Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in Bradyrhizobium sp. RD5-C2 Isolated from Non-Contaminated Soil

Abstract

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.

Since the 1940s, chlorophenoxy herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), have been widely used to control the growth of broadleaf weeds. These two herbicides were the main components of Agent Orange, which was sprayed during the Vietnam War. 2,4-D is still used worldwide, and is a model compound for studying the microbial acquisition of genes capable of degrading anthropogenic chemicals and the distribution of genes within a microbial genome. Diverse 2,4-D-degrading bacteria, belonging to Actinobacteria, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria phyla, have been isolated from various environments. Although 2,4,5-T has been prohibited worldwide due to its toxicity to humans, residues of 2,4,5-T have been reported in Canada, USA, and Vietnam (Rice et al., 2005; Donald et al., 2007; Huong et al., 2007b). Since 2,4,5-T is more persistent than 2,4-D, fewer studies have been conducted on 2,4,5-T-degrading bacteria than on 2,4-D degraders (Kilbane et al., 1982; Golovleva et al., 1990; Rice et al., 2005; Huong et al., 2007b).

2,4-D-degrading bacteria typically possess the tfdA (tfdAα), tftAB, and/or cadAB(C) genes, which catalyze the first step of the degradation pathway (Nojiri et al., 2014; Serbent et al., 2019). The proteins encoded by tfdA, tftAB, and cadABC catalyze the transformation of 2,4-D into 2,4-dichlorophenol (2,4-DCP). tfdA encodes an α-ketoglutarate-dependent dioxygenase (TfdA), tftA and cadA encode oxygenase large subunits (TftA and CadA, respectively), tftB and cadB encode small subunits (TftB and CadB, respectively), and cadC encodes a ferredoxin component (CadC). Dichlorophenol hydroxylase (TftB) then converts 2,4-DCP into 3,5-dichlorocatechol, which is further degraded via a modified ortho-cleavage pathway (Liu and Chapman, 1984; Perkins et al., 1990; Laemmli et al., 2000). Briefly, the cleavage of 3,5-dichlorocatechol by chlorocatechol 1,2-dioxygenase (TfdC) forms 2,4-dichloro-cis-cis-muconate. This is converted to 2-chlorodienelactone by chloromuconate cycloisomerase (TfdD), which is then transformed to 2-chloromaleylacetate by chlorodienelactone hydrolase (TfdE). 2-Chloromaleylacetate is converted by chloromalelylacetate reductase (TfdF), through the formation of maleylacetate, into beta-ketoadipate, which then enters the tricarboxylic acid cycle (Laemmli et al., 2000; Kumar et al., 2016b; Serbent et al., 2019).

Although the induction of cadABC remains unclear, cadR in the 2,4-D degrader Bradyrhizobium sp. HW13 was shown to be essential for the heterologous expression of cadA (Kitagawa et al., 2002). In contrast, in the non-2,4-D degrader Bradyrhizobium elkanii USDA94, cadR did not lead to the downstream induction of cadABC even when 2,4-D was present in the culture medium (Hayashi et al., 2016).

Various 2,4,5-T-degrading bacteria, including Burkholderia spp. (Kellogg et al., 1981; Danganan et al., 1994; Xun and Wagnon, 1995; Huong et al., 2007b), Nocardioides simplex 3E (Golovleva et al., 1990), Sphingomonas spp. (Huong et al., 2007b), and Bradyrhizobium spp. (Rice et al., 2005; Huong et al., 2007b), have been reported. Previous studies on degradation genes demonstrated that TftAB and CadABC have the ability to convert 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP) (Danganan et al., 1994; Kitagawa et al., 2002; Hayashi et al., 2016).

The multiplicities of cad genes responsible for 2,4-D and 2,4,5-T degradation have not yet been reported. However, previous studies showed the multiplicities of genes that encode degrading enzymes for xenobiotic compounds. For example, Cupriavidus pinatubonensis JMP134 contains duplicate tfdBCDEF gene clusters for chlorophenol degradation (Leveau et al., 1999), Sphingomonas sp. KA1 encodes two distinct car clusters for carbazole degradation (Urata et al., 2006), Rhodococcus jostii RHA1 possesses three chlorobiphenyl 2,3-dioxygenase genes (Iwasaki et al., 2006), and Mycobacterium spp. harbor several pyrene-degrading gene clusters (Sho et al., 2004; Kim et al., 2006; Zhang and Anderson, 2012). Two methods generally lead to multiplicities in catabolic genes-gene duplication in the genome and horizontal gene transfer from outside sources. The multiplicities of catabolic genes may promote adaption to the use of novel sources in the environment (Gevers et al., 2004). The study of genes involved in the degradation of xenobiotic compounds has provided insights into the acquisition of this ability in several environments. The information obtained has contributed to a more detailed understanding of how and why bacteria adapt and evolve to acquire the ability to degrade xenobiotic compounds. The multiplicities of related genes are considered to be one step in the process of the acquisition of this ability.

