- Email: [email protected]
Identification and functional study of an iif2 gene cluster for indole degradation in Burkholderia sp. IDO3
International Biodeterioration & Biodegradation 142 (2019) 36–42
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Identification and functional study of an iif2 gene cluster for indole degradation in Burkholderia sp. IDO3
T
Qiao Maa,∗, Bingyu Yangb, Hui Qua, Zhen Gaoa, Yuanyuan Qub, Yeqing Suna,∗∗ a
Institute of Environmental Systems Biology, College of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, China Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Burkholderia Indole degradation iif gene cluster Indole oxygenase Isatin hydrolase
Burkholderia sp. IDO3 is an indole-degrading bacterium isolated from activated sludge. A previous genomic clone library assay identified an iif1 gene cluster for indole metabolism in strain IDO3. To further explore the underlying indole degradation mechanisms, the complete genome of strain IDO3 was sequenced (8,003,806 bp). The genome contained three circular chromosomes and one plasmid, and 7550 genes were predicted. Interestingly, in addition to iif1 on chromosome 3, bioinformatic analyses identified a second indole oxygenase gene cluster, iif2, on chromosome 1. Both iif clusters were up-regulated in response to indole. Heterologous expression of iifC1D1 and iifC2D2 in Escherichia coli BL21(DE3) demonstrated that these genes were capable of oxidizing indole to indigo. Gene knockout assays provided additional evidence that iifC2 played crucial roles in indole metabolism. In addition, we identified a novel gene (iifF) in the iif2 cluster. This gene was shown to encode an isatin hydrolase. IifF was expressed in E. coli, and a purified his-tagged enzyme preparation was obtained. IifF converted isatin to isatinate with Km of 4.4 ± 0.7 μM and kcat of 95.5 ± 4 s−1. This is the first study to show that indole can be degraded by two iif gene clusters, and that isatin hydrolase is involved in indole metabolism, improving our understanding of indole metabolic processes.
1. Introduction Indole, which is a typical N-heterocyclic aromatic compound widespread in nature, is proven to be a versatile signal molecule that has recently aroused extensive concern (Lee and Lee, 2010). Indoles play important and complex roles in the health of human, animals, plants, and microorganisms (Lee et al., 2015). Meanwhile, high concentrations of indoles (mainly indole and 3-methylindole) have been identified in livestock wastes, intestinal tracts, and coal tar (Arora et al., 2015; Ma et al., 2018). Indoles are the key odor components in livestock waste causing environmental problems, and in the meat producing boar taint (Mackie et al., 1998). Thus, additional studies on indole biotransformation are required for environment protection and remediation in related industries. Many bacterial strains with the ability of degrading indole have been obtained over the past decades, among which Pseudomonas, Alcaligenes, Cupriavidus and Acinetobacter are the most investigated genera (Ma et al., 2018). Indole degradation pathways vary among different bacterial strains, but isatin and anthranilate are the two most
∗
common intermediates in both aerobically- and anaerobically-grown bacteria (Claus and Kutzner, 1983; Madsen and Bollag, 1989). Some strains, such as Cupriavidus sp. KK10 and Acinetobacter pittii L1, were also shown to metabolize indole carbocyclic-aromatic ring, expanding our knowledge on indole degradation process (Fukuoka et al., 2015; Yang et al., 2017). Although efforts have been made to elucidate the indole biotransformation process, the genetic mechanisms for indole degradation remain unclear, and the degradation pathways are debatable. As early as 1968, Fujioka and Wada (1968) attempted to purify the indole oxidation enzymes but failed due to the insolubility of the target protein. Only recently have the indole metabolic pathways and genes been identified in the genera Acinetobacter and Cupriavidus (Lin et al., 2015; Sadauskas et al., 2017; Qu et al., 2017). The iif (or ind) gene cluster was demonstrated to be responsible for indole oxidation in these genera. In the gene cluster, iifC and iifD are the functional genes encoding the flavin-dependent two-component indole oxygenase system. This enzyme system is capable of oxidizing indole, the first step in indole degradation. Burkholderia is a typical aromatic pollutant degrader with a wide
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q. Ma), [email protected] (Y. Sun).
∗∗
https://doi.org/10.1016/j.ibiod.2019.04.011 Received 18 January 2019; Received in revised form 26 April 2019; Accepted 26 April 2019 Available online 06 May 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
2.4. Heterologous expression of indole oxygenase genes
ecological niche, and we showed that this genus was predominant in indole wastewater treatment systems (Ma et al., 2015). However, the degradation of indole by Burkholderia has rarely been described (Kim et al., 2013). In an earlier study, we have successfully obtained an efficient indole-degrading bacterium, Burkholderia sp. IDO3, from activated sludge (Ma et al., 2015). Analysis of a genomic clone library successfully identified an iif gene cluster in strain IDO3, which was suggested as the functional gene cluster for indole degradation (Ma et al., 2019). Herein, the complete genomic sequence of strain IDO3 was obtained and functional studies were performed to identify and demonstrate the functionalities of the indole degradation genes. The present study should provide a deeper insight into the molecular mechanism of indole degradation and improve our understanding of indole fates in natural environments.
Genes iifC1D1 were cloned and expressed previously (Ma et al., 2019). In this study, genes iifC2D2 in strain IDO3 were amplified using 2 × Phanta® Master Mix (Vazyme, China). The primers and PCR program for amplification are provided in Table S2 and Table S4. The PCR products were purified and ligated with linearized pET-28a(+) plasmid using ClonExpress® II One Step Cloning Kit (Vazyme, China). Then the recombinant plasmid was successfully expressed in strain E. coli BL21(DE3). The culture conditions for both E. coli_IifC1D1 and E. coli_IifC2D2 were 30 °C and 150 r min−1 in tryptophan medium consisted of (g L−1) 1.0 tryptophan, 5.0 yeast extract and 10.0 NaCl. Kanamycin (50 mg L−1) was added to tryptophan medium before strain inoculation, and isopropyl-β-d-thiogalactoside (IPTG, 119 mg L−1) was added for strain E. coli_IifC2D2. After two days cultivation, the transformation products were collected, extracted by dimethylsulfoxide and analyzed by high performance liquid chromatography (HPLC) (Ma et al., 2019).
2. Materials and methods 2.1. Strains and culture media
2.5. Gene knockout and indole degradation assays
Burkholderia sp. IDO3 is maintained in our lab (Table S1). This strain is able to use indole as the sole carbon source (Ma et al., 2019). The culture medium for strain IDO3 was sodium citrate medium, containing (g L−1) 1.0 trisodium citrate dihydrate, 2.0 (NH4)2SO4, 2.0 KH2PO4, 1.3 Na2HPO4 and 0.00025 FeCl3. E. coli strains used in this study were cultured in Luria-Bertani (LB) medium with corresponding antibiotics (Table S1). Solid agar medium was obtained by the addition of 2.0% (w/v) agar to the liquid medium. Both liquid and agar media were autoclaved at 121 °C for 20 min. Indole was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), and other chemical reagents were of analytical grade purity. Indole was freshly dissolved in acetone and added to the medium when required.
Gene knockout manipulation is provided in Supplementary Material. We firstly knocked out genes iifC1 and iifC2, respectively, to obtain strains IDO3-ΔiifC1 and IDO3-ΔiifC2. Strain IDO3-ΔiifC1ΔiifC2 was derived from IDO3-ΔiifC1 with further mutation of gene iifC2. To examine the indole degradation abilities of wild strain IDO3 and its mutants, all strains were cultured in sodium citrate medium to stationary phase. Then the cells were collected and washed with the same medium and adjusted to OD600 0.5, and 500 μL feed culture was inoculated to fresh sodium citrate medium (50 mL in 250 mL conical flask) with 120 mg L−1 indole. Samples were taken at intervals to determine the residual indole concentration. Indole concentration was quantified by HPLC (Ma et al., 2019). All experiments were performed in triplicates.
