RESUMEN
Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: ⢠Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. ⢠Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. ⢠A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.
Asunto(s)
Dihidropteroato Sintasa , Sulfametoxazol , Sulfametoxazol/metabolismo , Dihidropteroato Sintasa/genética , Ecosistema , Antibacterianos/metabolismo , Sulfonamidas/metabolismo , Sulfanilamida , Biodegradación Ambiental , CarbonoRESUMEN
Several natural anaerobic fungus-methanogen co-cultures have been isolated from rumen and feces source of herbivores with strong fiber degrading ability. In this study, we isolated 7 Neocallimastix with methanogen co-cultures from the rumen of yaks grazing on the Qinghai Tibetan Plateau. Based on morphological characteristics and internal transcribed spacer 1 sequences (ITS1), all the fungi were identified as Neocallimastix frontalis. The co-cultures were confirmed as the one fungus - one methanogen pattern by the PCR-denatured gradient gel electrophoresis (DGGE) assay. All the methanogens were identified as Methanobrevibacter ruminantium by 16s rRNA gene sequencing. We investigated the biodegrading capacity of the co-culture (N. frontalis + M. ruminantium) Yaktz1 on wheat straw, corn stalk and rice straw in a 7 days-incubation. The in vitro dry matter digestibility (IVDMD), acid detergent fiber digestibility (ADFD) and neural detergent fiber digestibility (NDFD) values of the substrates in the co-culture were significantly higher than those in the mono-culture N. frontalis Yaktz1. The co-culture exhibited high polysaccharide hydrolase (xylanase and FPase) and esterase activities. The xylanase in the co-culture reached the highest activity of 12500 mU/ml on wheat straw at the day 3 of the incubation. At the end of the incubation, 3.00 mmol-3.29 mmol/g dry matter of methane were produced by the co-culture. The co-culture also produced high level of acetate (40.00 mM-45.98 mM) as the end-product during the biodegradation. Interestingly, the N. frontalis Yaktz1 mono-culture produced large amount of lactate (8.27 mM-11.60 mM) and ethanol (163.11 mM-242.14 mM), many times more than those recorded in the previously reported anaerobic fungi. Our data suggests that the (N. frontalis + M. ruminantium) Yaktz1 co-culture and the N. frontalis Yaktz1 mono-culture both have great potentials for different industrial use.
Asunto(s)
Fibras de la Dieta/metabolismo , Microbioma Gastrointestinal/fisiología , Methanobrevibacter/metabolismo , Neocallimastix/metabolismo , ARN Ribosómico 16S/genética , Rumen/microbiología , Ácido Acético/metabolismo , Anaerobiosis , Animales , Bovinos , Técnicas de Cocultivo , Endo-1,4-beta Xilanasas/metabolismo , Esterasas/metabolismo , Etanol/metabolismo , Ácido Láctico/metabolismo , Methanobrevibacter/genética , Methanobrevibacter/aislamiento & purificación , Neocallimastix/genética , Neocallimastix/aislamiento & purificación , Poaceae/metabolismo , Análisis de Secuencia de ADNRESUMEN
Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), Cd(2+) (0.01 mM), Mn(2+) (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals.
Asunto(s)
Corynebacterium glutamicum/fisiología , Cisteína/metabolismo , Expresión Génica , Glicopéptidos/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Inositol/metabolismo , Estrés Fisiológico , Corynebacterium glutamicum/efectos de los fármacos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Viabilidad Microbiana/efectos de los fármacosRESUMEN
NrdH redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. Although NrdH redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. In this study, we confirmed that the Corynebacterium glutamicum NrdH redoxin catalytically reduces the disulfides in the class Ib ribonucleotide reductases (RNR), insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), by exclusively receiving electrons from thioredoxin reductase. Overexpression of NrdH increased the resistance of C. glutamicum to multiple oxidative stresses by reducing ROS accumulation. Accordingly, elevated expression of the nrdH gene was observed when the C. glutamicum wild-type strain was exposed to oxidative stress conditions. It was discovered that the NrdH-mediated resistance to oxidative stresses was largely dependent on the presence of the thiol peroxidase Prx, as the increased resistance to oxidative stresses mediated by overexpression of NrdH was largely abrogated in the prx mutant. Furthermore, we showed that NrdH facilitated the hydroperoxide reduction activity of Prx by directly targeting and serving as its electron donor. Thus, we present evidence that the NrdH redoxin can protect against the damaging effects of reactive oxygen species (ROS) induced by various exogenous oxidative stresses by acting as a peroxidase cofactor.