We previously isolated Bradyrhizobium sp. RD5-C2, a 2,4-D-degrading strain, from arable soil in Japan with no history of exposure to 2,4-D (Itoh et al., 2000). This strain possesses a cadA gene (designated as cadA1 in the present study), which is highly similar to the cadA gene of another 2,4-D degrader, Bradyrhizobium sp. HW13 (Fig. 1) (Itoh et al., 2004). It also possesses the tfdAα gene, which exhibits weak 2,4-D dioxygenase activity when expressed in Escherichia coli (Itoh et al., 2002). The purpose of the present study was to obtain genetic information on the degradation genes present in Bradyrhizobium sp. RD5-C2 and elucidate the role of the aforementioned three degradation genes in the degradation of 2,4-D and 2,4,5-T.

Fig. 1.

Comparison of cad clusters and tfdAα genes from Bradyrhizobium sp. HW13, Bradyrhizobium sp. RD5-C2, and Bradyrhizobium elkanii USDA94. Each gene is represented by a large horizontal arrow containing its GC content (mol %). Similarity values (%) of the deduced amino acid sequences of corresponding proteins are represented with thin lines. Genes from Bradyrhizobium sp. RD5-C2 are indicated in bold.

Materials and Methods

Identification and phylogenetic analysis of cad clusters and tfdAα in Bradyrhizobium sp. RD5-C2

Draft genome sequencing of Bradyrhizobium sp. RD5-C2 was performed using Illumina HiSeq2000 equipment (Illumina). The de novo assembly of the resulting sequence data was performed using Velvet software version 1.2.08. Two distinct cad clusters, designated as cad1 and cad2, and tfdAα were identified in the genome using the sequences of cadA and tfdAα from Bradyrhizobium sp. RD5-C2 (accession no. AB119238 and AB074490) and B. elkanii USDA94 (AB119244), respectively, with the MUMmer 3.23 software program (Kurtz et al., 2004). Putative ORFs were identified using the Joint Genome Institute portal (http://jgi.doe.gov/), and phylogenetic trees were constructed using the neighbor-joining method with the MEGA7 software program (Kumar et al., 2016a). The nucleotide sequences identified in the present study have been annotated using DFAST and deposited in the DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp/index-j.html); accession numbers for the draft genome sequences are BOVL01000001 to BOVL01000073. The locus tags of cadR1A1B1K1C1, cadR2A2B2C2K2S, and tfdAα are BraRD5C2_67200 to BraRD5C2_67240, BraRD5C2_72670 to BraRD5C2_72620, and BraRD5C2_05110, respectively. All genes used in the present study are listed in Table S1.

Bacterial strains, plasmids, and growth conditions

All Bradyrhizobium strains, plasmids, and primers used in the present study are listed in Table 1, S2, and S3. Bradyrhizobium strains were cultivated in HM medium at 25°C (Minamisawa et al., 1998), and E. coli strains were manipulated as previously described (Sambrook and Russell, 2001). E. coli S17-1λpir (Mazodier et al., 1989) was used as the transconjugation donor, and E. coli transformants were grown in Luria-Bertani (LB) medium (Sambrook and Russell, 2001) with appropriate antibiotics at 37°C.

Table 1. Bradyrhizobium strains used in the present study.

Strain	Characteristics	Source or reference
Bradyrhizobium sp.
RD5-C2	Wild-type strain, TcR, KmS	(Itoh et al., 2000)
RD5-C2ΔcadA1	In-frame disruption mutant of cadA1 of RD5-C2	This study
RD5-C2ΔcadA2	In-frame disruption mutant of cadA2 of RD5-C2	This study
RD5-C2ΔtfdAα	In-frame disruption mutant of tfdAα of RD5-C2	This study
RD5-C2ΔcadA1ΔcadA2	In-frame disruption mutant of cadA1 and cadA2 of RD5-C2	This study
RD5-C2ΔcadA1ΔcadA2tfdAα	In-frame disruption mutant of cadA1, cadA2, and tfdAα of RD5-C2	This study
RD5-C2ΔcadR1	In-frame disruption mutant of cadR1 of RD5-C2	This study
RD5-C2ΔcadA1/BBR2-cadA1	RD5-C2ΔcadA1 harboring pBBR2-C2cadA1pro-cadA1, Kmr	This study
RD5-C2ΔcadA1/BBR2	RD5-C2ΔcadA1 harboring pBBR1MCS2_START (empty vector), Kmr	This study
Bradyrhizobium elkanii
USDA94	Wild-type strain, TcR, KmS	(Minamisawa et al., 2002)
USDA94BBR2C2cad1ABKC	USDA94 harboring pBBR2-C2cad1ABCK, Kmr	This study
USDA94BBR2C2cad2ABCK	USDA94 harboring pBBR2-C2cad2ABCK, Kmr	This study
USDA94BBR2	USDA94 harboring pBBR1MCS2_START (empty vector), Kmr	This study

