Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Park, Minjeong,Jeon, Yeji,Jang, Ho Hee,Ro, Hyun-Su,Park, Woojun,Madsen, Eugene L.,Jeon, Che Ok American Society for Microbiology 2007 Applied and environmental microbiology Vol.73 No.16

<B>ABSTRACT</B><P>Prior research revealed that <I>Polaromonas naphthalenivorans</I> CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising <I>nagRAaGHAbAcAdBFCQEDJI</I>′-<I>orf1</I>-<I>tnpA</I> and <I>nagR2</I>-<I>orf2I</I>″<I>KL</I> (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the <I>orf2</I> gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that <I>orf2</I> does not encode salicylate 5-hydroxylase. Instead, we have found that <I>orf2</I> encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed <I>orf2 nagX</I>. After expression in <I>Escherichia coli</I>, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of <I>P. naphthalenivorans</I> CJ2 defective in <I>nagX</I> failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because <I>nagX</I> and an adjacent MarR-type regulatory gene are both closely related to homologues in <I>Azoarcus</I> species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.</P>

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

Lee, Hyo Jung,Kim, Jeong Myeong,Lee, Se Hee,Park, Minjeong,Lee, Kangseok,Madsen, Eugene L.,Jeon, Che Ok Microbiology Society 2011 Microbiology Vol.157 No.10

<P>Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2δnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36-39 kDa, and their structures were deduced to be dimeric. The K(m) values of NagI2 and NagI3 were 31 and 10 ?M, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.</P>

오염지역에서 stable isotope probing을 이용한 나프탈렌 분해 미생물의 분포 분석

전체옥,Eugene L. Madsen 대한환경공학회 2004 대한환경공학회 학술발표논문집 Vol.2004 No.12

Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Yeom, Jinki,Jeon, Che Ok,Madsen, Eugene L.,Park, Woojun American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.5

<B>ABSTRACT</B><P><I>Pseudomonas putida</I> harbors two ferredoxin-NADP<SUP>+</SUP> reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of <I>P. putida</I>. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher <I>k</I>cat/<I>Km</I> value for reducing the ferric complex with free FMN. The growth rate of the <I>fprB</I> mutant was reduced more profoundly than that of the <I>fprA</I> mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the <I>fprB</I> mutant plus NADH were utilized, but not the <I>fprA</I> mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of <I>Escherichia coli</I> in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in <I>P. putida</I> as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.</P>

Microbiology : Protection of Polaromonas naphthalenivorans CJ2 from Naphthalene Toxicity by Extracellular Polysaccharide Capsules

Min Jeong Prak,Ye Ji Jeon,Eugene L. Madsen,Che Ok Jeon 한국응용생명화학회 2007 Journal of Applied Biological Chemistry (J. Appl. Vol.50 No.2

Role of Glyoxylate Shunt in Oxidative Stress Response

Ahn, Sungeun,Jung, Jaejoon,Jang, In-Ae,Madsen, Eugene L.,Park, Woojun American Society for Biochemistry and Molecular Bi 2016 The Journal of biological chemistry Vol.291 No.22

<P>The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.</P>

<i>Alteromonas</i> As a Key Agent of Polycyclic Aromatic Hydrocarbon Biodegradation in Crude Oil-Contaminated Coastal Sediment

Jin, Hyun Mi,Kim, Jeong Myeong,Lee, Hyo Jung,Madsen, Eugene L.,Jeon, Che Ok American Chemical Society 2012 Environmental science & technology Vol.46 No.14

<P>Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated <I>Alteromonas</I> (among others) were active. <I>Alteromonas</I> sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: <I>Alteromonas</I> and <I>Cycloclasticus</I>, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed <I>in situ</I> expression of only the <I>Alteromonas</I>-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 μM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring <I>Alteromonas</I>-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/esthag/2012/esthag.2012.46.issue-14/es3018545/production/images/medium/es-2012-018545_0003.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/es3018545'>ACS Electronic Supporting Info</A></P>

The Naphthalene Catabolic (nag) Genes of Polaromonas naphthalenivorans CJ2: Evolutionary Implications for Two Gene Clusters and Novel Regulatory Control

Jeon, Che Ok,Park, Minjeong,Ro, Hyun-Su,Park, Woojun,Madsen, Eugene L. American Society for Microbiology 2006 Applied and environmental microbiology Vol.72 No.2