2.2. Genome sequencing and analysis The genomic DNA of strain IDO3 was extracted using TIANamp Bacteria DNA Kit (TIANGEN, China), and sequenced using PacBio RS II (Pacific Biosciences, CA, USA) platform at the Beijing Novogene Bioinformatics Technology Co., Ltd. The low-quality reads were filtered by the SMRT Link v5.0.1, which were assembled to generate the complete genome of strain IDO3. GeneMarkS program was used to retrieve and predict the related coding genes, and the final genome was annotated by Rapid Annotation using Subsystem Technology (RAST) and NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Besemer et al., 2001; Overbeek et al., 2014). Function analyses of the genes were performed by BLAST search against GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), and NR (Non-Redundant Protein Database databases) with evalue less than 1e-5 and minimal coverage 40%.
2.6. Heterologous expression and characterization of the iifF gene Gene iifF was amplified and expressed in E. coli BL21(DE3) as described above (same with iifC2D2). The engineered strain was cultured in LB medium with kanamycin and IPTG at 20 °C overnight. The enzyme was purified from induced bacterial cells as previously reported (Bjerregaard-Andersen et al., 2014), which is summarized in Supplementary Material. The enzymatic activity of IifF was determined as a function of the isatin hydrolysis and isatinate production, which has a typical absorption peak at 368 nm (Bjerregaard-Andersen et al., 2014). Measurements were done in a UV-5500P spectrophotometer (Metash, China) with a cuvette light path of 1 cm. The enzyme reaction was performed in a reaction buffer (50 mM pH 8.0 Tris-HCl, 100 mM NaCl). 2 μL stock MnCl2 solution (0.2 M) was added to 1994 μL reaction buffer in the cuvette, and the reaction was quickly started by adding 4 μL enzyme stock solution (1 μM). The reaction was performed at room temperature (20 °C). An extinction coefficient of 4.5 × 103 cm−1 mol−1 L was used for isatinate (Olesen and Jochimsen, 1996). The data were fitted with the Michaelis-Menten equation using the software SigmaPlot 11.0.
2.3. RT-qPCR assay Strain IDO3 was cultured in sodium citrate medium (control group) and 100 mg L−1indole-sodium citrate medium (experiment group) for 24 h. Then the bacterial cells were collected and total RNA was extracted using the Bacteria RNA Extraction Kit (Vazyme, China). cDNA was synthesized by M-MLV Reverse Transcriptase (Invitrogen, USA) with the total RNA amount of 360 ng. The cDNA was diluted as the template for RT-qPCR analysis. RT-qPCR was conducted using Roche LightCycler® 480 system (Roche, Switzerland) with corresponding primers (Table S2) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). RT-qPCR program is provided in Table S3. Relative expression level was calculated using 2−ΔΔCT method with 16S rRNA gene as the reference gene (Livak and Schmittgen, 2001; Qu et al., 2017). The experiment was conducted using three biological replicates.
2.7. Nucleotide sequence accession numbers The complete genome sequence of Burkholderia sp. IDO3 has been deposited at GenBank under the accession numbers of CP028962, CP028963, CP028964, and CP028965. The accession numbers for protein IifC1, IifD1, IifC2, IifD2, and IifF are APT36898.1, APT36899.2, AXK62974.1, AXK62973.1, and WP_096501352, respectively.
37
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
2), 851,589 (chromosome 3) and 520,805 bp (plasmid) in length (Table 1, Fig. 1). A total of 7550 genes, including 90 RNAs, were predicted in the genome. KEGG pathway analysis indicated that most sequences were associated with membrane transport and metabolism pathways, including amino acid metabolism, carbohydrate metabolism, energy metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism (Fig. S1). Within the xenobiotics biodegradation and metabolism pathway, KEGG Orthology (KO) associated with aminobenzoate degradation (14 KOs), benzoate degradation (37 KOs), xylene degradation (13 KOs), chlorocyclohexane and chlorobenzene degradation (12 KOs), styrene degradation (10 KOs), toluene degradation (10 KOs), and naphthalene degradation (4 KOs) were identified (Table S5). Burkholderia occupies notably diverse ecological niches, such as contaminated soils, water, plant rhizospheres, and human respiratory tracts (Coenye and Vandamme, 2003). This genus has the potential to degrade many aromatic compounds (Pérez-Pantoja et al., 2012). In the previous study, we found that Burkholderia sp. IDO3 could effectively degrade indole, as well as other aromatic pollutants including skatole, phenol, and cresol (Ma et al., 2019). The numerous pathways identified
Table 1 Genome features of Burkholderia sp. IDO3. Features
Chr1
Chr2
Chr3
Plasmid
Genome
Size (bp) GC content (%) Number of genes rRNA tRNA Other RNA
3,139,806 67.0 2885 3 6 0
3,591,606 66.4 3444 12 60 4
851,589 66.33 784 3 2 0
420,805 61.57 437 0 0 0
8,003,806 66.4 7550 18 68 4
3. Results and discussions 3.1. Genome information of strain IDO3 The 140,176 (1,016,768,870 bp) obtained reads were de novo assembled into four polished contigs, with an average genome coverage of 127-fold. The genome of strain IDO3 had an overall GC content of 66.37% and consisted of three circular chromosomes and one plasmid, which were 3,139,806 bp (chromosome 1), 3,591,606 bp (chromosome
Fig. 1. Circular maps of the chromosomes and plasmid of Burkholderia sp. IDO3. A, chromosome 1; B, chromosome 2; C, chromosome 3; D, plasmid. From outside to center: predicted genes, COG categories, KEGG categories, GO categories, GC content, and GC skew. The iif1 gene cluster is located on Chr3 and iif2 on Chr1. Detailed KEGG categories are shown in Fig. S1. 38
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
Fig. 2. Indole oxidation gene cluster analysis in strain IDO3. Acinetobacter sp. O153 and Cupriavidus sp. SHE are two previously characterized indole-degrading strains.
here suggested that strain IDO3 might have potential applications in aromatic pollutants bioremediation. The genomic information of strain IDO3 will facilitate the molecular mechanism and bioremediation study of Burkholderia. 3.2. Identification of two iif gene clusters associated with indole metabolism The functional genes responsible for indole degradation were recently identified. The FAD-dependent oxygenase IifC and reductase IifD compose the two-component indole oxygenase system, which catalyzes the oxidation of indole in Acinetobacter (Sadauskas et al., 2017). To explore the functional genes for indole degradation in strain IDO3, the traditional genomic clone library method was applied to screen possible genes. An iif gene cluster (here designated iif1, Fig. 2) was previously identified and shown to be able to oxidize indole (Ma et al., 2019). To further explore the indole-degrading functional genes, a local BLAST comparing the indole oxygenase protein IifC from strain Acinetobacter sp. O153 to all protein sequences in strain IDO3 was conducted (Sadauskas et al., 2017). The protein IifC1 had a 39% similarity with the homolog IifC in strain O153. Interestingly, another indole oxygenase IifC2 was identified that displayed 62% similarities with IifC. Further analysis showed that iifC1 was located on chromosome 3 and iifC2 on chromosome 1, both of which clustered with the iifB and iifD genes. The evolutionary analyses of IifC1, IifC2 with other indole oxygenases and styrene monooxygenases were provided in Fig. S2. It was obvious that IifC2 was closely related to the indole oxygenases from Cupriavidus and Acinetobacter, whereas IifC1 formed a separate clade from the functionally similar indole oxygenases. IifD1 and IifD2, the reductase components of indole oxygenases, showed 45% and 47% similarities with corresponding proteins in strain O153. It was previously reported that iifA is a common component of the iif gene clusters. IifA is possibly a cofactor-independent oxygenase that catalyzes the transformation of 3-hydroxyindolin-2-one to anthranilic acid. However, we noted that iifA was absent from both the iif1 and iif2 clusters (Fig. 2). Therefore, the indole upstream transformation pathway in strain IDO3 may be distinct from that of Acinetobacter sp. O153 (Sadauskas et al., 2017). IifB encodes a short-chain dehydrogenase, which might convert indole oxidation products (possibly indole-2,3-dihydrodiol) to 3-hydroxyindolin-2-one. Here, IifB1 and IifB2 showed 52% and 59% similarities with IifB in strain O153. Furthermore, a novel gene, termed iifF, was identified in the iif2 gene cluster. Further analysis of this gene indicated that it encoded an isatin hydrolase gene, which might be responsible for isatin biotransformation in indole degradation process (Bjerregaard-Andersen et al., 2014).