Km: Kanamycin, Tc: Tetracycline

Cloning of cadA1B1K1C1 and cadA2B2C2K2 into B. elkanii USDA94

To examine the degradation activities of cadA1B1K1C1 and cadA2B2C2K2 in a related strain of Bradyrhizobium sp. RD5-C2, the clusters were expressed in the non-2,4-D degrader, B. elkanii USDA94. The cadA1B1K1C1 fragment was amplified with PCR using KOD plus DNA polymerase (Toyobo) with the primer set of C2cad1A-F-Nde/C2cad1C-R-Bam. The PCR amplification mixture was prepared according to the manufacturer’s instructions. The amplification reaction was as follows: 94°C for 2‍ ‍min, followed by 30 cycles at 95°C for 15‍ ‍s, 60°C for 15‍ ‍s, and 68°C for 6.5‍ ‍min. The amplified DNA fragment was then digested with NdeI and BamHI (FastDigest, Thermo Fisher Scientific) and inserted into the multiple cloning site of pBBR1MCS2_START (Obranić et al., 2013) to yield pBBR2-C2cad1ABKC. cadA2B2C2K2 and pBBR1MCS2_START fragments were amplified with PCR using KOD plus neo DNA polymerase (Toyobo) with the primer sets BBR2+C2cad2-F/BBR2+C2cad2-R and C2cad2+BBR2-F/C2cad2+BBR2-R, respectively. The amplification reaction was as follows: 94°C for 2‍ ‍min followed by 30 cycles at 98°C for 10‍ ‍s, 64°C (cadA2B2C2K2) or 62°C (pBBR1MCS2_START) for 30‍ ‍s, and 68°C for 2‍ ‍min (cadA2B2C2K2) or 2‍ ‍min and 45‍ ‍s (pBBR1MCS2_START). The amplified fragments were assembled to yield pBBR2-C2cad2ABKC using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions. The constructed plasmids were cloned into E. coli, and the transformants were cultivated on LB agar medium supplemented with kanamycin (30‍ ‍mg L^–1). The fidelity of inserts was confirmed with nucleotide sequencing. The extracted plasmid was transformed into E. coli S17-1λpir and subsequently introduced into B. elkanii USDA94 using conjugative transformation, as previously described (Hayashi et al., 2016).

Chlorophenoxyacetic acid-degrading activities of B. elkanii transformants

Bradyrhizobium transformants were cultivated in HM medium containing 100‍ ‍μM 2,4-D or 100‍ ‍μM 2,4,5-T and kanamycin (150‍ ‍mg L^–1) at 25°C with shaking. At appropriate intervals, the concentrations of compounds and degradation products (2,4-DCP and 2,4,5-TCP) in the supernatant were measured using a Prominence ultra-fast liquid chromatography system (Shimadzu) equipped with an SPD-M20A photodiode array (Shimadzu) and Shim-pack XR-ODS column (2.2‍ ‍μm, 100‍ ‍mm length×3.0‍ ‍mm i.d., Shimadzu), as previously described (Hayashi et al., 2016).

Construction of Bradyrhizobium sp. RD5-C2 cadA1, cadA2, tfdAα, and cadR1 deletion mutants

To produce in-frame deletion mutants of cadA1, cadA2, and tfdAα, insertional inactivation via double crossover was performed as previously described (Hayashi et al., 2016). The upstream and downstream regions of each gene were PCR-amplified using the following primer sets: Dcad1Aup5-Eco/Dcad1Aup3-Xba and Dcad1Adw5-Xba/Dcad1Adw3-Hind for cadA1; DcadAup5-Kpn/DcadAup3-Xba and DcadAdw5-Xba/DcadAdw3-Hind for cadA2; DtfdAαup5-Kpn/DtfdAαup3-Xba and DtfdAαdw5-Xba/DtfdAαdw3-Hind for tfdAα. The digested fragments were ligated into the multiple cloning sites of pK18mob (Schäfer et al., 1994) to generate pK18mob-C2cadA1updw, pK18mob-C2cadA2updw, and pK18mob-C2tfdAαupdw with in-frame deletions in cadA1, cadA2, and tfdAα, respectively. The resulting plasmids were introduced into Bradyrhizobium sp. RD5-C2 via E. coli S17-1λpir. Double-crossover mutants were screened from single crossover mutants based on kanamycin sensitivity. Successful in-frame deletions of 1,011 bp in cadA1, 1,038 bp in cadA2, and 634 bp in tfdAα were confirmed by the sequencing of new junction regions. To construct cadA1 and cadA2 double-deletion mutants, the 4-kb fragment in pK18mob-C2cadA1updw was amplified using the primers DcadAup5-Bam/DcadAdw3-Hind and then cloned into the multiple cloning sites of pK18mobsacB (Schäfer et al., 1994) to yield pK18mobsacB-C2cadA2updw, which was cloned into Bradyrhizobium sp. RD5-C2ΔcadA1 to delete the cadA2 gene. To generate cadA1, cadA2, and tfdAα triple-deletion mutants, the tfdAα gene of Bradyrhizobium sp. RD5-C2ΔcadA1ΔcadA2 was deleted using pK18mobsacB-C2tfdAαupdw, which contains the PCR-amplified fragment of pK18mob-94tfdAαupdw, using the primers DtfdAaup5-Bam/DtfdAαdw3-Hind. Double-crossover mutants were screened by culturing on HM medium containing 5% sucrose, which kills cells that containing the sacB (levansucrase) gene derived from pK18mobsacB (Schäfer et al., 1994), and using kanamycin sensitivity. To construct a cadR1 deletion mutant, the upstream and downstream regions of cadR1 were amplified using the primer sets DC2cad1up5-Hin/DC2cad1up3-Xba and DC2cad1Rdw5-Xba/DC2cad1Rdw3-Bam, respectively. Digested fragments were ligated into the multiple cloning sites of pK18mobsacB (Schäfer et al., 1994) to yield pK18mobsacB-C2cadR1updw. The deletion of cadR1 was conducted as described above for the cadA1, cadA2 and tfdAα deletions.