<B>ABSTRACT</B><P><I>Polaromonas naphthalenivorans</I> CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (C.-O. Jeon et al., Proc. Natl. Acad. Sci. USA 100:13591-13596, 2003), is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp <I>nagAc</I>-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway andadditional flanking regions. We found that the naphthalene catabolic genes in <I>P. naphthalenivorans</I> CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster (<I>nagRAaGHAbAcAdBFCQEDJI′ORF1tnpA</I>) is bounded by a LysR-type regulator (<I>nagR</I>). The small cluster (<I>nagR2ORF2I'KL</I>) is bounded by a MarR-type regulator (<I>nagR2</I>). The catabolic genes of <I>P. naphthalenivorans</I> CJ2 were homologous to many of those of <I>Ralstonia</I> U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (<I>nagY</I>, <I>nagM</I>, and <I>nagN</I>), present in <I>Ralstonia</I> U2, were absent. Also, <I>P. naphthalenivorans</I> carries two copies of gentisate dioxygenase (<I>nagI</I>) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in <I>Ralstonia</I> sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that <I>nagR2</I> is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative <I>Azoarcus</I>-related transposases with the large cluster and one <I>Azoarcus</I>-related putative salicylate 5-hydroxylase gene (<I>ORF2</I>) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in <I>P. naphthalenivorans</I>.</P>

Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and¹H Nuclear Magnetic Resonance Spectroscopy

Lee, Hyo Jung,Jung, Ji Young,Oh, Young Kyoon,Lee, Sang-Suk,Madsen, Eugene L.,Jeon, Che Ok American Society for Microbiology 2012 Applied and environmental microbiology Vol.78 No.17

<B>ABSTRACT</B><P>Pyrosequencing of 16S rRNA genes (targetingBacteriaandArchaea) and<SUP>1</SUP>H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla-Bacteroidetes,Firmicutes,Fibrobacteres, andProteobacteria-in all ruminants. However, interestingly, the bacterial reads belonging toFibrobactereswere present at a very low abundance (@@<@@0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.</P>

Overexpressing antioxidant enzymes enhances naphthalene biodegradation in Pseudomonas sp. strain As1

Kang, Yoon-Suk,Lee, Yunho,Jung, Hyungil,Jeon, Che Ok,Madsen, Eugene L.,Park, Woojun Microbiology Society 2007 Microbiology Vol.153 No.10

<P>We tested the hypothesis that during metabolism of naphthalene and other substrates by Pseudomonas sp. strain As1 oxidative stress arises and can be reduced by antioxidant enzymes. Our approach was to prepare plasmid constructs that conferred expression of two single antioxidant enzymes [Fpr (ferredoxin-NADP(+) reductase) and SOD (superoxide dismutase)] and the pair of enzymes SOD plus AhpC (alkyl hydroperoxide reductase). The fpr, sodA and ahpC genes were placed under the transcriptional control of both the constitutive lac promoter and their respective native promoters. Both HPLC and growth-rate analyses showed that naphthalene metabolism was enhanced in the recombinant strains. All antioxidant-overexpressing recombinant strains, with the exception of one with an upregulated sodA gene due to the lac promoter [strain As1(sodA)], exhibited resistance to the superoxide generating agent paraquat (PQ). The growth of strain As1(sodA) was inhibited by PQ, but this growth defect was rapidly overcome by the simultaneous overproduction of AhpC, which is a known hydrogen peroxide scavenger. After PQ-induced oxidative damage of the [Fe-S] enzyme aconitase, recovery of enzyme activity was enhanced in the recombinant strains. Reporter strains to monitor oxidative stress in strain As1 were prepared by fusing gfp (encoding green fluorescent protein, GFP) to the fpr promoter. Growth on salicylate and naphthalene boosted the GFP fluorescent signal 21- and 14-fold, respectively. Using these same oxidative stress reporters, overexpression of fpr and sodA was found to considerably reduce PQ-induced stress. Taken together, these data demonstrate that the overproduction of Fpr or SodA contributes to oxidative tolerance during naphthalene degradation; however, elevated SOD activity may trigger the generation of excess hydrogen peroxide, resulting in cell death.</P>