GenR, an IclR-Type Regulator, Activates and Represses the Transcription of<i>gen</i>Genes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum

Chao, Han-Chieh; Zhou, Ning‐Yi

doi:10.1128/jb.02216-12

Cited by 28 publications

(25 citation statements)

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“…Total RNA from strain RKJ300 was isolated using the hot phenol method (19) and reverse transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). Reverse transcription (RT)-PCR was carried out with the primers listed in Table 2.…”

Section: Methodsmentioning

confidence: 99%

A Two-Component para -Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

Min

Zhang

Zhou

2016

Appl Environ Microbiol

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bRhodococcus imtechensis RKJ300 (DSM 45091) grows on 2-chloro-4-nitrophenol (2C4NP) and para-nitrophenol (PNP) as the sole carbon and nitrogen sources. In this study, by genetic and biochemical analyses, a novel 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with hydroxyquinol (hydroxy-1,4-hydroquinone or 1,2,4-benzenetriol [BT]) as the ring cleavage substrate. Real-time quantitative PCR analysis indicated that the pnp cluster located in three operons is likely involved in the catabolism of both 2C4NP and PNP. The oxygenase component (PnpA1) and reductase component (PnpA2) of the two-component PNP monooxygenase were expressed and purified to homogeneity, respectively. The identification of chlorohydroquinone (CHQ) and BT during 2C4NP degradation catalyzed by PnpA1A2 indicated that PnpA1A2 catalyzes the sequential denitration and dechlorination of 2C4NP to BT and catalyzes the conversion of PNP to BT. Genetic analyses revealed that pnpA1 plays an essential role in both 2C4NP and PNP degradations by gene knockout and complementation. In addition to catalyzing the oxidation of CHQ to BT, PnpA1A2 was also found to be able to catalyze the hydroxylation of hydroquinone (HQ) to BT, revealing the probable fate of HQ that remains unclear in PNP catabolism by Gram-positive bacteria. This study fills a gap in our knowledge of the 2C4NP degradation mechanism in Gram-positive bacteria and also enhances our understanding of the genetic and biochemical diversity of 2C4NP catabolism.C hloronitrophenols, such as 2-chloro-4-nitrophenol (2C4NP), 4-chloro-2-nitrophenol (4C2NP), and 2-chloro-5-nitrophenol (2C5NP), with high toxicity to human beings and animals have been widely used in the pharmaceutical, agricultural, and chemical industries (1). The natural formation of chloronitrophenols is rare, and most of these compounds in the environment result from anthropogenic activity. Apparently, the introduction of chloronitrophenols into the environment has selected microorganisms to develop the ability to degrade these compounds. So far, several strains able to degrade chloronitrophenols have been isolated, including the 2C4NP-degrading strains Burkholderia sp. strain SJ98 (2), Burkholderia sp. strain RKJ800 (3), Rhodococcus imtechensis RKJ300 (4), and Arthrobacter sp. strain SJCon (5), the 4C2NP-degrading strain Exiguobacterium sp. strain PMA (6), and the 2C5NP-degrading strain Ralstonia eutropha JMP134 (7).Structurally, chloronitrophenols are chemical analogs of nitrophenols. The microbial degradation of nitrophenols has been extensively investigated at genetic and biochemical levels (8-13). In contrast to nitrophenols, chloronitrophenols are more resistant to microbial degradation due to the simultaneous existence of electron-withdrawing chloro and nitro groups, and the knowledge of their microbial degradation is thus very limited. Previously, the partially purified enzymes involved in meta-nitrophenol catabolism were reported to be able to catalyze 2C5NP transformation in Ralstonia eutroph...

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Section: Methodsmentioning

confidence: 99%

A Two-Component para -Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

Min

Zhang

Zhou

2016

Appl Environ Microbiol

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“…Recently, C. glutamicum ATCC 13032 has been found to utilize a number of aromatic compounds as its sole carbon and energy source, including 3-HBA and GEN (22). Its catabolic pathway of 3-HBA degradation via GEN was catalyzed by genD-, genF-, genM-, and genHencoded enzymes (1,(23)(24)(25). In addition to these catabolic genes, GenK was identified to actively transport gentisate to facilitate GEN utilization (23).…”

Section: T He Gentisate (25-dihydroxybenzoate [Gen]mentioning

confidence: 99%

“…In addition to these catabolic genes, GenK was identified to actively transport gentisate to facilitate GEN utilization (23). More recently, IclR-type regulator GenR has been characterized to be a dual-function protein that acts as an activator of the transcription of genDFM and genKH operons for 3-HBA and GEN catabolism in response to 3-HBA and GEN and as a repressor of its own expression (25).…”

Section: T He Gentisate (25-dihydroxybenzoate [Gen]mentioning

confidence: 99%

“…The GlxR binding sites DFMx, R-KHx01, and R-KHx02 were mutated using the In-Fusion Advantage PCR cloning method with the templates of pZWHJ006, pZWHJ007, and pZWHJ008, respectively, as described previously (25). To mutate site DFMx, 5-bp substitutions (GTGN 8 TTC to CACN 8 GTG) were generated using the mutagenic primers pDFMxmut01 and pDFMxmut02 to generate pZWHJ025.…”

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confidence: 99%

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Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

Chao

Zhou

2014

Appl Environ Microbiol

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Corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of genDFM and genKH operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three gen operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located ؊13 to ؉8 bp upstream of the genDFM promoter, which was involved in negative regulation of genDFM transcription. The GlxR binding site R-KHx01 (located between positions ؊11 to ؉5) was upstream of the genKH promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to genR promoter to repress its expression, was found within a footprint extending from positions ؊71 to ؊91 bp. These results reveal that GlxR represses the transcription of all three gen operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.

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“…Unlike the LysR family of transcriptional regulators that possess a conserved operator sequence motif (TN 11 A; Maddocks & Oyston, 2008), operator sequences of genes regulated by the IclR family proteins are more variable in their base composition and secondary structures, which may include palindromes, pseudopalindromes and direct repeats. Moreover, the regulatory mechanisms through which IclR proteins regulate transcription are also diverse, as they act as either repressors or activators, and some family members possess dual functions including autoregulation (Chao & Zhou, 2013). It is therefore challenging to predict the operator sequences, as well as regulatory mechanisms, important for the function of uncharacterized proteins in the IclR family of transcriptional regulators.…”

Section: Introductionmentioning

confidence: 99%

Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Shimizu

Nakamura

2014

Microbiology

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Five genes encoding enzymes required for L-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of L-gluconate or D-idonate, a catabolite of L-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. D-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the L-gluconate catabolic pathway and Dgalactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

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GenR, an IclR-Type Regulator, Activates and Represses the Transcription ofgenGenes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum

Cited by 28 publications

References 50 publications

A Two-Component para -Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

A Two-Component para -Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

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