To construct the complementary strain of the cadA1 deletion mutant, cadA1 was expressed under the control of the cadA1 promoter because our preliminary experiments indicated that the lac promoter did not induce the expression of downstream genes in Bradyrhizobium sp. RD5-C2 (data not shown). The fragment containing the cadA1 and cadA1 promoter regions was amplified with PCR using KOD plus DNA polymerase (Toyobo) with the primer set C2cad1P-F-Mph/C2cadA1-Bam-R. The resultant fragment was digested with Mph11031 and BamHI (FastDigest, Thermo Fisher Scientific), and ligated into pBBR1MCS2_START to yield pBBR2-C2cadA1pro-cadA1. The constructed plasmid was cloned into E. coli, amplified, and extracted. The plasmid was then transformed into E. coli S17-1λpir and introduced into the cadA1 deletion mutant to generate the complementary strain, as described above for the introduction of cad clusters into B. elkanii USDA94. The complementary strain was cultivated in HM medium supplemented with kanamycin (150‍ ‍mg L^–1).

Analysis of expression levels of degradation genes using quantitative reverse transcription-PCR (qRT-PCR)

After Bradyrhizobium sp. RD5-C2 was precultivated in HM medium to reach the stationary phase, it was exposed to 100‍ ‍μM 2,4-D (1 day) and 100‍ ‍μM 2,4,5-T (1 and 3 days) with shaking. Total RNA was extracted using ISOGEN-LS (Nippon Gene) according to the manufacturer’s instructions. RNA samples were then treated with DNase I (Takara Bio), and 1‍ ‍μg of each treated sample was used for cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche Diagnostics) and LightCycler Nano Instrument (Roche Diagnostics) according to the manufacturer’s instructions. The sig gene for sigma factor was used as an internal control. Primer sets were C2cad1A227-f/C2cad1A436-r (cadA1, 210‍ ‍bp), C2cad2A548-f/C2cad2A779-r (cadA2, 232‍ ‍bp), C2tfdAa308-f/C2tfdAa488-r (tfdAa, 181‍ ‍bp), and C2sig1288-f/C2sig1454-r (sig, 167‍ ‍bp). The PCR reaction was as follows: 95°C for 10‍ ‍min followed by 45 cycles at 95°C for 10‍ ‍s, 57°C for 10‍ ‍s, and 72°C for 15 s. The expression level of each gene was normalized to that of the sig (sigma factor) gene using the 2^–ΔCT (ΔCT=Ct target gene–CT sig) calculation for statistical analyses (Dunnett’s test [P<0.05]). Three biological experiments were conducted for each treatment, and three real-time PCR reactions were performed for each experiment.

Results

Two cad clusters and tfdAα identified in Bradyrhizobium sp. RD5-C2

The genome of Bradyrhizobium sp. RD5-C2 was sequenced using the Illumina HiSeq2000 platform. The preprocessing and assembly of 31,473,568 paired reads yielded 73 contigs, with a combined size of 8,259,668 bp and GC content of 64.2%. The completeness value of the draft genome was found to be 99.3% using CheckM (Parks et al., 2015). The 16S ribosomal RNA, tRNA-Ile, tRNA-Ala, and 23S ribosomal RNA sequences of Bradyrhizobium sp. RD5-C2 showed 99% similarities to those of Bradyrhizobium elkanii USDA4341 (JQ911628).

The nucleotide sequence around cadA1 was elucidated, and a cad1 cluster was identified. Additionally, a cad2 cluster was identified within a different contig containing the cad1 cluster. Based on the deduced amino acid sequences of putative ORFs, both cad clusters contained genes encoding a transcriptional regulator (cadR), the oxygenase large and small subunits (cadA and cadB, respectively), a ferredoxin component (cadC), and a transporter (cadK) (Fig. 1). An additional gene (cadS) encoding a transcriptional regulator was observed within the downstream region of cadK2. Amino acid sequence similarities were moderate (24–71%) between the corresponding Cad1 and Cad2 proteins. CadR1A1B1K1C1 showed high similarities (99–100%) with the corresponding proteins of the 2,4-D degrader Bradyrhizobium sp. HW13, while the sequences of CadR2A2B2C2K2S were similar (91–98%) to the non-2,4-D degrader B. elkanii USDA94. Similar results were observed for the GC contents of all genes, except cadK. The GC content of cadR1A1B1C1 (55–58%) was lower than the average GC content (64.2%) of the genome. In the hierarchical cluster analysis of the codon usage of cad and 34 housekeeping genes, cadR1A1B1K1 separated from other genes (Table S4 and Fig. S1). In contrast to the representative tfdA clustered with other tfd genes (Don and Pemberton, 1981; Leveau et al., 1999), a corresponding gene was not detected around tfdAα, similar to B. elkanii USDA94 (Hayashi et al., 2016). The tfdAα GC contents, tfdAα codon usage, and TfdAα sequences of the three Bradyrhizobium strains were equivalent (Fig. 1).