Fig. 3. RT-qPCR verification of the iif gene clusters. The treatment group was cultured in sodium citrate medium with 100 mg L−1 indole, and the control group was cultured in sodium citrate medium.
5.3 ± 0.67, 5.04 ± 2.7, 3.71 ± 2.06, 2.66 ± 0.86, 1.53 ± 0.27, and 3.08 ± 1.20 folds, respectively. This suggested that both gene clusters were likely to participate in indole metabolism.
3.4. Heterologous expression of indole oxygenase genes The iifC1D1 genes were previously heterologously expressed in E. coli strain (Ma et al., 2019). In this study, iifC2D2 genes were also expressed in E. coli BL21(DE3) strain. As E. coli strain harbors tryptophanase, which can convert tryptophan to indole endogenously but is unable to convert indole to indigo (Lee and Lee, 2010), the recombinant strains were cultured in a tryptophan-rich medium to examine their indigo production ability. It was found that the recombinant strains carrying the iifC1D1 and iifC2D2 genes could both produce indigo in tryptophan medium, while the E. coli strain carrying the empty pET-28a (+) plasmid did not produce indigo. This proved that IifC1D1 and IifC2D2 were able to oxidize indole (Fig. S3). The indigo yields for strains E. coli_IifC1D1 and E. coli_IifC2D2 were 37.1 ± 8.5 and 78.1 ± 2.0 mg L−1, respectively (Fig. 4), implying that the indole oxygenases obtained here may be applied for indigo production studies. The indigo bio-production abilities of several other oxygenases have previously been investigated. For example, the E. coli strain carrying the phenol hydroxylase gene from Acinetobacter sp. ST-550 could produce 52 mg L−1 indigo in a water-organic solvent two-phase system (Doukyu et al., 2003). Over-expression of the styrene monooxygenase in Pseudomonas putida led to the production of 52.1 mg L−1 indigo (Cheng et al., 2016). The most investigated naphthalene dioxygenase expressed in E. coli yielded 135 mg L−1 indigo directly from glucose in laboratory experiment after a series of genetic manipulations (Murdock et al., 1993). Furthermore, the recombinant E. coli strain expressing the flavin
3.3. Transcriptomic analysis of strain IDO3 in response to indole RT-qPCR assays were conducted to determine the mRNA expression levels of both gene clusters in response to indole. It was shown that indole increased the expression of all chosen genes (Fig. 3). Genes iifB1, iifC1, iifD1, iifB2, iifC2, iifD2, and iifF were up-regulated by 3.3 ± 0.70, 39
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
3.5. Mutations study of the two indole oxygenase genes To further elucidate the indole degradation mechanism in strain IDO3, gene knockout experiments were performed. We successfully obtained three mutant strains, i.e. IDO3-ΔiifC1, IDO3-ΔiifC2, and IDO3ΔiifC1ΔiifC2 (Table S1), after extensive genetic manipulations. A comparison of the PCR products of the iifC1 and iifC2 genes among the three mutant strains and the wildtype strain clearly showed the disruption of the relevant genes (Fig. S4). The indole degradation abilities of these strains were carefully investigated. The indole degradation curves of the four strains were shown in Fig. 5. It was obvious that indole degradation process of strain IDO3-ΔiifC1 was similar to that of wildtype IDO3, both of which removed ∼120 mg L−1 indole within 12 h. This suggested that gene iifC1 played a limited role in indole degradation under the tested condition. The indole removal capacities of strains IDO3-ΔiifC2 and IDO3-ΔiifC1ΔiifC2 were significantly reduced as compared to the wildtype. Strain IDO3-ΔiifC2 removed the indole in ∼60 h, indicating that gene iifC2 was crucial for indole metabolism. It was worth noting that strain IDO3ΔiifC1ΔiifC2 could not completely remove the indole and almost 80% indole remained in the medium. This result indicated that gene iifC1 also participated in the indole degradation process in strain IDO3, especially when the iifC2 gene was absent from strain IDO3. Therefore, above data indicate that iifC2 rather than iifC1 plays crucial roles in indole degradation. As two copies of the indole oxygenase genes are not common in other bacterial strains, the evolutional significance of this duplication requires further investigation. The iif gene cluster has been identified in Acinetobacter, Pseudomonas, Cupriavidus, Alcaligenes, Burkholderia and Ralstonia, and these genera are the most reported indole-degrading bacteria (Sadauskas et al., 2017). This suggests that the iif-dependent pathway is the most common indole degradation pathway. However, some strains (e.g. Pseudomonas aeruginosa PAO1) lacking the iif gene cluster can also rapidly degrade indole (Lee et al., 2009), suggesting that other functional genes should be involved in indole biodegradation.
Fig. 4. Production of indigo in E. coli strains expressing iifC1D1 and iifC2D2. From left to right are E. coli strain with empty pET-28a(+) plasmid, strain E. coli_IifC1D1, and E. coli_IifC2D2.
3.6. Heterologous expression and functional study of the iifF gene
Fig. 5. Indole degradation curves of strain IDO3 and its mutant strains.
Isatin hydrolase is reported to be involved in indole-3-acetic acid degradation, which can hydrolyze isatin to isatinate (Olesen and Jochimsen, 1996). This new class of metalloenzyme has received much recent attention because isatin plays a neurological role in human and acts as a signal molecule in bacteria. The crystal structures of the isatin hydrolases have been revealed (Bjerregaard-Andersen et al., 2014).
monooxygenase gene from Methylophaga aminisulfidivorans MPT produced up to 920 mg L−1 indigo under optimal conditions (Han et al., 2008). Compared with these oxygenases in favor of indigo production, the indigo yield in our study should be further improved.
Fig. 6. Phylogenetic analysis and expression of IifF. (A) Phylogram of IifF and its homologs. IHA/B, isatin hydrolase A/B; KynB; kynurenine formamidas; AHD; amidohydrolase. The evolutionary history was inferred using the Neighbor-Joining method with bootstrap test of 1000 replicates, and the figure was produced using MEGA7 (Kumar et al., 2016). (B) Amplification of iifF and the pET-28a(+) plasmid. From left to right are DNA marker, iifF gene PCR product, and pET-28a(+) PCR product. Primers used for PCR are given in Table S2. (C) SDS-PAGE analysis of purified IifF. From left to right are protein marker and IifF protein bands. 40
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
Fig. 7. Catalytic performance of IifF. (A) The full wavelength spectra of isatin and its catalytic products by IifF. (B) Michaelis-Menten curves for verification of IifF kinetic model.
Fig. 8. The predicted indole metabolic pathway of strain IDO3.
first time, we are showing that the isatin hydrolase gene (iifF) is a component of the iif gene cluster. The function of iifF is to catalyze isatin hydrolysis to produce isatinate, demonstrating that isatin is a real metabolic intermediate of the indole degradation process. We also tested the growth ability of strain IDO3 with isatin as the carbon source. Data showed that strain IDO3 could grow within two days (Fig. S6). As isatin can be hydrolyzed in the aqueous medium, whether strain IDO3 uses isatin or its transformation products (such as isatinate) as the growth substrate is to be confirmed. A proposed indole metabolic pathway in strain IDO3 is provided in Fig. 8. Indole is oxidized to oxidation products (such as indole-2,3-dihydrodiol, indoxyl, or indole epoxide) via IifC1D1 and IifC2D2, of which IifC2D2 plays the crucial role in this process (Heine et al., 2019). The oxidation products are converted to 3-hydroxyindolin-2-one by IifB1 and IifB2 based on the previous study (Sadauskas et al., 2017). Isatin is formed by oxidation or dehydrogenation process, which is further hydrolyzed to isatinate by IifF. Finally, the anthranilate might be produced from 3-hydroxyindolin-2-one, isatin, or isatinate by oxygenases or decarboxylases. However, some issues in indole degradation process, such as isatin formation, isatinate conversion and anthranilate degradation, will require further investigation.