Asunto(s)
Coenzimas/metabolismo , Corynebacterium glutamicum/enzimología , Corynebacterium glutamicum/fisiología , Estrés Oxidativo , Peroxidasa/metabolismo , Tiorredoxinas/metabolismo , Coenzimas/genética , Corynebacterium glutamicum/efectos de los fármacos , Corynebacterium glutamicum/metabolismo , Perfilación de la Expresión Génica , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Tiorredoxinas/genéticaRESUMEN
Mycothiol (MSH) plays important roles in maintaining cytosolic redox homeostasis and in adapting to reactive oxygen species in the high-(G + C)-content Gram-positive Actinobacteria. However, its physiological roles are ill defined compared to glutathione, the functional analog of MSH in Gram-negative bacteria and most eukaryotes. In this research, we explored the impact of intracellular MSH on cellular physiology by using MSH-deficient mutants in the model organism Corynebacterium glutamicum. We found that intracellular MSH contributes significantly to resistance to alkylating agents, glyphosate, ethanol, antibiotics, heavy metals and aromatic compounds. In addition, intracellular MSH is beneficial for withstanding oxidative stress induced by various oxidants in C. glutamicum. This study greatly expanded our current knowledge on the physiological functions of mycothiol in C. glutamicum and could be applied to improve the robustness of this scientifically and commercially important species in the future.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Cisteína/metabolismo , Glicopéptidos/metabolismo , Microbiología Industrial , Inositol/metabolismo , Corynebacterium glutamicum/efectos de los fármacos , Cisteína/biosíntesis , Etanol/farmacología , Glicopéptidos/biosíntesis , Inactivación Metabólica , Inositol/biosíntesis , Metales Pesados/farmacología , Naftalenos/metabolismo , Naftalenos/farmacología , Oxidación-Reducción , Resorcinoles/metabolismo , Resorcinoles/farmacologíaRESUMEN
With the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by Corynebacterium glutamicum has been emerging. This review summarizes recent findings on degradation of aromatic compounds by C. glutamicum. Among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in C. glutamicum. Other important knowledge derived from C. glutamicum would be the discovery of linkages among aromatic degradation and primary metabolisms such as gluconeogenesis and central carbon metabolism. Various transporters in C. glutamicum have also been identified, and they play an essential role in microbial assimilation of aromatic compounds. Regulation on aromatic degradation occurs mainly at transcription level via pathway-specific regulators, but global regulator(s) is presumably involved in the regulation. It is concluded that C. glutamicum is a very useful model organism to disclose new knowledge of biochemistry, physiology, and genetics of the catabolism of aromatic compounds in high GC content Gram-positive bacteria, and that the new physiological properties of aromatic degradation and assimilation are potentially important for industrial applications of C. glutamicum.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrocarburos Aromáticos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biotecnología/métodos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/fisiología , Gentisatos/química , Gentisatos/metabolismo , Hidrocarburos Aromáticos/químicaRESUMEN
This study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected patients in Hubei, China, from 2004 to 2006, all of whom had received anti-HIV-1 therapy. The presence of NRTI- and NNRTI-associated mutations were established by sequencing; genotypic and predicted phenotypic drug resistance were evaluated using HIVdb Program version 5.0.1 (http://hivdb.stanford.edu/pages/algs/HIVdb.html ). Genotypic drug resistance analysis showed significant increases in percentages of patients carrying HIV-1 strains with M41L, T215Y/F, D67N, K103N, G190A/S, Y181C/F or L210W mutations. Of the variants' predicted phenotypic drug resistance, highly significant increases were detected in percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs, and to delavirdine (DLV), efavirenz (EFV) or nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC), AZT, D4T, didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs, and to etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine (3TC), ABC, emtricitabine (FTC) or TDF in NRTIs, and to ETR in NNRTIs.