Phylogenetic trees were generated for Cad proteins and related enzymes, including Cad homologs in the genomes of Bradyrhizobium strains (Fig. 2). All corresponding Cad1 and Cad2 proteins were separated; the former were grouped into clades with those of Bradyrhizobium sp. HW13, while the latter formed distinct clades with other Bradyrhizobium strains. CadA1 and CadB1 in 2,4-D-degrading Bradyrhizobium were grouped with the corresponding Cad proteins in 2,4-D-degrading Sphingomonas (Müller et al., 2004; Shimojo et al., 2009; Nielsen et al., 2013). CadA2 and CadB2 belonged to the clades containing related dioxygenases, which were annotated as benzoate/toluene 1,2-dioxygenase large and small subunits, respectively.

Fig. 2.

Phylogenetic tree analysis of CadA(A), CadB(B), CadC(C), CadK(D), and CadR(E). Phylogenetic trees were constructed for the amino acid sequences of CadA and CadB with related oxygenase large and small subunits, respectively, CadC with related ferredoxin components of oxygenases, CadK with related transporters, and CadR with related regulators using the Neighbor Joining method and 1,000 bootstrap replicates, constructed using the MEGA7 software program. Bootstrap values above 60% are shown at the nodes. Sequences from Bradyrhizobium sp. RD5-C2 are indicated in bold. Closed circles indicate homologs of Cad enzymes in the genomes of Bradyrhizobium strains. The representative substrates for each enzyme are indicated to the right of the figures. Scale bars indicate substitutions per site. The numbers on the right are accession numbers.

CadA1 and CadB1 exhibited 57 and 46% amino acid sequence similarities with the TftA and TftB proteins of Burkholderia cepacia AC1100 (Kellogg et al., 1981), respectively, which degrade 2,4,5-T into 2,4,5-TCP (Danganan et al., 1994; Xun and Wagnon, 1995). CadA2 and CadB2 were 54 and 48% similar to TftA and TftB, respectively. CadA and CadB in Bradyrhizobium spp. were separated from oxygenases involved in the degradation of aromatic compounds, including BenA and BenB (benzoate) of Acinetobacter sp. ADP1 (Neidle et al., 1991), NahAc and NahAd (naphthalene) of Pseudomonas putida NCIB9816-4 (Dennis and Zylstra, 2004), TodC1 and TodC2 (toluene) of P. putida F1 (Zylstra and Gibson, 1989), and CarAa (carbazole) of Nocardioides aromaticivorans (Inoue et al., 2006) (Fig. 2A and B).

In the phylogenetic tree of CadC, CadC1 and CadC of Bradyrhizobium sp. HW13 formed an independent clade with CarAcII, a ferredoxin component of carbazole 1,9a-dioxygenase from Norosphingomonas sp. KA1 (Urata et al., 2006) (Fig. 2C). They were separated from the CadC of Sphingomonas sp. ERG5 (Nielsen et al., 2013) and the related ferredoxins involved in the degradation of aromatic compounds. CadK1 formed a distinct clade with TfdK from Cupriavidus necator JMP134, Sphingomonas sp. ERG5 (Nielsen et al., 2013), and Sphingomonas herbicidovorans MH (Müller et al., 2004) (Fig. 2D). CadR1 was grouped in an independent clade with XylS from Pseudomonas spp. (Gomada et al., 1992; Stover et al., 2000) and belonged to an AraC-type transcriptional regulator (Fig. 2E). On the other hand, CadR2 formed a clade containing BenM from Acinetobacter sp. ADP1 (Collier et al., 1998), NahR from P. putida NCIB9816-4 (Dennis and Zylstra, 2004), and NagR from Ralstonia sp. U2 (Zhou et al., 2001), which are LysR-type transcriptional regulators.

Genes for the degradation of 2,4-DCP and 2,4,5-TCP

tfdBaFRDEC genes were identified upstream of the cad1 cluster and three ORFs were detected between tfdBa and other tfd genes (Fig. S2). An analysis of the deduced amino acid sequences of tfdBaFRDEC indicated that they were 2,4-DCP 6-monooxygenase, maleylacetate reductase, LysR family transcriptional regulator, TfdD, TftB, and TfdC. There were five catabolic genes for the conversion of chlorophenol before it entered the tricarboxylic acid cycle. Their GC contents (54–56%) were similar to those of cad1 genes and different from the average GC content of the entire genome.

Degradation of chlorophenoxyacetic acids by cad cluster transformants

B. elkanii USDA94-BBR2C2cad1ABKC degraded 30 and 20% of 2,4-D and 2,4,5-T, respectively, in 7 days, and the corresponding degradation products were detected (Fig. 3). B. elkanii USDA94-BBR2C2cad2ABCK degraded 30 and 8% of 2,4-D and 2,4,5-T, respectively. The 2,4-D degradation rate of B. elkanii USDA94-BBR2C2cad1ABKC was similar to that of B. elkanii USDA94-BBR2C2cad2ABCK. The 2,4,5-T degradation rate of the former was faster than that of the latter. The control strain, B. elkanii USDA94-BBR2, showed negligible or no degrading activity for 2,4-D or 2,4,5-T, as previously reported (Hayashi et al., 2016).

Fig. 3.

