Reversible accumulation of p-hydroxybenzoate and catechol determines the sequential decomposition of phenolic compounds in mixed substrate cultivations in pseudomonads

cCupriavidus pinatubonensis JMP134, like many other environmental bacteria, uses a range of aromatic compounds as carbon sources. Previous reports have shown a preference for benzoate when this bacterium grows on binary mixtures composed of this aromatic compound and 4-hydroxybenzoate or phenol. However, this observation has not been extended to other aromatic mixtures resembling a more archetypal context. We carried out a systematic study on the substrate preference of C. pinatubonensis JMP134 growing on representative aromatic compounds channeled through different catabolic pathways described in aerobic bacteria. Growth tests of nearly the entire set of binary combinations and in mixtures composed of 5 or 6 aromatic components showed that benzoate and phenol were always the preferred and deferred growth substrates, respectively. This pattern was supported by kinetic analyses that showed shorter times to initiate consumption of benzoate in aromatic compound mixtures. Gene expression analysis by real-time reverse transcription-PCR (RT-PCR) showed that, in all mixtures, the repression by benzoate over other catabolic pathways was exerted mainly at the transcriptional level. Additionally, inhibition of benzoate catabolism suggests that its multiple repressive actions are not mediated by a sole mechanism, as suggested by dissimilar requirements of benzoate degradation for effective repression in different aromatic compound mixtures. The hegemonic preference for benzoate over multiple aromatic carbon sources is not explained on the basis of growth rate and/or biomass yield on each single substrate or by obvious chemical or metabolic properties of these aromatic compounds.A romatic compounds (AC) are widespread in the environment, displaying a heterogeneous structural diversity. They can be naturally originated by biotic and abiotic processes or released as pollutants into the environment. AC primarily can be found as aromatic amino acids, secondary products abundantly generated by plants, structural components of the very complex lignin heteropolymer in woody plants, and xenobiotic compounds: biocides, industrial by-products, and petroleum derivatives, among others. Microorganisms may degrade hundreds of different AC using specialized biochemical pathways that allow them to grow on these carbon sources (1-3). Typically, bacteria deal with AC as part of complex mixtures in naturally occurring organic compounds, such as those found in plant exudates (4), in soils (5), and even in dissolved organic matter from freshwater and seawater (6). Therefore, microorganisms are concurrently exposed to several structurally heterogeneous AC as potential substrates, which raises the question of whether the components of these mixtures are used simultaneously or in a sequential manner. In the case of the sequential utilization pattern, characterized by diauxic growth, one compound inhibits degradation of the other by exerting metabolite toxicity (7), competitive inhibition of enzymes (8, 9), depletion of electron acceptors (10, 11),...

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confidence: 99%

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

Pérez‐Pantoja

Leiva-Novoa

Donoso

et al. 2015

“…The incubation of enzyme extract with 2C4NBA in an 18 O 2 -enriched atmosphere yielded a product whose retention time and mass spectrum were identical to those of 2H4NBA. However, when enzyme extract was incubated with H 2 18 O and 2C4NBA, the product yielded had the same retention time as 2H4NBA but a mass spectrum with major peaks that were 2 mass units larger than those of untreated 2H4NBA, which is consistent with the incorporation of one atom of 18 O into 2H4NBA, indicating that mono-oxygenase catalyzed the initial hydrolytic dechlorination reaction (6,36,52,53). Likewise, the incubation of enzyme extract with 2H4NBA in an atmosphere of 18 O 2 or H 2 18 O separately yielded a product with molecular ion peaks and fragments 2 mass units larger than those of untreated 2H4NBA, which is consistent with the incorporation of one atom of 18 O into 2,4-DHBA under an atmosphere enriched with 18 O 2 but not under an atmosphere enriched with H 2 18 O, suggesting that the denitration was an oxygenolytic reaction (23,52,53).…”

Section: Resultsmentioning

confidence: 58%

“…The 2C4NBA-grown whole-cell lysate of RKJ12 was incubated with catechol. The development of redbrown color with the formation of cis,cis-muconate in the reaction mixture suggested the mode of ortho ring cleavage in the 2C4NBA degradation pathway (18).…”

Section: Resultsmentioning

confidence: 99%

Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12

Prakash

Kumar

Jain

et al. 2011

The organism Acinetobacter sp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidative ortho dehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O 2 per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H 2 18 O and 18 O 2 indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by the ortho ring cleavage catechol-1,2-dioxygenase to cis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA؊ derivative and a 2C4NBA ؉ transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ϳ55-kb transmissible plasmid present in RKJ12.

“…This type of ordered catabolism has been noted for strains of Acinetobacter, Pseudomonas, and Ralstonia (1,15,19,25). While reminiscent of catabolite repression, which has been well studied in some enteric and gram-positive bacteria (32,35), the method by which the gram-negative bacterium Acinetobacter sp.…”

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confidence: 90%

“…1) (18). However, in P. fluorescens strains PC24 and PC18, which also degrade benzoate and 4HB through the ␤-ketoadipate pathway, benzoate is not the preferred carbon source (19). Strain PC24 consumes both substrates simultaneously, while PC18 degrades 4HB in preference to benzoate.…”

mentioning

confidence: 99%

Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the β-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the Acinetobacter sp. Strain ADP1 pobA Gene

Brzostowicz

Reams

Clark

et al. 2003

Transcriptional control of carbon source preferences by Acinetobacter sp. strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates. The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression. In each case, pobA expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the ␤-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate. These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the ␤-ketoadipate pathway.