Asunto(s)
Donantes de Sangre/estadística & datos numéricos , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Vigilancia de Guardia , Adulto , China , Femenino , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
Rhesus monkey TRIM5α (TRIM5α(rh)), a member of the tripartite motif (TRIM) family, was identified as the main restriction factor responsible for resistance of old world monkey cells to HIV-1 infection. However, the precise mechanism of HIV-1 infection inhibition by TRIM5α remains elusive and appears to be related to some cellular cofactors. Here we reported that TRIM5α(rh) can significantly reduce the infection efficiency of VSV-G pseudotyped HIV-1/MA-YFP virus in human epithelial carcinoma (HeLa) cells, moderately reduce in porcine kidney (PK-15) cells and have no effect on the pseudotyped virus infection in Madin-Darby canine kidney (MDCK) cells. Furthermore, we found that the different HIV-1 restriction activities have no relation with the intracellular localization of TRIM5α(rh). These results indicate that the cellular environment is very important for the efficient anti-HIV-1 activity of TRIM5α(rh). We speculate that some unknown factors required for HIV-1 infection inhibition activity are adequately expressed in HeLa cells, inadequately expressed in PK-15 cells and absent in MDCK cells.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Proteínas/inmunología , Secuencia de Aminoácidos , Animales , Callithrix , Clonación Molecular , Perros , Variación Genética , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , Humanos , Macaca mulatta , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas/genética , Especificidad de la Especie , Transducción Genética , Ubiquitina-Proteína LigasasRESUMEN
TRIM5α has been identified as the main restriction factor responsible for resistance of Old World monkey cells to HIV-1 infection. The precise mechanism of viral inhibition by TRIM5α remains elusive but appears to occur in multiple ways. Here, we report that rhesus monkey TRIM5α (TRIM5α(rh)) can represses HIV-1 LTR promoter activity by negatively regulating TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation when TRIM5α(rh) is overexpressed. We show that the overexpressed TRIM5α(rh) can interact with the TAK1/TAB1/TAB2/TAB3 complex by binding to TAB1 and promotes the degradation of TAB2 within the complex via the lysosomal degradation pathway. Subsequently, TRIM5α(rh) lowers the IKKα protein level and inhibits NF-κB p65 phosphorylation, and knockdown of TRIM5α(rh) expression by small interfering RNA in TRIM5α(rh)-overexpressing cells can abolish this inhibition. Finally, the inhibition of p65 phosphorylation results in the repression of HIV-1 LTR promoter activity. Taken together, these findings indicate that TRIM5α(rh) plays a previously unrecognized role in repressing HIV-1 transcription by inhibiting TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation when TRIM5α(rh) is overexpressed.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación hacia Abajo , Infecciones por VIH/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/genética , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Quinasas Quinasa Quinasa PAM/genética , Macaca mulatta , FN-kappa B/genética , Fosforilación , Unión Proteica , Transducción de SeñalRESUMEN
The human immunodeficiency virus type 1 (HIV-1) can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase (MAPK) signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase (ERK) pathway inhibitor, PD98059, the Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus and HIV-1(NL4-3) virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus inhibition activity but no HIV-1(NL4-3) virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.
Asunto(s)
Regulación hacia Abajo , Infecciones por VIH/enzimología , VIH-1/fisiología , Sistema de Señalización de MAP Quinasas , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA ( Cg ) (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA (Cg) was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA(Cg). The pobA (Cg) is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.