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) (A) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (B) by Bradyrhizobium elkanii USDA94 harboring cadA1B1K1C1 and cadA2B2C2K2 from Bradyrhizobium sp. RD5-C2. Solid and dashed lines indicate substrates and their corresponding degradation products (2,4-dichlorophenol [2,4-DCP] and 2,4,5-trichlorophenol [2,4,5-TCP]), respectively. Error bars indicate standard deviations based on triplicate cultures. If not visible, error bars are smaller than symbols.

Degradation of chlorophenoxyacetic acids by cadA1, cadA2, and tfdAα deletion mutants

The wild-type strain Bradyrhizobium sp. RD5-C2 degraded 2,4-D and 2,4,5-T, and no degradation products were detected (Fig. 4). While 2,4-D disappeared within 1 day of the incubation, 2,4,5-T concentrations began to decrease after 3 days, and a small amount of the compound was detected after 7 days. Bradyrhizobium sp. RD5-C2ΔcadA2 (Fig. 4) and Bradyrhizobium sp. RD5-C2ΔtfdAα (Fig. S3) degraded 2,4-D and 2,4,5-T similar to the wild-type strain. On the other hand, degradation by Bradyrhizobium sp. RD5-C2ΔcadA1 was negligible. The double-deletion mutant, Bradyrhizobium sp. RD5-C2ΔcadA1ΔcadA2 (Fig. 4), and the triple-deletion mutant, Bradyrhizobium sp. RD5-C2ΔcadA1ΔcadA2ΔtfdAα (Fig. S3), did not degrade 2,4-D or 2,4,5-T. Bradyrhizobium sp. RD5-C2ΔcadR1 only slightly degraded 2,4-D, similar to Bradyrhizobium sp. RD5-C2ΔcadA1. Negligible and no degradation were confirmed in comparisons with non-inoculated samples (data not shown). Although the degradation rate of the complementary strain did not equal that of the wild-type strain, it was faster than Bradyrhizobium sp. RD5-C2ΔcadA1/pBBR2, which had the empty vector introduced in Bradyrhizobium sp. RD5-C2ΔcadA1, indicating that 2,4-D-degrading activity was complemented by the introduction of cadA1 under the control of the cadA1 promoter (Fig. S4).

Fig. 4.

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) (A) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (B) by cadA1 and/or cadA2 deletion mutants of Bradyrhizobium sp. RD5-C2. Error bars indicate standard deviations based on triplicate cultures. If not visible, error bars are smaller than symbols.

Expression of degradation genes in Bradyrhizobium sp. RD5-C2 following exposure to chlorophenoxyacetic acids

A significantly higher cadA1 expression level was detected following exposure to 2,4-D than under the control condition (Fig. 5). The average relative expression of cadA1 was more than 1,000-fold higher than that under the control condition. cadA1 expression after 1 day of exposure to 2,4,5-T did not significantly differ from that under the control condition; however, its average relative expression was more than 10-fold higher. The expression levels of cadA2 and tfdAα after 1 day of exposure to 2,4-D and 2,4,5-T did not significantly differ from those under the control condition. A significantly higher expression level of cadA1 and lower expression levels of cadA2 and tfdAα were detected 3 days after exposure to 2,4,5-T than under the control condition (Fig. 5). cadA1 expression levels did not markedly vary in the three biological replicants. No specific fragment was obtained without RT-PCR, indicating that the samples were not contaminated with DNA (data not shown).

Fig. 5.

Effects of a 1- and 3-day exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the expression of cadA1 (A and D), cadA2 (B and E), and tfdAα (C and F) in Bradyrhizobium sp. RD5-C2. The expression level of each gene was normalized to that of the sig (sigma factor) gene. CTL indicates the control condition. Error bars indicate standard deviations based on triplicate cultures. The means of the control condition in triplicate cultures are shown as 1. Statistical analyses were performed for comparisons with the control (Dunnett’s test, n=3, *P<0.05).

Discussion

Roles of cad1, cad2, and tfdAα in the degradation of chlorophenoxyacetic acids

In the present study, two cad clusters with distinctly different phylogenies were identified in the genome of Bradyrhizobium sp. RD5-C2. Although both cad clusters possessed the ability to degrade 2,4-D and 2,4,5-T, the cad1 cluster was mainly responsible for their degradation. qRT-PCR analyses revealed that the contribution of the cad1 cluster to the degradation of 2,4-D was attributed to the high induction of the cad1 cluster following the exposure to 2,4-D. The expression of the cad1 cluster exposed to 2,4,5-T was significantly higher than that under the control condition after 3 days of exposure, suggesting that the induction of the cad1 cluster by 2,4,5-T required a longer time. This coincides with the 3-day lag before the initiation of 2,4,5-T degradation (Fig. 4B). CadR1 is assumed to be important for the expression of cad1-degrading genes and their functions in the degradation of chlorophenoxyacetic acids.

Although the degradation rate of the complementary strain did not equal that of the wild-type strain, it was faster than that of RD5-C2ΔcadA1/BBR2 (Fig. S4). A previous study reported that the benA complementary strain of Rhodococcus sp. RHA1, which uses benzoate as a sole carbon source, grew on benzoate; however, its growth rate was lower than that of the wild type (Kitagawa et al., 2001). The gene benA encodes the benzoate dioxygenase large subunit and forms an operon with benB, which encodes the benzoate dioxygenase small subunit. The genes cadA1 and cadB1 are most likely transcribed as a single operon when the start codon of cadB1 overlaps with the upstream region of the termination codon of cadA1, and no promoter sequence was detected upstream of cadB1. To avoid a polar effect, the cadA1 deletion mutant was constructed without changing the triplet sequences downstream. Based on the recovery of 2,4-D degradation activity following the introduction of cadA1 and construction of deletion mutants, we concluded that the cadA1 deletion significantly decreased the 2,4-D degradation rate.