BLAST analysis of the IifF amino acid sequence indicated that it shared 49% identity with isatin hydrolase A (LaIHA) from Labrenzia aggregate, 58% identity with isatin hydrolase B (LaIHB) from L. aggregate, and 84% identity with isatin hydrolase (RsIHA) from Ralstonia solanacearum (Fig. 6A). Alignment analysis showed that IifF was similar to other isatin hydrolases containing the central metal binding motif HxG[T/A] HxDxPxH as shown in Fig. S5. In addition, IifF also contained the other conserved GLQC motif for isatin hydrolase activity (Sommer et al., 2018). To further explore the characteristics of IifF, gene iifF was amplified from strain IDO3 DNA and successfully expressed in E. coli (Fig. 6B). The purified his-tagged IifF was obtained using Ni-NTA agarose with elution buffer (50 mM pH 8.0 Tris-HCl, 0.5 M NaCl, 10 mM Na2EDTA and 50 mM imidazole). The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Fig. 6C). The calculated molecular weight of IifA was 32.7 kDa, consistent with the SDS-PAGE results. Activity measurements confirmed that IifF harbored isatin hydrolase activity. It was shown in Fig. 7A that isatin was converted to a product with the characteristic absorption peak at 368 nm, identical to isatinate. Isatin hydrolysis followed Michaelis-Menten kinetics. The determined Km was 4.4 ± 0.7 μM, similar to that of LaIHB (Fig. 7B, Sommer et al., 2018). The kcat value was 95.5 ± 4 s−1, similar to that of LaIHA and RsIHA (Sommer et al., 2018). These results indicated that IifA had similar enzymatic characteristics to previously reported isatin hydrolases. Although isatin has been identified as a possible indole degradation intermediate in several studies (Ma et al., 2018), the functional gene has not yet been identified. As previously reported iif gene clusters did not contain the isatin hydrolase iifF gene, it was debatable whether isatin participated to the indole degradation process or whether it was just an experimental artifact (Sadauskas et al., 2017). Herein, for the
4. Conclusion The complete genome of an indole-degrading strain Burkholderia sp. IDO3 was sequenced. Analysis showed that strain IDO3 contained two indole oxygenase gene clusters on Chr1 and Chr3. A combination of RTqPCR heterologous expression and gene knockout assays showed that both iif1 and iif2 clusters were involved in indole degradation process. The indole degradation ability of strain IDO3-ΔiifC2 was remarkably reduced, suggesting that iifC2 gene was crucial for indole oxidation. A 41
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
novel iifF gene was identified in the iif2 gene cluster. Bioinformatic analyses and gene expression studies proved that iifF encoded an isatin hydrolase, which was responsible for isatin hydrolysis to isatinate.
aromatic ring cleavage and production of indigoids. Int. Biodeterior. Biodegrad. 97, 13–24. Han, G.H., Shin, H.J., Kim, S.W., 2008. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene. Enzym. Microb. Technol. 42, 617–623. Heine, T., Großmann, C., Hofmann, S., Tischler, D., 2019. Indigoid dyes by group E monooxygenases: mechanism and biocatalysis. Biol. Chem. https://doi.org/10.1515/ hsz-2019-0109. Kim, D., Rahman, A., Sitepu, I.R., Hashidoko, Y., 2013. Accelerated degradation of exogenous indole by Burkholderia unamae strain CK43B exposed to pyrogallol-type polyphenols. Biosc. Biotech. Biochem. 77, 1722–1727. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Lee, J.H., Lee, J., 2010. Indole as an intercellular signal in microbial communities. FEMS Microbiol. Rev. 34, 426–444. Lee, J., Attila, C., Cirillo, S.L.G., Cirillo, J.D., Wood, T.K., 2009. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence. Microbial Biotechnology 2, 75–90. Lee, J.H., Wood, T.K., Lee, J., 2015. Roles of indole as an interspecies and interkingdom signaling molecule. Trends Microbiol. 23, 707–718. Lin, G.H., Chen, H.P., Shu, H.Y., 2015. Detoxification of indole by an indole-induced flavoprotein oxygenase from Acinetobacter baumannii. PLoS One 10, e0138798. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408. Ma, Q., Qu, Y., Zhang, X., Liu, Z., Li, H., Zhang, Z., Wang, J., Shen, W., Zhou, J., 2015. Systematic investigation and microbial community profile of indole degradation processes in two aerobic activated sludge systems. Sci. Rep. 5, 17674. Ma, Q., Zhang, X., Qu, Y., 2018. Biodegradation and biotransformation of indole: advances and perspectives. Front. Microbiol. 9, 2625. Ma, Q., Liu, Z., Yang, B., Dai, C., Qu, Y., 2019. Characterization and functional gene analysis of a newly isolated indole-degrading bacterium Burkholderia sp. IDO3. J. Hazard Mater. 367, 144–151. Mackie, R.I., Stroot, P.G., Varel, V.H., 1998. Biochemical identification and biological origin of key odor components in livestock waste. J. Anim. Sci. 76, 1331–1342. Madsen, E.L., Bollag, J.M., 1989. Pathway of indole metabolism by a denitrifying microbial community. Arch. Microbiol. 151, 71–76. Murdock, D., Ensley, B.D., Serdar, C., Thalen, M., 1993. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli. Nat. Biotechnol. 11, 381–386. Olesen, M.R., Jochimsen, B.U., 1996. Identification of enzymes involved in indole-3acetic acid degradation. Plant Soil 186, 143–149. Overbeek, R., Olson, R., Pusch, G.D., Olsen, G.J., Davis, J.J., Disz, T., et al., 2014. The SEED and the Rapid annotation of microbial genomes using subsystems Technology (RAST). Nucleic Acids Res. 42, D206–D214. Qu, Y., Ma, Q., Liu, Z., Wang, W., Tang, H., Zhou, J., Xu, P., 2017. Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain. Mol. Microbiol. 106, 905–918. Sadauskas, M., Vaitekūnas, J., Gasparavičiūtė, R., Meškys, R., 2017. Indole biodegradation in Acinetobacter sp. strain O153, genetic and biochemical characterization. Appl. Environ. Microbiol. 83 e01453-17. Sommer, T., Bjerregaard-Andersen, K., Uribe, L., Etzerodt, M., Diezemann, G., Gauss, J., Cascella, et al., 2018. A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase. Sci. Rep. 8, 13104. Yang, Z., Zhou, J., Xu, Y., Zhang, Y., Luo, H., Chang, K., 2017. Analysis of the metabolites of indole degraded by an isolated Acinetobacter pittii L1. BioMed Res. Int. 2017, 2564363.
Conflicts of interest The authors declare no conflicts of interest. Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 31800091), the Doctoral Scientific Research Foundation of Liaoning Province (No. 20180540072), and the Open Foundation of Key Laboratory of Industrial Ecology and Environmental Engineering, MOE (No. KLIEEE-17-01). Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.ibiod.2019.04.011. References Pérez‐Pantoja, D., Donoso, R., Agulló, L., 2012. Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales. Environ. Microbiol. 14, 1091–1117. Arora, P.K., Sharma, A., Bae, H., 2015. Microbial degradation of indole and its derivatives. J. Chem. 2015, 129–159. Besemer, J., Lomsadze, A., Borodovsky, M., 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29, 2607–2618. Bjerregaard-Andersen, K., Sommer, T., Jensen, J.K., Jochimsen, B., Etzerodt, M., Morth, J.P., 2014. A proton wire and water channel revealed in the crystal structure of isatin hydrolase. J. Biol. Chem. 289, 21351–21359. Cheng, L., Yin, S., Chen, M., Sun, B., Hao, S., Wang, C., 2016. Enhancing indigo production by over-expression of the styrene monooxygenase in Pseudomonas putida. Curr. Microbiol. 73, 248–254. Claus, G., Kutzner, H.J., 1983. Degradation of indole by Alcaligenes spec. Syst. Appl. Microbiol. 4, 169–180. Coenye, T., Vandamme, P., 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol. 5, 719–729. Doukyu, N., Toyoda, K., Aono, R., 2003. Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp. strain ST-550 in a water-organic solvent two-phase system. Appl. Microbiol. Biotechnol. 60, 720–725. Fujioka, M., Wada, H., 1968. The bacterial oxidation of indole. Biochim. Biophys. Acta. 158, 70–78. > Fukuoka, K., Tanaka, K., Ozeki, Y., Kanaly, R.A., 2015. Biotransformation of indole by Cupriavidus sp. strain KK10 proceeds through N-heterocyclic-and carbocyclic-
42
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Identification and functional study of an iif2 gene cluster for indole degradation in Burkholderia sp. IDO3
T
Qiao Maa,∗, Bingyu Yangb, Hui Qua, Zhen Gaoa, Yuanyuan Qub, Yeqing Suna,∗∗ a
Institute of Environmental Systems Biology, College of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, China Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Burkholderia Indole degradation iif gene cluster Indole oxygenase Isatin hydrolase
Burkholderia sp. IDO3 is an indole-degrading bacterium isolated from activated sludge. A previous genomic clone library assay identified an iif1 gene cluster for indole metabolism in strain IDO3. To further explore the underlying indole degradation mechanisms, the complete genome of strain IDO3 was sequenced (8,003,806 bp). The genome contained three circular chromosomes and one plasmid, and 7550 genes were predicted. Interestingly, in addition to iif1 on chromosome 3, bioinformatic analyses identified a second indole oxygenase gene cluster, iif2, on chromosome 1. Both iif clusters were up-regulated in response to indole. Heterologous expression of iifC1D1 and iifC2D2 in Escherichia coli BL21(DE3) demonstrated that these genes were capable of oxidizing indole to indigo. Gene knockout assays provided additional evidence that iifC2 played crucial roles in indole metabolism. In addition, we identified a novel gene (iifF) in the iif2 cluster. This gene was shown to encode an isatin hydrolase. IifF was expressed in E. coli, and a purified his-tagged enzyme preparation was obtained. IifF converted isatin to isatinate with Km of 4.4 ± 0.7 μM and kcat of 95.5 ± 4 s−1. This is the first study to show that indole can be degraded by two iif gene clusters, and that isatin hydrolase is involved in indole metabolism, improving our understanding of indole metabolic processes.
