potential regulatory role for the RNA. Consistent with this observation, we found that cadA and galE are repressed by base pairing with the RNA (denoted AzuCR). Interestingly, translation of AzuC interferes with the observed repression of cadA and galE by AzuCR and base pairing interferes with AzuC translation, demonstrating that the translation and base pairing functions compete.

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Section: Resultsmentioning

confidence: 99%

AzuCR RNA modulates carbon metabolism as a dual-function RNA inEscherichia coli

Raina

Aoyama

Bhatt

et al. 2021

Preprint

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“…Manipulation of GlpD levels on the other hand, had strong negative effects on both growth rate and biomass. GlpD is a key enzyme in glycerol metabolism as it was shown that growth on glycerol strictly depends on the presence of GlpD [ 46 ]. Hence, the availability of physiological levels of the phospholipid precursor glycerol-3P appears to be an important prerequisite for a healthy lipid homeostasis.…”

Section: Discussionmentioning

confidence: 99%

Control of n-Butanol Induced Lipidome Adaptations in E. coli

et al. 2021

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The versatile compound n-butanol is one of the most promising biofuels for use in existing internal combustion engines, contributing to a smooth transition towards a clean energy society. Furthermore, n-butanol is a valuable resource to produce more complex molecules such as bioplastics. Microbial production of n-butanol from waste materials is hampered by the biotoxicity of n-butanol as it interferes with the proper functioning of lipid membranes. In this study we perform a large-scale investigation of the complete lipid-related enzyme machinery and its response to exposure to a sublethal concentration of n-butanol. We profiled, in triplicate, the growth characteristics and phospholipidomes of 116 different genetic constructs of E. coli, both in the presence and absence of 0.5% n-butanol (v/v). This led to the identification of 230 lipid species and subsequently to the reconstruction of the network of metabolites, enzymes and lipid properties driving the homeostasis of the E. coli lipidome. We were able to identify key lipids and biochemical pathways leading to altered n-butanol tolerance. The data led to new conceptual insights into the bacterial lipid metabolism which are discussed.

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“…It contains a chromosomal deletion of the glpD gene, and, therefore, allows to use a plasmid-bound glpD gene as a reporter gene without any background. To enable the simultaneous use of the gusA gene as a reporter gene, the chromosomal gusA copy has also been deleted (Wegener et al, 2016). E. coli was grown in SOB + medium as described (Huber et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

Unidirectional gene pairs in archaea and bacteria require overlaps or very short intergenic distances for translational coupling via termination-reinitiation and often encode subunits of heteromeric complexes

Huber,

Vogel,

Borst

et al. 2023

Front. Microbiol.

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Genomes of bacteria and archaea contain a much larger fraction of unidirectional (serial) gene pairs than convergent or divergent gene pairs. Many of the unidirectional gene pairs have short overlaps of −4 nt and −1 nt. As shown previously, translation of the genes in overlapping unidirectional gene pairs is tightly coupled. Two alternative models for the fate of the post-termination ribosome predict either that overlaps or very short intergenic distances are essential for translational coupling or that the undissociated post-termination ribosome can scan through long intergenic regions, up to hundreds of nucleotides. We aimed to experimentally resolve the contradiction between the two models by analyzing three native gene pairs from the model archaeon Haloferax volcanii and three native pairs from Escherichia coli. A two reporter gene system was used to quantify the reinitiation frequency, and several stop codons in the upstream gene were introduced to increase the intergenic distances. For all six gene pairs from two species, an extremely strong dependence of the reinitiation efficiency on the intergenic distance was unequivocally demonstrated, such that even short intergenic distances of about 20 nt almost completely abolished translational coupling. Bioinformatic analysis of the intergenic distances in all unidirectional gene pairs in the genomes of H. volcanii and E. coli and in 1,695 prokaryotic species representative of 49 phyla showed that intergenic distances of −4 nt or −1 nt (= short gene overlaps of 4 nt or 1 nt) were by far most common in all these groups of archaea and bacteria. A small set of genes in E. coli, but not in H. volcanii, had intergenic distances of around +10 nt. Our experimental and bioinformatic analyses clearly show that translational coupling requires short gene overlaps, whereas scanning of intergenic regions by the post-termination ribosome occurs rarely, if at all. Short overlaps are enriched among genes that encode subunits of heteromeric complexes, and co-translational complex formation requiring precise subunit stoichiometry likely confers an evolutionary advantage that drove the formation and conservation of overlapping gene pairs during evolution.

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The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA

Cited by 10 publications

References 31 publications

AzuCR RNA modulates carbon metabolism as a dual-function RNA inEscherichia coli

AzuCR RNA modulates carbon metabolism as a dual-function RNA inEscherichia coli

Control of n-Butanol Induced Lipidome Adaptations in E. coli

Unidirectional gene pairs in archaea and bacteria require overlaps or very short intergenic distances for translational coupling via termination-reinitiation and often encode subunits of heteromeric complexes

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