The effects of the cadA2 deletion were only observed in the cadA1 and cadA2 double-deletion mutant (Fig. 4), suggesting that although the cad2 cluster exhibits similar degradation activity to the cad1 cluster (Fig. 3), the contribution of the cad2 cluster was very small. The expression of cadA2 after a 1-day exposure to 2,4-D and 2,4,5-T did not significantly differ from that under the control condition, while a 3-day exposure to 2,4,5-T significantly reduced the expression of cadA2. This result indicates that the cad2 cluster was not induced by 2,4-D or 2,4,5-T and also that a longer exposure to 2,4,5-T inhibited the expression of the cad2 cluster, which may explain why the cad2 cluster was not primarily responsible for degradation. B. elkanii USDA94-BBR2C2cad2ABCK degraded 2,4-D similar to B. elkanii USDA94-BBR2C2cad1ABKC (Fig. 3A). Therefore, if the cad2 cluster is expressed at a similar level to the cad1 cluster in Bradyrhizobium sp. RD5-C2, it may play an equivalent role in degradation to the cad1 cluster.

The effects of the tfdAα deletion were not detected in the degradation of 2,4-D or 2,4,5-T (Fig. S3), and the tfdAα deletion mutant degraded 4-chlorophenoxyacetic acid and phenoxyacetic acid similar to the wild-type strain (data now shown). The TfdAα protein expressed in E. coli exhibited degradation activities for 2,4-D, 4-chlorophenoxyacetate, and phenoxyacetate in vitro (Itoh et al., 2002). The forced expression of tfdAα in B. elkanii USDA94, which is very similar to tfdAα in Bradyrhizobium sp. RD5-C2, did not lead to an increase in 2,4-D degradation (Hayashi et al., 2016). The expression level of tfdAα after a 3-day exposure to 2,4,5-T was significantly lower than that under the control condition, indicating that 2,4,5-T inhibited the expression of tfdAα. These results suggest that tfdAα does not play a significant role in the xenobiotic degradation activity of Bradyrhizobium sp. RD5-C2, whereas the TfdAα protein degrades (chloro)phenoxyacetic acids in vitro.

The 2,4-DCP conversion by TfdBa (Huong et al., 2007a) and the results of the analysis of the deduced amino acid sequence of tfdBaFDEC in the genome of Bradyrhizobium sp. RD5-C2 indicate that the tfd genes play a role in the degradation of 2,4-DCP by the same pathway as in C. pinatubonensis JMP134. This is supported by the finding showing that Bradyrhizobium sp. RD5-C2 uses 2,4-D as a sole carbon and energy source (Itoh et al., 2000). The protein encoded by tfdR is a transcriptional regulator that controls the expression of other tfd genes. tfdBaCDEF is presumed to play a role in the degradation of 2,4,5-TCP because 2,4,5-TCP was not detected in the culture during 2,4,5-T degradation.

Multiplicities of chlorophenoxyacetic acid degradation genes

A previous study reported that R. jostii RHA1 possessed three chlorobiphenyl 2,3-dioxygenase genes, bphA1, etbA1, and ebdA1 (Iwasaki et al., 2006). The 4-chlorobiphenyl-degrading activity of the single insertion mutants of dioxygenase genes indicated that all were involved in degradation. Sho et al. (2004) reported that Mycobacterium sp. S65 possessed two pyrene- and phenanthrene-degrading gene clusters (nid and pdo clusters), and both clusters were transcribed with pyrene and phenanthrene. In contrast to these strains, only one of the three genes, the cad1 cluster, was expressed, and it degraded chlorophenoxyacetic acids in Bradyrhizobium sp. RD5-C2. The multiplicities of the degradation gene homologues of xenobiotic compounds in a strain have been reported for chlorophenol in C. pinatubonensis JMP134 (Leveau et al., 1999), carbazole in Norosphingomonas sp. KA1 (Urata et al., 2006), and dihydroxybiphenyl in Rhodococcus spp. (Maeda et al., 1995; Kosono et al., 1997; Taguchi et al., 2004). In addition to these examples, multiplicities in cad genes responsible for 2,4-D and 2,4,5-T degradation were observed in the present study.

Acquisition of the cad1 cluster via horizontal gene transfer

cad1 and cad2 clusters appear to be of distinct origins, with the former being acquired via horizontal gene transfer based on analyses of GC contents, codon usage, and phylogenetic properties (Fig. 1, 2, and S1 and Table S4). tfdBaFRDEC present upstream of the cad1 cluster is presumed to have been acquired via horizontal gene transfer with the cad1 cluster. On the other hand, the cad2 cluster and tfdAα were evolutionarily acquired by Bradyrhizobium without any recent horizontal transfer. Therefore, Bradyrhizobium sp. RD5-C2 evolved from a non-2,4-D degrader that harbored the cad2 cluster via the acquisition of the cad1 cluster, thereby becoming a 2,4-D degrader. The GC contents of the car-I (52.6–62.7%) and car-II (59.4–71.3%) gene clusters in Norosphingomonas sp. KA1 were previously reported to differ (Urata et al., 2006) from those of the cad1 and cad2 clusters. The GC contents of the nid (AF546904, 63.0–67.6%) and pdo (AF546905, 62.5–66.9%) clusters in Mycobacterium sp. S65 were similar. The duplication of degradation genes within a bacterial genome may lead to similarities in the GC contents of these genes. Differences in the GC contents of degradation genes may be attributed to the process of gene acquisition.