1. Introduction Indole, which is a typical N-heterocyclic aromatic compound widespread in nature, is proven to be a versatile signal molecule that has recently aroused extensive concern (Lee and Lee, 2010). Indoles play important and complex roles in the health of human, animals, plants, and microorganisms (Lee et al., 2015). Meanwhile, high concentrations of indoles (mainly indole and 3-methylindole) have been identified in livestock wastes, intestinal tracts, and coal tar (Arora et al., 2015; Ma et al., 2018). Indoles are the key odor components in livestock waste causing environmental problems, and in the meat producing boar taint (Mackie et al., 1998). Thus, additional studies on indole biotransformation are required for environment protection and remediation in related industries. Many bacterial strains with the ability of degrading indole have been obtained over the past decades, among which Pseudomonas, Alcaligenes, Cupriavidus and Acinetobacter are the most investigated genera (Ma et al., 2018). Indole degradation pathways vary among different bacterial strains, but isatin and anthranilate are the two most
∗
common intermediates in both aerobically- and anaerobically-grown bacteria (Claus and Kutzner, 1983; Madsen and Bollag, 1989). Some strains, such as Cupriavidus sp. KK10 and Acinetobacter pittii L1, were also shown to metabolize indole carbocyclic-aromatic ring, expanding our knowledge on indole degradation process (Fukuoka et al., 2015; Yang et al., 2017). Although efforts have been made to elucidate the indole biotransformation process, the genetic mechanisms for indole degradation remain unclear, and the degradation pathways are debatable. As early as 1968, Fujioka and Wada (1968) attempted to purify the indole oxidation enzymes but failed due to the insolubility of the target protein. Only recently have the indole metabolic pathways and genes been identified in the genera Acinetobacter and Cupriavidus (Lin et al., 2015; Sadauskas et al., 2017; Qu et al., 2017). The iif (or ind) gene cluster was demonstrated to be responsible for indole oxidation in these genera. In the gene cluster, iifC and iifD are the functional genes encoding the flavin-dependent two-component indole oxygenase system. This enzyme system is capable of oxidizing indole, the first step in indole degradation. Burkholderia is a typical aromatic pollutant degrader with a wide
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q. Ma), [email protected] (Y. Sun).
∗∗
https://doi.org/10.1016/j.ibiod.2019.04.011 Received 18 January 2019; Received in revised form 26 April 2019; Accepted 26 April 2019 Available online 06 May 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
2.4. Heterologous expression of indole oxygenase genes
ecological niche, and we showed that this genus was predominant in indole wastewater treatment systems (Ma et al., 2015). However, the degradation of indole by Burkholderia has rarely been described (Kim et al., 2013). In an earlier study, we have successfully obtained an efficient indole-degrading bacterium, Burkholderia sp. IDO3, from activated sludge (Ma et al., 2015). Analysis of a genomic clone library successfully identified an iif gene cluster in strain IDO3, which was suggested as the functional gene cluster for indole degradation (Ma et al., 2019). Herein, the complete genomic sequence of strain IDO3 was obtained and functional studies were performed to identify and demonstrate the functionalities of the indole degradation genes. The present study should provide a deeper insight into the molecular mechanism of indole degradation and improve our understanding of indole fates in natural environments.
Genes iifC1D1 were cloned and expressed previously (Ma et al., 2019). In this study, genes iifC2D2 in strain IDO3 were amplified using 2 × Phanta® Master Mix (Vazyme, China). The primers and PCR program for amplification are provided in Table S2 and Table S4. The PCR products were purified and ligated with linearized pET-28a(+) plasmid using ClonExpress® II One Step Cloning Kit (Vazyme, China). Then the recombinant plasmid was successfully expressed in strain E. coli BL21(DE3). The culture conditions for both E. coli_IifC1D1 and E. coli_IifC2D2 were 30 °C and 150 r min−1 in tryptophan medium consisted of (g L−1) 1.0 tryptophan, 5.0 yeast extract and 10.0 NaCl. Kanamycin (50 mg L−1) was added to tryptophan medium before strain inoculation, and isopropyl-β-d-thiogalactoside (IPTG, 119 mg L−1) was added for strain E. coli_IifC2D2. After two days cultivation, the transformation products were collected, extracted by dimethylsulfoxide and analyzed by high performance liquid chromatography (HPLC) (Ma et al., 2019).
2. Materials and methods 2.1. Strains and culture media
2.5. Gene knockout and indole degradation assays
Burkholderia sp. IDO3 is maintained in our lab (Table S1). This strain is able to use indole as the sole carbon source (Ma et al., 2019). The culture medium for strain IDO3 was sodium citrate medium, containing (g L−1) 1.0 trisodium citrate dihydrate, 2.0 (NH4)2SO4, 2.0 KH2PO4, 1.3 Na2HPO4 and 0.00025 FeCl3. E. coli strains used in this study were cultured in Luria-Bertani (LB) medium with corresponding antibiotics (Table S1). Solid agar medium was obtained by the addition of 2.0% (w/v) agar to the liquid medium. Both liquid and agar media were autoclaved at 121 °C for 20 min. Indole was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), and other chemical reagents were of analytical grade purity. Indole was freshly dissolved in acetone and added to the medium when required.
Gene knockout manipulation is provided in Supplementary Material. We firstly knocked out genes iifC1 and iifC2, respectively, to obtain strains IDO3-ΔiifC1 and IDO3-ΔiifC2. Strain IDO3-ΔiifC1ΔiifC2 was derived from IDO3-ΔiifC1 with further mutation of gene iifC2. To examine the indole degradation abilities of wild strain IDO3 and its mutants, all strains were cultured in sodium citrate medium to stationary phase. Then the cells were collected and washed with the same medium and adjusted to OD600 0.5, and 500 μL feed culture was inoculated to fresh sodium citrate medium (50 mL in 250 mL conical flask) with 120 mg L−1 indole. Samples were taken at intervals to determine the residual indole concentration. Indole concentration was quantified by HPLC (Ma et al., 2019). All experiments were performed in triplicates.
2.2. Genome sequencing and analysis The genomic DNA of strain IDO3 was extracted using TIANamp Bacteria DNA Kit (TIANGEN, China), and sequenced using PacBio RS II (Pacific Biosciences, CA, USA) platform at the Beijing Novogene Bioinformatics Technology Co., Ltd. The low-quality reads were filtered by the SMRT Link v5.0.1, which were assembled to generate the complete genome of strain IDO3. GeneMarkS program was used to retrieve and predict the related coding genes, and the final genome was annotated by Rapid Annotation using Subsystem Technology (RAST) and NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Besemer et al., 2001; Overbeek et al., 2014). Function analyses of the genes were performed by BLAST search against GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), and NR (Non-Redundant Protein Database databases) with evalue less than 1e-5 and minimal coverage 40%.
