MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Xu, Ying; Gao, Xiaoli; Wang, Songhe; Liu, Hong; Williams, Peter; Zhou, Ning‐Yi

doi:10.1128/aem.01511-12

Cited by 27 publications

(24 citation statements)

References 48 publications

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“…S6 in the supplemental material). At low pH, GRA most likely exists in the form of undissociated molecules and is transported through the membrane by passive diffusion, similar to other aromatic acids (24,(26)(27)(28). This feature might underlie the growth of the tsdT mutant on GRA at low pH.…”

Section: Discussionmentioning

confidence: 99%

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γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

Kasai

Araki

Motoi

et al. 2015

Appl Environ Microbiol

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The Rhodococcus jostii RHA1 gene cluster required for ␥-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.

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Section: Discussionmentioning

confidence: 99%

“…S6 in the supplemental material). Aromatic acids in their undissociated form enter cells by passive diffusion at lower pH (24,(26)(27)(28); therefore, the growth of DTT cells was examined at low pH. When the pH of the medium was adjusted to 6.0 or 5.5, the growth retardation of DTT was partially suppressed.…”

Section: Identification Of Gra Catabolism Genesmentioning

confidence: 99%

γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

Kasai

Araki

Motoi

et al. 2015

Appl Environ Microbiol

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“…Accumulating evidence indicates that the active transport of aromatic acids is widespread among bacteria (10,13,15,16,(32)(33)(34). In addition to E. coli K-12 (7), Rhodococcus globerulus PWD1 (4), Comamonas testosteroni TA441 (5), and Cupriavidus necator JMP134 (6) were also reported to be able to grow on 3HPP.…”

Section: Discussionmentioning

confidence: 99%

“…Among the various categories of transporters classified in the Transporter Classification Database (TCDB; http://www.tcdb.org/) (12), the functionally identified aromatic acid transporters (13)(14)(15)(16)(17)(18) belong to the aromatic acid/H ϩ symporter (AAHS) family (cluster 2.A.1.15 in TCDB), except for the 4-hydroxyphenylacetate transporter HpaX in E. coli W (18). In this study, we report that MhpT, which shares 27 to 37% identities with other AAHS transporters, is specifically involved in 3HPP catabolism of E. coli K-12 and transports 14 C-labeled 3HPP but not 14 C-labeled benzoate, 3-hydroxybenzoate, or gentisate into cells.…”

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mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Chen

Chao

et al. 2013

Appl Environ Microbiol

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Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with 14 C-labeled substrates demonstrated that strain W3110 and strain W3110⌬mhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110⌬mhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions. Microbes play a major role in the degradation of phenylpropanoid compounds, which result largely from the degradation of lignin and other aromatic constituents of plants (1, 2). Among these phenylpropanoid compounds, phenylpropionate and its hydroxylated derivatives have been reported to be degraded by various bacterial strains (3-7). In particular, a number of Escherichia coli strains (strains B, C, W, K-12, and NCTC 5928) were shown to be able to utilize 3-(3-hydroxyphenyl)propionate (3HPP), a major member of the phenylpropanoids, as their sole carbon source (7). A 9.8-kb mhp cluster conferred the ability to grow on 3HPP to E. coli MC4100, Salmonella enterica serovar Typhimurium LT-2, Pseudomonas putida KT2442, and Rhizobium meliloti Rm1021Rif, suggesting that this cluster contained all of the catabolic genes necessary for the catabolism of this aromatic acid into Krebs cycle intermediates and also the regulatory element of the pathway (8). The expression of mhp catabolic genes was shown to be induced by 3HPP (9). In addition to the genes mhpRABCDFE encoding the regulatory function and 3HPP catabolism (8, 9), mhpT (formerly orfT) was tentatively proposed to encode a potential transporter (8). Nevertheless, the function of MhpT has so far not been identified genetically or biochemically. As a matter of fact, no report on the involvement of any transporter in the microbial degradation of phenylpropionate and its hydroxylated derivatives can be found in the literature.The robust ability of microorganisms to grow on a variety of aromatic compounds partially relies on multiple transporters for their uptake into cells (10, 11). Among the various categories of transporters classified in the Transpor...

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“…strain U2 and Polaromonas naphthalenivorans CJ2 are all organized in a single operon, and a LysR-type transcriptional regulator NagR, divergently transcribed from its structural genes, activates the expression of nag catabolic genes (including those for gentisate catabolism) in the presence of the inducer salicylate (18,19). The degradation of 3-HBA in Klebsiella pneumoniae M5a1 is accomplished by enzymes encoded by the mhbTDHIM operon, which is positively regulated by a LysR-type regulator, MhbR (20,21).…”

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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

Chao

Zhou

2013

J Bacteriol

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bThe genes required for 3-hydroxybenzoate and gentisate catabolism in Corynebacterium glutamicum are closely clustered in three operons. GenR, an IclR-type regulator, can activate the transcription of genKH and genDFM operons in response to 3-hydroxybenzoate and gentisate, and it can repress its own expression. Footprinting analyses demonstrated that GenR bound to four sites with different affinities. Two GenR-binding sites (DFMn01 and DFMn02) were found to be located between positions ؊41 and ؊84 upstream of the ؊35 and ؊10 regions of the genDFM promoter, which was involved in positive regulation of genDFM transcription. The GenR binding site R-KHn01 (located between positions ؊47 and ؊16) overlapped the ؊35 region of the genKH promoter sequence and is involved in positive regulation of its transcription. The binding site R-KHn02, at which GenR binds to its own promoter, was found within a footprint extending from position ؊44 to ؊67. It appeared to be involved in negative regulation of the activity of the genR promoter. A consensus motif with a 5-bp imperfect palindromic sequence [ATTCC-N 7(5) -GGAAT] was identified among all four GenR binding sites and found to be necessary to GenR regulation through site-directed mutagenesis. The results reveal a new regulatory function of the IclR family in the catabolism of aromatic compounds.

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MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Abstract: ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. Four of the six gene… Show more

Cited by 27 publications

References 48 publications

γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

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

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