Bradyrhizobium sp. RD5-C2 was isolated from soil with no previous history of exposure to 2,4-D or 2,4,5-T. Bradyrhizobium sp. HW13 and Bradyrhizobium sp. BTH, which contain the homologous gene of cadA1, were isolated from pristine soil with no 2,4-D contamination in Hawaii and Canada, respectively (Kamagata et al., 1997). This suggests that chlorophenoxyacetic acids are not a selective pressure for the acquisition of the cad1 cluster in 2,4-D degraders. Additionally, no Bradyrhizobium strain that harbors the homologous cadA1 gene from 2,4-D-contaminated environments has been reported to date. Although there is no experimental data to exclude the possibility that the acquisition of the cad1 cluster occurred under the selective pressure of 2,4-D in enrichment processes, the acquisition of the cad1 cluster via horizontal gene transfer may be related to unknown factors, except for chlorophenoxyacetic acids, in the original soil in which the microbes evolved.

Original substrates of cad clusters

The isolation source of Bradyrhizobium sp. RD5-C2 indicated that 2,4-D and 2,4,5-T were not the original substrates of the cad1 and cad2 clusters, although cadA1 was strongly induced by 2,4-D and both clusters were capable of degrading 2,4-D and 2,4,5-T. The presence of the homologs of the cad2 cluster in Bradyrhizobium strains (Fig. 2) indicates that these enzymes play important roles in the oxygenation of other unknown natural compound(s). The multiplication of degradation genes generally enables an organism to utilize novel carbon and energy sources for survival. Bradyrhizobium sp. RD5-C2 may exhibit additional degrading activity for a wider range of compound(s) by acquiring the cad1 cluster.

Cad proteins in two clusters

Although CadA1 belongs to a distinct clade with CadA in Bradyrhizobium sp. HW13, it shares a common ancestor with the CadA2 lineage (Fig. 2A). Similarly, the two CadB of Bradyrhizobium sp. RD5-C2 fell into a branch that contained no known oxygenases of other substances. These phylogenies indicate that the cadA and cadB genes provide chlorophenoxyacetic acid substrate specificity. In contrast, the CadC1 protein was located in different branches of CadC2 in the phylogenetic tree (Fig. 2C). Since cadC and its homologous genes are predicted to encode ferredoxin components, they do not possess high substrate specificity.

Regarding regulators, the two cad clusters were located in different contigs and both of them contained cadR. CadR1 and CadR2 exhibited distinct amino acid sequences, indicating that the expression of the two cadABCK genes was independently regulated. Based on the inhibition of 2,4-D degradation by the cadR1 deletion (Fig. 4), we conclude that cadR1 is necessary for the downstream expression of genes, and CadR1 appears to induce downstream cad1-degrading genes in the presence of 2,4-D. cadR1 is presumed to be specifically adapted to induce the expression of downstream degradation genes in response to 2,4-D, and this is supported by the phylogenetic property of CadR1, which belongs to a distinct clade with CadR in 2,4-D degraders (Fig. 2E). The GC content of cadK1 is distinct from that of the remaining cad1 genes, but is similar to that of cad2 genes, implying that cadK1 may have been inserted into the region between cadB1 and cadC1 after the acquisition of cadR1A1B1C1.

Conclusion

The present results demonstrated that Bradyrhizobium sp. RD5-C2 possessed two distinct cad clusters, which have the ability to degrade chlorophenoxyacetic acids when expressed. The degradation of these compounds by Bradyrhizobium sp. RD5-C2 is primarily mediated by the cad1 cluster, which is induced at high levels. The results of the phylogenetic analysis imply that Bradyrhizobium sp. RD5-C2 evolved from a non-2,4-D degrader that harbored the cad2 cluster and subsequently acquired the cad1 cluster via horizontal gene transfer, thereby becoming a 2,4-D degrader.

Citation

Hayashi, S., Tanaka, S., Takao, S., Kobayashi, S., Suyama, K., and Itoh, K. (2021) Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in Bradyrhizobium sp. RD5-C2 Isolated from Non-Contaminated Soil. Microbes Environ 36: ME21016.

https://doi.org/10.1264/jsme2.ME21016

Acknowledgements

The authors would like to express their gratitude to associate professor Gordana Maravić-Vlahoviček at the University of Zagreb for the kind gift of pBBR1MCS2_START. This research was supported in part by a Grant-in-Aid for Pesticide Science from the Pesticide Science Society of Japan (to Shohei Hayashi). The authors thank the Faculty of Life and Environmental Science in Shimane University for the financial support to publish this study. We would like to thank Editage (www.editage.jp) for English language editing.

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