Asunto(s)
Corynebacterium glutamicum/enzimología , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Parabenos/metabolismo , Secuencia de Bases , Sitios de Unión , Corynebacterium glutamicum/genética , Dimerización , Electroforesis en Gel de Poliacrilamida , Orden Génico , Genes Bacterianos , Hidroxibenzoatos/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/metabolismo , NADP/metabolismo , Operón , Unión Proteica , Especificidad por SustratoRESUMEN
Genome-wide data mining indicated that six genes (ncgl1031, ncgl2302, ncgl2325, ncgl2326, ncgl2922 and ncgl2953) encoding putative transport proteins are involved in uptake of various aromatic compounds that are further degraded through the beta-ketoadipate, gentisate and resorcinol pathways in Corynebacterium glutamicum. The gentisate (GenK/NCgl2922) and vanillate (VanK/NCgl2302) transporters have been identified previously. In this study, physiological functions of the remaining four putative transporters as well as the vanillate transporter (VanK/NCgl2302) were examined by genetic disruption/complementation and uptake assays. Results indicated that ncgl1031 encodes PcaK for 4-hydroxybenzoate and protocatechuate transport, and ncgl2302 encodes VanK for vanillate transport. Genetic studies and uptake assays indicated that both ncgl2325/benK and ncgl2326/benE are involved in benzoate transport in C. glutamicum. When growth rates were compared for two benzoate transporter mutants, benK and benE, a high growth rate was observed for the benE mutant. Sequence alignments revealed that PcaK, VanK, BenK and GenK belong to the major facilitator superfamily (MFS). Modelling of secondary structures based on previously characterized MFS members revealed that NCgl1031, NCgl2302, NCgl2325 and NCgl2922 are typical 12 helix transmembrane proteins but NCgl2326 contains only 11 alpha-helices. Thus the functionally identified NCgl2326 belongs to a novel type of benzoate transporters. Attempts to identify the phenotype of a hydK/ncgl2953 mutant failed, so the function of ncgl2953 remains unclear.
Asunto(s)
Proteínas Portadoras/genética , Corynebacterium glutamicum/metabolismo , Genoma Bacteriano/genética , Hidrocarburos Aromáticos/metabolismo , Benzoatos/metabolismo , Proteínas Portadoras/fisiología , Corynebacterium glutamicum/genética , Eliminación de Gen , Orden Génico , Prueba de Complementación Genética , Hidroxibenzoatos/metabolismo , Parabenos/metabolismo , Filogenia , Homología de Secuencia de Aminoácido , Ácido Vanílico/metabolismoRESUMEN
Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Regulación Bacteriana de la Expresión Génica , Resorcinoles/metabolismo , Corynebacterium glutamicum/enzimología , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Familia de Multigenes , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismoRESUMEN
Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Disacáridos/química , Regulación Bacteriana de la Expresión Génica , Gentisatos/metabolismo , Pirazoles/química , Compuestos de Sulfhidrilo/química , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Carbono/química , Carbono/metabolismo , Cromatografía Líquida de Alta Presión , Cisteína , Cartilla de ADN/química , Disacáridos/biosíntesis , Disacáridos/metabolismo , Escherichia coli/metabolismo , Eliminación de Gen , Genes Bacterianos , Glicopéptidos , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Inositol , Iones , Cinética , Espectrometría de Masas , Modelos Químicos , Datos de Secuencia Molecular , Peso Molecular , Mutación , Fenotipo , Ácidos Pimélicos/metabolismo , Plásmidos/metabolismo , Pirazoles/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Compuestos de Sulfhidrilo/metabolismo , Factores de TiempoRESUMEN
Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Gentisatos/metabolismo , Glutatión/metabolismo , Clonación Molecular , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrolasas/genética , Hidrolasas/metabolismo , Hidroxibenzoatos/metabolismo , Familia de Multigenes , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismoRESUMEN
Corynebacterium glutamicum assimilated phenol, benzoate, 4-hydroxybenzoate p-cresol and 3,4-dihydroxybenzoate. Ring cleavage was by catechol 1,2-dioxygenase when phenol or benzoate was used and by protocatechuate 3,4-dioxygenase when the others were used as substrate. The locus ncg12319 of its genome was cloned and expressed in Escherichia coli. Enzyme assays showed that ncg12319 encodes a catechol 1,2-dioxygenase. This catechol 1,2-dioxygenase was purified and accepted catechol, 3-, or 4-methylcatechols, but not chlorinated catechols, as substrates. The optimal temperature and pH for catechol cleavage catalyzed by the enzyme were 30 degrees C and 9, respectively, and the Km and Vmax were determined to be 4.24 micromol l(-1) and 3.7 micromol l(-1) min(-1) mg(-1) protein, respectively.