Benzoate Metabolism Intermediate Benzoyl Coenzyme A Affects Gentisate Pathway Regulation in Comamonas testosteroni

Abstract

A previous study showed that benzoate was catabolized via a coenzyme A (CoA)-dependent epoxide pathway in Azoarcus evansii (R. Niemetz, U. Altenschmidt, S. Brucker, and G. Fuchs, Eur. J. Biochem. 227:161-168, 1995), but gentisate 1,2-dioxygenase was induced. Similarly, we found that the Comamonas testosteroni strain CNB-1 degraded benzoate via a CoA-dependent epoxide pathway and that gentisate 1,2-dioxygenase (GenA) was also induced when benzoate or 3-hydroxybenzoate served as a carbon source for growth. Genes encoding the CoA-dependent epoxide (box genes) and gentisate (gen genes) pathways were identified. Genetic disruption revealed that the gen genes were not involved in benzoate and 3-hydroxybenzoate degradation. Hence, we investigated gen gene regulation in the CNB-1 strain. The P genA promoter, a MarR-type regulator (GenR), and the GenR binding site were identified. We found that GenR took gentisate, 3-hydroxybenzoate, and benzoyl-CoA as effectors and that binding of GenR to its target DNA sequence was prohibited when these effectors were present. In vivo studies showed that the CNB-1 mutant that lost benzoyl-CoA synthesis was not able to activate P genA promoter, while transcription of genA was upregulated in another CNB-1 mutant that lost the ability to degrade benzoyl-CoA. The finding that benzoyl-CoA (a metabolic intermediate of benzoate degradation) and 3-hydroxybenzoate function as GenR effectors explains why GenA was induced when CNB-1 grew on benzoate or 3-hydroxybenzoate. Regulation of gentisate pathways by MarR-, LysR-, and IclR-type regulators in diverse bacterial groups is discussed in detail.

Hierarchy of Carbon Source Utilization in Soil Bacteria: Hegemonic Preference for Benzoate in Complex Aromatic Compound Mixtures Degraded by Cupriavidus pinatubonensis Strain JMP134

“…Only the ben/cat pathway is active under the conditions used in this work, since the C. pinatubonensis ⌬benA mutant does not grow on Bz and accumulates Bz (24). This dismisses possibilities such as benzoyl-CoA-type intermediates (generated during Box pathway operation) repressing the expression of genes involved in the degradation of some of the other AC tested here, which has been demonstrated for Box intermediates controlling the degradation of gentisate in Comamonas testosteroni (51).…”

Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

“…To investigate if our system can be used to analyze chemotactic responses of indigenous microbial species in a non-invasive and label-free manner, non-labelled Comamonas testosteroni CNB-1 was selected and tested with 4-hydroxybenzoic acid ( p -HBA) as its chemo-attractant. CNB-1 was isolated from sewage sludge of a wastewater-treatment plant from a chemical factory producing chloronitrobenzene 36 . Previous studies using swarm plate assays showed that CNB-1 exhibited strong chemotactic responses toward various aromatic compounds, including p -HBA 37 .…”

“…E. coli strains were cultivated at 37 °C in Luria Bertani (LB) medium with 100 μg/mL ampicillin. CNB-1 was cultivated at 30 °C in mineral salt broth (MSB) as previously described 36 . All the cells in the seed culture were harvested at the exponential phase, centrifuged and suspended in 1 × PBS containing 0.1 mmol/L EDTA as the pre-sample.…”

Unraveling the Specific Regulation of the Central Pathway for Anaerobic Degradation of 3-Methylbenzoate

“…There is an increasing number of regulators, i.e. PaaR ( 63 ) (TetR family), CouR, FerC, HcaR, FerR, and GenR (MarR family) ( 27 , 64 – 67 ), PaaX (GntR family) ( 38 ), and BzdR and BoxR(XRE family) ( 17 , 20 ), that control aromatic degradation pathways and recognize aromatic CoA thioesters as inducers. Thus, FerR/FerC recognize feruloyl-CoA; CouR/HcaR recognize p -coumaroyl-CoA; BzdR/BoxR/GenR recognize benzoyl-CoA; and PaaX/PaaR recognize phenylacetyl-CoA.…”