Finely Tuned Regulation of the Aromatic Amine Degradation Pathway in Escherichia coli

ABSTRACTPseudomonassp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis ofmcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm ofEscherichia coli. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis ofE. coliexpressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparentKmandVmaxin the range of 98 to 111 μM and 69 to 73 μmol · min−1· mg−1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm ofPseudomonasspp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCEProteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm ofPseudomonasspp. Predicted transmembrane domain and signal peptide sequences ofPseudomonaswere found to be functional inEscherichia coliand to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

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Physiological, Genetic, and Transcriptomic Analysis of Alcohol-Induced Delay ofEscherichia coliDeath

Applied and Environmental Microbiology ◽

10.1128/aem.02113-18 ◽

2018 ◽

Vol 85 (2) ◽

Cited By ~ 1

Author(s):

Christina M. Ferraro ◽

Steven E. Finkel

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Degradation Pathway ◽

Signaling Molecules ◽

Threshold Concentration ◽

Content Type ◽

E Coli ◽

Alcohol Effect ◽

Death Phase ◽

K 12

ABSTRACTWhenEscherichia coliK-12 is inoculated into rich medium in batch culture, cells experience five phases. While the lag and logarithmic phases are mechanistically fairly well defined, the stationary phase, death phase, and long-term stationary phase are less well understood. Here, we characterize a mechanism of delaying death, a phenomenon we call the “alcohol effect,” where the addition of small amounts of certain alcohols prolongs stationary phase for at least 10 days longer than in untreated conditions. We show that the stationary phase is extended when ethanol is added above a minimum threshold concentration. Once ethanol levels fall below a threshold concentration, cells enter the death phase. We also show that the effect is conferred by the addition of straight-chain alcohols 1-propanol, 1-butanol, 1-pentanol, and, to a lesser degree, 1-hexanol. However, methanol, isopropanol, 1-heptanol, and 1-octanol do not delay entry into death phase. Though modulated by RpoS, the alcohol effect does not require RpoS activity or the activities of the AdhE or AdhP alcohol dehydrogenases. Further, we show that ethanol is capable of extending the life span of stationary-phase cultures for non-K-12E. colistrains and that this effect is caused in part by genes of the glycolate degradation pathway. These data suggest a model where ethanol and other shorter 1-alcohols can serve as signaling molecules, perhaps by modulating patterns of gene expression that normally regulate the transition from stationary phase to death phase.IMPORTANCEIn one of the most well-studied organisms in the life sciences,Escherichia coli, we still do not fully understand what causes populations to die. This is largely due to the technological difficulties of studying bacterial cell death. This study provides an avenue to studying how and whyE. colipopulations, and perhaps other microbes, transition from stationary phase to death phase by exploring how ethanol and other alcohols delay the onset of death. Here, we demonstrate that alcohols are acting as signaling molecules to achieve the delay in death phase. This study not only offers a better understanding of a fundamental process but perhaps also provides a gateway to studying the dynamics between ethanol and microbes in the human gastrointestinal tract.

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Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli

Applied Microbiology and Biotechnology ◽

10.1007/s00253-007-0902-3 ◽

2007 ◽

Vol 75 (5) ◽

pp. 1095-1101 ◽

Cited By ~ 7

Author(s):

Anett Kirschner ◽

Josef Altenbuchner ◽

Uwe T. Bornscheuer

Keyword(s):

Escherichia Coli ◽

Pseudomonas Fluorescens ◽

Secondary Alcohol ◽

Degradation Pathway ◽

Engineered Escherichia Coli

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Knockout of acetoacetate degradation pathway gene atoDA enhances the toxicity tolerance of Escherichia coli to isopropanol and acetone

3 Biotech ◽

10.1007/s13205-019-1867-5 ◽

2019 ◽

Vol 9 (9) ◽

Cited By ~ 2

Author(s):

Jia Zhou ◽

Xiaoqing Lu ◽

Baoxia Tian ◽

Chonglong Wang ◽

Hao Shi ◽

...

Keyword(s):

Escherichia Coli ◽

Degradation Pathway ◽

Pathway Gene

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Radical new paradigm for heme degradation in Escherichia coli O157:H7

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603209113 ◽

2016 ◽

Vol 113 (43) ◽

pp. 12138-12143 ◽

Cited By ~ 41

Author(s):

Joseph W. LaMattina ◽

David B. Nix ◽

William Nicholas Lanzilotta

Keyword(s):

Escherichia Coli ◽

Enteric Bacteria ◽

Degradation Pathway ◽

Escherichia Coli O157 ◽

Heme Uptake ◽

New Paradigm ◽

Heme Degradation ◽

Lower Intestine ◽

Uptake And Transport

All of the heme-degrading enzymes that have been characterized to date require molecular oxygen as a cosubstrate. Escherichia coli O157:H7 has been shown to express heme uptake and transport proteins, as well as use heme as an iron source. This enteric pathogen colonizes the anaerobic space of the lower intestine in mammals, yet no mechanism for anaerobic heme degradation has been reported. Herein we provide evidence for an oxygen-independent heme-degradation pathway. Specifically, we demonstrate that ChuW is a radical S-adenosylmethionine methyltransferase that catalyzes a radical-mediated mechanism facilitating iron liberation and the production of the tetrapyrrole product we termed “anaerobilin.” We further demonstrate that anaerobilin can be used as a substrate by ChuY, an enzyme that is coexpressed with ChuW in vivo along with the heme uptake machinery. Our findings are discussed in terms of the competitive advantage this system provides for enteric bacteria, particularly those that inhabit an anaerobic niche in the intestines.

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Genetic Characterization of the Phenylacetyl-Coenzyme A Oxygenase from the Aerobic Phenylacetic Acid Degradation Pathway of Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01550-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7422-7426 ◽

Cited By ~ 27

Author(s):

Cristina Fernández ◽

Abel Ferrández ◽

Baltasar Miñambres ◽

Eduardo Díaz ◽

José L. García

Keyword(s):

Escherichia Coli ◽

Primary Structure ◽

Phenylacetic Acid ◽

Coenzyme A ◽

Genetic Characterization ◽

Distinct Group ◽

Degradation Pathway ◽

Acid Degradation

ABSTRACT We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.

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