2.6. Heterologous expression and characterization of the iifF gene Gene iifF was amplified and expressed in E. coli BL21(DE3) as described above (same with iifC2D2). The engineered strain was cultured in LB medium with kanamycin and IPTG at 20 °C overnight. The enzyme was purified from induced bacterial cells as previously reported (Bjerregaard-Andersen et al., 2014), which is summarized in Supplementary Material. The enzymatic activity of IifF was determined as a function of the isatin hydrolysis and isatinate production, which has a typical absorption peak at 368 nm (Bjerregaard-Andersen et al., 2014). Measurements were done in a UV-5500P spectrophotometer (Metash, China) with a cuvette light path of 1 cm. The enzyme reaction was performed in a reaction buffer (50 mM pH 8.0 Tris-HCl, 100 mM NaCl). 2 μL stock MnCl2 solution (0.2 M) was added to 1994 μL reaction buffer in the cuvette, and the reaction was quickly started by adding 4 μL enzyme stock solution (1 μM). The reaction was performed at room temperature (20 °C). An extinction coefficient of 4.5 × 103 cm−1 mol−1 L was used for isatinate (Olesen and Jochimsen, 1996). The data were fitted with the Michaelis-Menten equation using the software SigmaPlot 11.0.
2.3. RT-qPCR assay Strain IDO3 was cultured in sodium citrate medium (control group) and 100 mg L−1indole-sodium citrate medium (experiment group) for 24 h. Then the bacterial cells were collected and total RNA was extracted using the Bacteria RNA Extraction Kit (Vazyme, China). cDNA was synthesized by M-MLV Reverse Transcriptase (Invitrogen, USA) with the total RNA amount of 360 ng. The cDNA was diluted as the template for RT-qPCR analysis. RT-qPCR was conducted using Roche LightCycler® 480 system (Roche, Switzerland) with corresponding primers (Table S2) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). RT-qPCR program is provided in Table S3. Relative expression level was calculated using 2−ΔΔCT method with 16S rRNA gene as the reference gene (Livak and Schmittgen, 2001; Qu et al., 2017). The experiment was conducted using three biological replicates.
2.7. Nucleotide sequence accession numbers The complete genome sequence of Burkholderia sp. IDO3 has been deposited at GenBank under the accession numbers of CP028962, CP028963, CP028964, and CP028965. The accession numbers for protein IifC1, IifD1, IifC2, IifD2, and IifF are APT36898.1, APT36899.2, AXK62974.1, AXK62973.1, and WP_096501352, respectively.
37
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
2), 851,589 (chromosome 3) and 520,805 bp (plasmid) in length (Table 1, Fig. 1). A total of 7550 genes, including 90 RNAs, were predicted in the genome. KEGG pathway analysis indicated that most sequences were associated with membrane transport and metabolism pathways, including amino acid metabolism, carbohydrate metabolism, energy metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism (Fig. S1). Within the xenobiotics biodegradation and metabolism pathway, KEGG Orthology (KO) associated with aminobenzoate degradation (14 KOs), benzoate degradation (37 KOs), xylene degradation (13 KOs), chlorocyclohexane and chlorobenzene degradation (12 KOs), styrene degradation (10 KOs), toluene degradation (10 KOs), and naphthalene degradation (4 KOs) were identified (Table S5). Burkholderia occupies notably diverse ecological niches, such as contaminated soils, water, plant rhizospheres, and human respiratory tracts (Coenye and Vandamme, 2003). This genus has the potential to degrade many aromatic compounds (Pérez-Pantoja et al., 2012). In the previous study, we found that Burkholderia sp. IDO3 could effectively degrade indole, as well as other aromatic pollutants including skatole, phenol, and cresol (Ma et al., 2019). The numerous pathways identified
Table 1 Genome features of Burkholderia sp. IDO3. Features
Chr1
Chr2
Chr3
Plasmid
Genome
Size (bp) GC content (%) Number of genes rRNA tRNA Other RNA
3,139,806 67.0 2885 3 6 0
3,591,606 66.4 3444 12 60 4
851,589 66.33 784 3 2 0
420,805 61.57 437 0 0 0
8,003,806 66.4 7550 18 68 4
3. Results and discussions 3.1. Genome information of strain IDO3 The 140,176 (1,016,768,870 bp) obtained reads were de novo assembled into four polished contigs, with an average genome coverage of 127-fold. The genome of strain IDO3 had an overall GC content of 66.37% and consisted of three circular chromosomes and one plasmid, which were 3,139,806 bp (chromosome 1), 3,591,606 bp (chromosome
Fig. 1. Circular maps of the chromosomes and plasmid of Burkholderia sp. IDO3. A, chromosome 1; B, chromosome 2; C, chromosome 3; D, plasmid. From outside to center: predicted genes, COG categories, KEGG categories, GO categories, GC content, and GC skew. The iif1 gene cluster is located on Chr3 and iif2 on Chr1. Detailed KEGG categories are shown in Fig. S1. 38
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
Fig. 2. Indole oxidation gene cluster analysis in strain IDO3. Acinetobacter sp. O153 and Cupriavidus sp. SHE are two previously characterized indole-degrading strains.
here suggested that strain IDO3 might have potential applications in aromatic pollutants bioremediation. The genomic information of strain IDO3 will facilitate the molecular mechanism and bioremediation study of Burkholderia. 3.2. Identification of two iif gene clusters associated with indole metabolism The functional genes responsible for indole degradation were recently identified. The FAD-dependent oxygenase IifC and reductase IifD compose the two-component indole oxygenase system, which catalyzes the oxidation of indole in Acinetobacter (Sadauskas et al., 2017). To explore the functional genes for indole degradation in strain IDO3, the traditional genomic clone library method was applied to screen possible genes. An iif gene cluster (here designated iif1, Fig. 2) was previously identified and shown to be able to oxidize indole (Ma et al., 2019). To further explore the indole-degrading functional genes, a local BLAST comparing the indole oxygenase protein IifC from strain Acinetobacter sp. O153 to all protein sequences in strain IDO3 was conducted (Sadauskas et al., 2017). The protein IifC1 had a 39% similarity with the homolog IifC in strain O153. Interestingly, another indole oxygenase IifC2 was identified that displayed 62% similarities with IifC. Further analysis showed that iifC1 was located on chromosome 3 and iifC2 on chromosome 1, both of which clustered with the iifB and iifD genes. The evolutionary analyses of IifC1, IifC2 with other indole oxygenases and styrene monooxygenases were provided in Fig. S2. It was obvious that IifC2 was closely related to the indole oxygenases from Cupriavidus and Acinetobacter, whereas IifC1 formed a separate clade from the functionally similar indole oxygenases. IifD1 and IifD2, the reductase components of indole oxygenases, showed 45% and 47% similarities with corresponding proteins in strain O153. It was previously reported that iifA is a common component of the iif gene clusters. IifA is possibly a cofactor-independent oxygenase that catalyzes the transformation of 3-hydroxyindolin-2-one to anthranilic acid. However, we noted that iifA was absent from both the iif1 and iif2 clusters (Fig. 2). Therefore, the indole upstream transformation pathway in strain IDO3 may be distinct from that of Acinetobacter sp. O153 (Sadauskas et al., 2017). IifB encodes a short-chain dehydrogenase, which might convert indole oxidation products (possibly indole-2,3-dihydrodiol) to 3-hydroxyindolin-2-one. Here, IifB1 and IifB2 showed 52% and 59% similarities with IifB in strain O153. Furthermore, a novel gene, termed iifF, was identified in the iif2 gene cluster. Further analysis of this gene indicated that it encoded an isatin hydrolase gene, which might be responsible for isatin biotransformation in indole degradation process (Bjerregaard-Andersen et al., 2014).
Fig. 3. RT-qPCR verification of the iif gene clusters. The treatment group was cultured in sodium citrate medium with 100 mg L−1 indole, and the control group was cultured in sodium citrate medium.
