Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high‐cell‐density cultures of <i>Escherichia coli</i>

Lin, Hongying; Hoffmann, Frank; Rozkov, Aleksei; Enfors, Sven‐Olof; Rinas, Ursula; Neubauer, Peter

doi:10.1002/bit.20152

Cited by 29 publications

(23 citation statements)

References 51 publications

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“…Intracellular cAMP. The method was adapted from Lin et al (2004) and modified as follows. After centrifugation of the samples taken from the reactor, intracellular cAMP was determined in the pellet, removing all visible liquid residues by vacuum pump.…”

Section: Extracellular and Intracellular Camp Concentrationmentioning

confidence: 99%

Impairing and Monitoring Glucose Catabolite Repression in l‐Carnitine Biosynthesis

Sevilla¹,

Cánovas²,

Keller³

et al. 2007

Biotechnology Progress

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Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of L-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of L-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the L-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the L-carnitine production when glucose was used as C-source. Additionally, a way to monitor the L-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.

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Section: Extracellular and Intracellular Camp Concentrationmentioning

confidence: 99%

Impairing and Monitoring Glucose Catabolite Repression in l‐Carnitine Biosynthesis

Sevilla¹,

Cánovas²,

Keller³

et al. 2007

Biotechnology Progress

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“…It should be noted that for the quantitative evaluation of the NIR‐Raman data MVDA is essential . In case of cAMP, addition of Ag NPs further improved the prediction results from PLSR, however, for glucose, acetate and AMP, no comparable effect was observed with SERS although the spectra exhibited more spectral lines and details . Remarkably, the application of complex media might lead to different conclusions about the benefits of SERS pretreatment and Raman spectroscopy evaluation.…”

Section: Resultsmentioning

confidence: 99%

“…61 In case of cAMP, addition of Ag NPs further improved the prediction results from PLSR, however, for glucose, acetate and AMP, no comparable effect was observed with SERS although the spectra exhibited more spectral lines and details. 62 Remarkably, the application of complex media might lead to different conclusions about the benefits of SERS pretreatment and Raman spectroscopy evaluation. Despite the advantages of SERS nanostructures to drastically enhance the Raman signal and quench fluorescence, a careful planned set-up is necessary to carry out reproducible measurement results and neglect effects of signal variations due to aggregation.…”

Section: Resultsmentioning

confidence: 99%

Comparison of time‐gated surface‐enhanced raman spectroscopy (TG‐SERS) and classical SERS based monitoring of Escherichia coli cultivation samples

Kögler

Paul

Anane

et al. 2018

Biotechnology Progress

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The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP, and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids, and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., :1-10, 2018.

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“…in the virulence of Bulkhorderia dolosa ) [97, 98]. Cyclic AMPs, on the other hand, are involved mainly in intracellular signaling and were recently shown to participate in generic extracellular signaling, particularly during stress conditions [99, 100]. These two signaling modes were upregulated under the TBZ treatment and participated in the correlation network between the Sphingomonas MAG 3X21F and MAG 23F of Hydrogenophaga .…”

Section: Discussionmentioning

confidence: 99%

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on aSphingomonasfor the degradation of the fungicide thiabendazole

Vasileiadis

Perruchon

Scheer

et al. 2020

Preprint

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22Background: Thiabendazole (TBZ), is a benzimidazole fungicide and anthelminthic whose high 23 persistence and toxicity pose a serious environmental threat. In our quest for environmental mitigation 24 we previously isolated the first TBZ-degrading bacterial consortium and provided preliminary evidence 25 for its composition and the degrading role of a Sphingomonas. Here, we employed a multi-omic 26 approach combined with DNA-stable isotope probing (SIP) to determine the genetic make-up of the 27 key consortium members, to disentangle nutritional and metabolic interdependencies, to identify the 28 transformation pathway of TBZ and to understand the genetic network driving its transformation. 29Results: Time-series SIP in combination with amplicon sequencing analysis verified the key role of 30 Sphingomonas in TBZ degradation by assimilating over 80% of the 13 C-labelled phenyl moiety of TBZ. 31Non-target mass spectroscopy (MS) analysis showed the accumulation of thiazole-4-carboxamidine as 32 a single dead-end transformation product and no phenyl-containing derivative, in line with the phenyl 33 moiety assimilation in the SIP analysis. Time series metagenomic analysis of the consortium 34 supplemented with TBZ or succinate led to the assembly of 18 metagenome-assembled genomes 35 (MAGs) with >80% completeness, six (Sphingomonas 3X21F, γ-Proteobacterium 34A, 36Bradyrhizobiaceae 9B and Hydrogenophaga 19A, 13A, and 23F) being dominant. Meta-transcriptomic 37 and -proteomic analysis suggested that Sphingomonas mobilize a carbazole dioxygenase (car) operon 38 during the initial cleavage of TBZ to thiazole-4-carboxamidine and catechol, the latter is further 39 transformed by enzymes encoded in a catechol ortho-cleavage (cat) operon; both operons being up-40 regulated during TBZ degradation. Computational docking analysis of the terminal oxygenase 41 component of car, CarAa, showed high affinity to TBZ, comparable to carbazole, reinforcing its high 42 potency for TBZ transformation. These results suggest no interactions between consortium members in 43 TBZ transformation, performed solely by Sphingomonas. In contrast, gene expression network analysis 44 revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, 45with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin 46 salvage pathway along TBZ degradation. 47 Conclusions:Our findings suggest interactions between consortium members which align with the 48 "black queen hypothesis": Sphingomonas detoxifies TBZ, releasing consortium members by a toxicant; 49 in return for this, Hydrogenophaga 23F provides cobalamin to the auxotrophic Sphingomonas. 50 Background 54 Thiabendazole (TBZ) is a benzimidazole compound which is used as a post-harvest fungicide to control 55 fungal infestations on fruits during storage [1] and as a broad spectrum anthelminthic to control 56 endoparasites in livestock farming [2]. It acts by binding to tubulin monomers inhibiting the 57 polymerization of microtubules and, thus,...

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Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high‐cell‐density cultures of Escherichia coli

Cited by 29 publications

References 51 publications

Impairing and Monitoring Glucose Catabolite Repression in l‐Carnitine Biosynthesis

Impairing and Monitoring Glucose Catabolite Repression in l‐Carnitine Biosynthesis

Comparison of time‐gated surface‐enhanced raman spectroscopy (TG‐SERS) and classical SERS based monitoring of Escherichia coli cultivation samples

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on aSphingomonasfor the degradation of the fungicide thiabendazole

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