Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in <i>Bradyrhizobium</i> 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.

Evolution end classification oftfdgene clusters mediating bacterial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D)

“…This group combines non-traditional clusters that have variable gene structures and significantly differed from the canonical tfd gene clusters of the I and II types. The published clusters were annotated as dccEAIDI, dccAIIDII, dccEAIDI (Müller et al, 2004), tfdC2E2, tfdC2E2F2, tfdDRFCE, tfdDRF (Thiel et al, 2005Nielsen et al, 2017), cnbCDEF (Liu et al, 2011), tfdBCDEFKR (Nielsen et al, 2013), tfdEC, tfdCE (Nguyen et al, 2021a), tfdBaFRDEC (Hayashi et al, 2021), tfdEICIFIRDI,K,BI and tfdDII,FIIEIICII,BII (Zhang et al, 2023). Nevertheless, comparative genomic analysis showed that the genetic structure of this tfdIV type cluster could be divided into three main subtypes -A, B and C (fig.…”

“…TFD44 (tfdIVBFEC cluster) encoded 2,4-D degradation and had the tfdD gene in another place in the genome (Thiel et al, 2005) respectively. The first one was designated as tfdBaFRDEC and involved in 2,4-D degradation from dichlorophenol with the same degradation pathway as that of C. pinatubonensis JMP134 (Hayashi et al, 2021). The S. histidinilytica BT1 5.2 which capable to degrade of 2,4-D possessed the second cluster, tfdIVcCE,D,R,F, which was annotated by Nguyen and colleagues (2021a) as tfdF,S,D,EC.…”

“…The cad clusters were identified in members of all tfdIV subtypes. It was assumed that they were involved in the initial stages of 2,4-D degradation (Nielsen et al, 2013;Nielsen et al, 2017;Hayashi et al, 2021;Nguyen et al, 2021a). Nevertheless, some members of Sphingomonas and Bradyrhizobium could have their own versions of the first system, the tfdA gene and its homologs, named tfdA-like and tfdAα, respectively (Zharikova et al, 2018a).…”

“…They encode for enzymes involved in the degradation of 2,4dichlorophenoxyacetic acid (2,4-D) which has human health and ecological risks (Peterson et al, 2016;ATSDR, 2020), but is still used worldwide as an herbicide in agriculture (Islam et al, 2018). Continued exploration of microbial 2,4-D degradation in various environments in China (Xia et al, 2017;Xiang et al, 2020), Brazil (Brucha et al, 2021), Vietnam (Nguyen et al, 2021a;Nguyen et al, 2021b), Russia (Zharikova et al, 2021) and Japan (Hayashi et al, 2021) suggests that this problem is still in the spotlight.…”

“…In the past three decades, all explorations of tfd gene clusters, from a comparative genomic point of view, have been focused primarily on their tandem matching with the canonical gene structure of tfdI и tfdII gene clusters. A lot of both canonical and non-canonical clusters have been identified, amongst them: tfdT-CDEF (Liu et al, 2001), tfdDRCEBKAF (Poh et al, 2002), tfdCDEF and tfdCIIEIIBKA (Delftia acidovorans P4a), tfdRCEBKA (Vedler et al, 2004), tfdC2E2F2 and tfdDRFCE (Thiel et al, 2005), tfdAKBECR (Sen et al 2011), tfdRDCEFBKA (Kim et al, 2013), tfdBCDEFKR (Nielsen et al, 2013), tfdFAKBIEICIDIRI and tfdCIIEIIBII (Sakai et al, 2014), tfdFEDCT (Ricker et al, 2016), tfdBIFIEIDICIT and variations of the tfdKBIIFEIICIIDIIR cluster with deletions (Nguyen et al, 2019), tfdBFET, tfdAKBFECDR and tfdBFEDC (Xiang et al, 2020), tfdBFEDCS, tfdFE(tctC)DCS (Nguyen et al, 2021a), tfdBaFRDEC (Hayashi et al, 2021), tfdCDEF (Yamamoto-Tamura et al, 2021), tfdEICIFIRDI,K,BI, tfdDII,FIIEIICII,BII (Zhang et al, 2023) and other cluster structures.…”