5.3 ± 0.67, 5.04 ± 2.7, 3.71 ± 2.06, 2.66 ± 0.86, 1.53 ± 0.27, and 3.08 ± 1.20 folds, respectively. This suggested that both gene clusters were likely to participate in indole metabolism.
3.4. Heterologous expression of indole oxygenase genes The iifC1D1 genes were previously heterologously expressed in E. coli strain (Ma et al., 2019). In this study, iifC2D2 genes were also expressed in E. coli BL21(DE3) strain. As E. coli strain harbors tryptophanase, which can convert tryptophan to indole endogenously but is unable to convert indole to indigo (Lee and Lee, 2010), the recombinant strains were cultured in a tryptophan-rich medium to examine their indigo production ability. It was found that the recombinant strains carrying the iifC1D1 and iifC2D2 genes could both produce indigo in tryptophan medium, while the E. coli strain carrying the empty pET-28a (+) plasmid did not produce indigo. This proved that IifC1D1 and IifC2D2 were able to oxidize indole (Fig. S3). The indigo yields for strains E. coli_IifC1D1 and E. coli_IifC2D2 were 37.1 ± 8.5 and 78.1 ± 2.0 mg L−1, respectively (Fig. 4), implying that the indole oxygenases obtained here may be applied for indigo production studies. The indigo bio-production abilities of several other oxygenases have previously been investigated. For example, the E. coli strain carrying the phenol hydroxylase gene from Acinetobacter sp. ST-550 could produce 52 mg L−1 indigo in a water-organic solvent two-phase system (Doukyu et al., 2003). Over-expression of the styrene monooxygenase in Pseudomonas putida led to the production of 52.1 mg L−1 indigo (Cheng et al., 2016). The most investigated naphthalene dioxygenase expressed in E. coli yielded 135 mg L−1 indigo directly from glucose in laboratory experiment after a series of genetic manipulations (Murdock et al., 1993). Furthermore, the recombinant E. coli strain expressing the flavin
3.3. Transcriptomic analysis of strain IDO3 in response to indole RT-qPCR assays were conducted to determine the mRNA expression levels of both gene clusters in response to indole. It was shown that indole increased the expression of all chosen genes (Fig. 3). Genes iifB1, iifC1, iifD1, iifB2, iifC2, iifD2, and iifF were up-regulated by 3.3 ± 0.70, 39
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
3.5. Mutations study of the two indole oxygenase genes To further elucidate the indole degradation mechanism in strain IDO3, gene knockout experiments were performed. We successfully obtained three mutant strains, i.e. IDO3-ΔiifC1, IDO3-ΔiifC2, and IDO3ΔiifC1ΔiifC2 (Table S1), after extensive genetic manipulations. A comparison of the PCR products of the iifC1 and iifC2 genes among the three mutant strains and the wildtype strain clearly showed the disruption of the relevant genes (Fig. S4). The indole degradation abilities of these strains were carefully investigated. The indole degradation curves of the four strains were shown in Fig. 5. It was obvious that indole degradation process of strain IDO3-ΔiifC1 was similar to that of wildtype IDO3, both of which removed ∼120 mg L−1 indole within 12 h. This suggested that gene iifC1 played a limited role in indole degradation under the tested condition. The indole removal capacities of strains IDO3-ΔiifC2 and IDO3-ΔiifC1ΔiifC2 were significantly reduced as compared to the wildtype. Strain IDO3-ΔiifC2 removed the indole in ∼60 h, indicating that gene iifC2 was crucial for indole metabolism. It was worth noting that strain IDO3ΔiifC1ΔiifC2 could not completely remove the indole and almost 80% indole remained in the medium. This result indicated that gene iifC1 also participated in the indole degradation process in strain IDO3, especially when the iifC2 gene was absent from strain IDO3. Therefore, above data indicate that iifC2 rather than iifC1 plays crucial roles in indole degradation. As two copies of the indole oxygenase genes are not common in other bacterial strains, the evolutional significance of this duplication requires further investigation. The iif gene cluster has been identified in Acinetobacter, Pseudomonas, Cupriavidus, Alcaligenes, Burkholderia and Ralstonia, and these genera are the most reported indole-degrading bacteria (Sadauskas et al., 2017). This suggests that the iif-dependent pathway is the most common indole degradation pathway. However, some strains (e.g. Pseudomonas aeruginosa PAO1) lacking the iif gene cluster can also rapidly degrade indole (Lee et al., 2009), suggesting that other functional genes should be involved in indole biodegradation.
Fig. 4. Production of indigo in E. coli strains expressing iifC1D1 and iifC2D2. From left to right are E. coli strain with empty pET-28a(+) plasmid, strain E. coli_IifC1D1, and E. coli_IifC2D2.
3.6. Heterologous expression and functional study of the iifF gene
Fig. 5. Indole degradation curves of strain IDO3 and its mutant strains.
Isatin hydrolase is reported to be involved in indole-3-acetic acid degradation, which can hydrolyze isatin to isatinate (Olesen and Jochimsen, 1996). This new class of metalloenzyme has received much recent attention because isatin plays a neurological role in human and acts as a signal molecule in bacteria. The crystal structures of the isatin hydrolases have been revealed (Bjerregaard-Andersen et al., 2014).
monooxygenase gene from Methylophaga aminisulfidivorans MPT produced up to 920 mg L−1 indigo under optimal conditions (Han et al., 2008). Compared with these oxygenases in favor of indigo production, the indigo yield in our study should be further improved.
Fig. 6. Phylogenetic analysis and expression of IifF. (A) Phylogram of IifF and its homologs. IHA/B, isatin hydrolase A/B; KynB; kynurenine formamidas; AHD; amidohydrolase. The evolutionary history was inferred using the Neighbor-Joining method with bootstrap test of 1000 replicates, and the figure was produced using MEGA7 (Kumar et al., 2016). (B) Amplification of iifF and the pET-28a(+) plasmid. From left to right are DNA marker, iifF gene PCR product, and pET-28a(+) PCR product. Primers used for PCR are given in Table S2. (C) SDS-PAGE analysis of purified IifF. From left to right are protein marker and IifF protein bands. 40
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
Fig. 7. Catalytic performance of IifF. (A) The full wavelength spectra of isatin and its catalytic products by IifF. (B) Michaelis-Menten curves for verification of IifF kinetic model.
Fig. 8. The predicted indole metabolic pathway of strain IDO3.
first time, we are showing that the isatin hydrolase gene (iifF) is a component of the iif gene cluster. The function of iifF is to catalyze isatin hydrolysis to produce isatinate, demonstrating that isatin is a real metabolic intermediate of the indole degradation process. We also tested the growth ability of strain IDO3 with isatin as the carbon source. Data showed that strain IDO3 could grow within two days (Fig. S6). As isatin can be hydrolyzed in the aqueous medium, whether strain IDO3 uses isatin or its transformation products (such as isatinate) as the growth substrate is to be confirmed. A proposed indole metabolic pathway in strain IDO3 is provided in Fig. 8. Indole is oxidized to oxidation products (such as indole-2,3-dihydrodiol, indoxyl, or indole epoxide) via IifC1D1 and IifC2D2, of which IifC2D2 plays the crucial role in this process (Heine et al., 2019). The oxidation products are converted to 3-hydroxyindolin-2-one by IifB1 and IifB2 based on the previous study (Sadauskas et al., 2017). Isatin is formed by oxidation or dehydrogenation process, which is further hydrolyzed to isatinate by IifF. Finally, the anthranilate might be produced from 3-hydroxyindolin-2-one, isatin, or isatinate by oxygenases or decarboxylases. However, some issues in indole degradation process, such as isatin formation, isatinate conversion and anthranilate degradation, will require further investigation.
BLAST analysis of the IifF amino acid sequence indicated that it shared 49% identity with isatin hydrolase A (LaIHA) from Labrenzia aggregate, 58% identity with isatin hydrolase B (LaIHB) from L. aggregate, and 84% identity with isatin hydrolase (RsIHA) from Ralstonia solanacearum (Fig. 6A). Alignment analysis showed that IifF was similar to other isatin hydrolases containing the central metal binding motif HxG[T/A] HxDxPxH as shown in Fig. S5. In addition, IifF also contained the other conserved GLQC motif for isatin hydrolase activity (Sommer et al., 2018). To further explore the characteristics of IifF, gene iifF was amplified from strain IDO3 DNA and successfully expressed in E. coli (Fig. 6B). The purified his-tagged IifF was obtained using Ni-NTA agarose with elution buffer (50 mM pH 8.0 Tris-HCl, 0.5 M NaCl, 10 mM Na2EDTA and 50 mM imidazole). The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Fig. 6C). The calculated molecular weight of IifA was 32.7 kDa, consistent with the SDS-PAGE results. Activity measurements confirmed that IifF harbored isatin hydrolase activity. It was shown in Fig. 7A that isatin was converted to a product with the characteristic absorption peak at 368 nm, identical to isatinate. Isatin hydrolysis followed Michaelis-Menten kinetics. The determined Km was 4.4 ± 0.7 μM, similar to that of LaIHB (Fig. 7B, Sommer et al., 2018). The kcat value was 95.5 ± 4 s−1, similar to that of LaIHA and RsIHA (Sommer et al., 2018). These results indicated that IifA had similar enzymatic characteristics to previously reported isatin hydrolases. Although isatin has been identified as a possible indole degradation intermediate in several studies (Ma et al., 2018), the functional gene has not yet been identified. As previously reported iif gene clusters did not contain the isatin hydrolase iifF gene, it was debatable whether isatin participated to the indole degradation process or whether it was just an experimental artifact (Sadauskas et al., 2017). Herein, for the
4. Conclusion The complete genome of an indole-degrading strain Burkholderia sp. IDO3 was sequenced. Analysis showed that strain IDO3 contained two indole oxygenase gene clusters on Chr1 and Chr3. A combination of RTqPCR heterologous expression and gene knockout assays showed that both iif1 and iif2 clusters were involved in indole degradation process. The indole degradation ability of strain IDO3-ΔiifC2 was remarkably reduced, suggesting that iifC2 gene was crucial for indole oxidation. A 41
International Biodeterioration & Biodegradation 142 (2019) 36–42
Q. Ma, et al.
novel iifF gene was identified in the iif2 gene cluster. Bioinformatic analyses and gene expression studies proved that iifF encoded an isatin hydrolase, which was responsible for isatin hydrolysis to isatinate.
aromatic ring cleavage and production of indigoids. Int. Biodeterior. Biodegrad. 97, 13–24. Han, G.H., Shin, H.J., Kim, S.W., 2008. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene. Enzym. Microb. Technol. 42, 617–623. Heine, T., Großmann, C., Hofmann, S., Tischler, D., 2019. Indigoid dyes by group E monooxygenases: mechanism and biocatalysis. Biol. Chem. https://doi.org/10.1515/ hsz-2019-0109. Kim, D., Rahman, A., Sitepu, I.R., Hashidoko, Y., 2013. Accelerated degradation of exogenous indole by Burkholderia unamae strain CK43B exposed to pyrogallol-type polyphenols. Biosc. Biotech. Biochem. 77, 1722–1727. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Lee, J.H., Lee, J., 2010. Indole as an intercellular signal in microbial communities. FEMS Microbiol. Rev. 34, 426–444. Lee, J., Attila, C., Cirillo, S.L.G., Cirillo, J.D., Wood, T.K., 2009. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence. Microbial Biotechnology 2, 75–90. Lee, J.H., Wood, T.K., Lee, J., 2015. Roles of indole as an interspecies and interkingdom signaling molecule. Trends Microbiol. 23, 707–718. Lin, G.H., Chen, H.P., Shu, H.Y., 2015. Detoxification of indole by an indole-induced flavoprotein oxygenase from Acinetobacter baumannii. PLoS One 10, e0138798. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408. Ma, Q., Qu, Y., Zhang, X., Liu, Z., Li, H., Zhang, Z., Wang, J., Shen, W., Zhou, J., 2015. Systematic investigation and microbial community profile of indole degradation processes in two aerobic activated sludge systems. Sci. Rep. 5, 17674. Ma, Q., Zhang, X., Qu, Y., 2018. Biodegradation and biotransformation of indole: advances and perspectives. Front. Microbiol. 9, 2625. Ma, Q., Liu, Z., Yang, B., Dai, C., Qu, Y., 2019. Characterization and functional gene analysis of a newly isolated indole-degrading bacterium Burkholderia sp. IDO3. J. Hazard Mater. 367, 144–151. Mackie, R.I., Stroot, P.G., Varel, V.H., 1998. Biochemical identification and biological origin of key odor components in livestock waste. J. Anim. Sci. 76, 1331–1342. Madsen, E.L., Bollag, J.M., 1989. Pathway of indole metabolism by a denitrifying microbial community. Arch. Microbiol. 151, 71–76. Murdock, D., Ensley, B.D., Serdar, C., Thalen, M., 1993. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli. Nat. Biotechnol. 11, 381–386. Olesen, M.R., Jochimsen, B.U., 1996. Identification of enzymes involved in indole-3acetic acid degradation. Plant Soil 186, 143–149. Overbeek, R., Olson, R., Pusch, G.D., Olsen, G.J., Davis, J.J., Disz, T., et al., 2014. The SEED and the Rapid annotation of microbial genomes using subsystems Technology (RAST). Nucleic Acids Res. 42, D206–D214. Qu, Y., Ma, Q., Liu, Z., Wang, W., Tang, H., Zhou, J., Xu, P., 2017. Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain. Mol. Microbiol. 106, 905–918. Sadauskas, M., Vaitekūnas, J., Gasparavičiūtė, R., Meškys, R., 2017. Indole biodegradation in Acinetobacter sp. strain O153, genetic and biochemical characterization. Appl. Environ. Microbiol. 83 e01453-17. Sommer, T., Bjerregaard-Andersen, K., Uribe, L., Etzerodt, M., Diezemann, G., Gauss, J., Cascella, et al., 2018. A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase. Sci. Rep. 8, 13104. Yang, Z., Zhou, J., Xu, Y., Zhang, Y., Luo, H., Chang, K., 2017. Analysis of the metabolites of indole degraded by an isolated Acinetobacter pittii L1. BioMed Res. Int. 2017, 2564363.
Conflicts of interest The authors declare no conflicts of interest. Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 31800091), the Doctoral Scientific Research Foundation of Liaoning Province (No. 20180540072), and the Open Foundation of Key Laboratory of Industrial Ecology and Environmental Engineering, MOE (No. KLIEEE-17-01). Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.ibiod.2019.04.011. References Pérez‐Pantoja, D., Donoso, R., Agulló, L., 2012. Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales. Environ. Microbiol. 14, 1091–1117. Arora, P.K., Sharma, A., Bae, H., 2015. Microbial degradation of indole and its derivatives. J. Chem. 2015, 129–159. Besemer, J., Lomsadze, A., Borodovsky, M., 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29, 2607–2618. Bjerregaard-Andersen, K., Sommer, T., Jensen, J.K., Jochimsen, B., Etzerodt, M., Morth, J.P., 2014. A proton wire and water channel revealed in the crystal structure of isatin hydrolase. J. Biol. Chem. 289, 21351–21359. Cheng, L., Yin, S., Chen, M., Sun, B., Hao, S., Wang, C., 2016. Enhancing indigo production by over-expression of the styrene monooxygenase in Pseudomonas putida. Curr. Microbiol. 73, 248–254. Claus, G., Kutzner, H.J., 1983. Degradation of indole by Alcaligenes spec. Syst. Appl. Microbiol. 4, 169–180. Coenye, T., Vandamme, P., 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol. 5, 719–729. Doukyu, N., Toyoda, K., Aono, R., 2003. Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp. strain ST-550 in a water-organic solvent two-phase system. Appl. Microbiol. Biotechnol. 60, 720–725. Fujioka, M., Wada, H., 1968. The bacterial oxidation of indole. Biochim. Biophys. Acta. 158, 70–78. > Fukuoka, K., Tanaka, K., Ozeki, Y., Kanaly, R.A., 2015. Biotransformation of indole by Cupriavidus sp. strain KK10 proceeds through N-heterocyclic-and carbocyclic-
42