Common origin of methylenedioxy ring degradation and demethylation in bacteria

Plants produce many specific secondary metabolites as a response to environmental stress, especially biological stress. These compounds show strong biological activities and high stability against degradation by microbes and animals. Berberine, a benzylisoquinoline alkaloid, is found in many plant species and has strong antimicrobial activity, and is often included in traditional herbal medicines. We previously investigated how berberine is degraded in nature and we isolated two berberine-utilizing bacteria. In this study, we characterized the gene encoding the enzyme that degrades the 2,3-methylenedioxy ring of berberine; this ring is important for its activity and stability. Further characterization of several other berberine-utilizing bacteria and the genes encoding key demethylenation enzymes revealed that these enzymes are tetrahydrofolate dependent and similar to demethylation enzymes such as GcvT. Because the degradation of O-methyl groups or the methylenedioxy ring in phenolic compounds such as lignin, lignan and many other natural products, including berberine, is the key step for the catabolism of these compounds, our discovery reveals the common origin of the catabolism of these stable chemicals in bacteria.


Plasmid construction
[pEbrdA] The 1,317 bp DNA fragment carrying brdA of BD7100 was prepared by PCR with primers ETNde6194Fw and ETHin6194Rv (Supplementary Table 4), adding NdeI and HindIII sites, respectively. The PCR product was cloned into the SmaI site of pUC19 to generate pUbrdAE. pUbrdAE was digested with NdeI and HindIII, and the fragment carrying brdA was cloned into the NdeI and HindIII sites of pET-28a to generate pEbrdA.
[pE1201] The 1,412 bp DNA fragment carrying brdA1 of BD3100 was produced by PCR with primers ETBam1201Fw and ETHin1201Rv (Supplementary Table 4), adding BamHI and HindIII sites, respectively. The PCR product was cloned into the SmaI site of pUC19 to generate pU1201. pU1201 was digested with BamHI and HindIII, and the fragment carrying brdA1 was cloned into the BamHI and HindIII sites of pET-28a to generate pE1201.
[pE1137] The 1,383 bp DNA fragment carrying brdA2 of BD3100 was produced by PCR with primers ETBam1137Fw and ETHin1137Rv (Supplementary Table 4), adding BamHI and HindIII sites, respectively. The PCR product was cloned into the SmaI site of pUC19 to generate pU1137. pU1137 was digested with BamHI and HindIII, and the fragment carrying brdA2 was cloned into the BamHI and HindIII sites of pET-28a to generate pE1137.
[pE4430] The 1,301 bp DNA fragment carrying brdA of GBD-1 was produced by PCR with primers ETEco4430Fw and ETHin4430Rv (Supplementary Table 4). The PCR product was cloned into the SmaI site of pUC19 to generate pU4430. pU4430 was digested with EcoRI and HindIII, and the fragment carrying brdA was cloned into the EcoRI and HindIII sites of pET-28a to generate pE4430.
[pE4435] The 1,419 bp DNA fragment carrying CDS4435 of GBD-1 was produced by PCR with primers ETNde4435Fw and ETHin4435Rv (Supplementary Table 4), in which ETNde4435Fw added an NdeI site. The PCR product was cloned into the SmaI site of pUC19 to generate pU4435. pU4435 was digested with NdeI and HindIII, and the fragment carrying CDS4435 was cloned into the NdeI and HindIII sites of pET-28a to generate pE4435.
[pE7326] The 1,320 bp DNA fragment carrying brdA1 of CJ1 was produced by PCR with primers ETNde7326Fw and ETHin7326Rv (Supplementary Table 4), in which ETNde7326Fw added an NdeI site. The PCR product was cloned into the SmaI site of pUC19 to generate pU7326. pU7326 was digested with NdeI and HindIII, and the fragment carrying brdA1 of CJ1 was cloned into NdeI and HindIII sites of pET-28a to generate pE7326.
[pE7349] The 1,293 bp DNA fragment carrying brdA2 of CJ1 was produced by PCR with primers ETNde7349Fw and ETHin7349Rv (Supplementary Table 4), in which ETNde7349Fw added an NdeI site. The PCR product was cloned into the SmaI site of pUC19 to generate pU7349. pU7349 was digested with NdeI and HindIII, and the fragment carrying brdA2 of CJ1 was cloned into the NdeI and HindIII sites of pET-28a to generate pE7349. Please substitute either "Plasmid construction" or "Plasmid construct" here.
[pK18∆1201] The 869 bp PCR fragment carrying the 5'-upstream region of brdA1 of BD3100 was amplified with primer set 1201UPFw and 1201UPRv (Supplementary Table S4), which provided additional EcoRI and XbaI sites, respectively. The PCR fragment was cloned into the SmaI site of pUC19 to produce pU1201UP. The 807 bp PCR fragment carrying the 3'downstream region of brdA1 was amplified with primer set 1201DOWNFw and 1201DOWNRv (Supplementary Table S4), which provided additional XbaI and HindIII sites, respectively. The PCR fragment was cloned into the SmaI site of pUC19 to produce pU1201DOWN. The EcoRI-XbaI fragment of pU1201UP was cloned into the EcoRI-XbaI site of pK18mobsacB to produce pK18-1201UP. The XbaI-HindIII fragment of pU1201DOWN was cloned into the XbaI-HindIII site of pK18-1201UP to produce pK18∆1201.
[pK18∆1137] The 1,127 bp PCR fragment carrying the 5'-upstream region of brdA2 of BD3100 was amplified with primer set 1137UPFw and 1137UPRv (Supplementary Table S4), which provided additional EcoRI and XbaI sites, respectively. The PCR fragment was cloned into the SmaI site of pUC19 to produce pU1137UP. The 943 bp PCR fragment carrying the 3'downstream region of brdA2 was amplified with primer set 1137DOWNFw and 1137DOWNRv (Supplementary Table S4), which provided additional XbaI and HindIII sites, respectively. The PCR fragment was cloned into the SmaI site of pUC19 to produce pU1137DOWN. The EcoRI-XbaI fragment of pU1137UP was cloned into the EcoRI-XbaI site of pK18mobsacB to produce pK18-1137UP. The XbaI-HindIII fragment of pU1137DOWN was cloned into the XbaI-HindIII site of pK18-1137UP to produce pK18∆1137.

Supplementary Figure 1. Transcription of brdA in BD7100.
Transcription of brdA in BD7100 was analysed by RT-PCR. Total RNA from Rhodococcus sp. BD7100 cells grown on LB or LB containing 0.5 mM BBR was reverse-transcribed. RNA samples were concurrently analysed in PCR mixtures with (+) and without (-) reverse transcriptase (RT) to verify the absence of total DNA. Primer sets (RT6194Fw and RT6194Rv) indicated in Supplementaly Table 4 were designed to amplify cDNAs in the internal region of brdA. The lane of the DNA ladder marker is indicated by M. RT-PCR products were detected and corresponded to the predicted size of 884 bp. Since RT-PCR products were only detected in the cells grown with BBR, transcription of brdA is induced by BBR in BD7100. Resting-cell assays of BBR were carried out using BD7100 and TA140. The blue and orange lines show BD7100 and TA140, respectively. Each value is the average of at least three measurements. The vertical lines indicate the standard deviations from the means. TA140 lost the ability to degrade BBR. The 16S rRNA sequence of GBD-1 was aligned by the Sequence Match application on the RDP website (http://rdp.cme.msu.edu/). Data Set Options were selected as follows: strain, Type; source, Isolates; size, >1200; quality, good; KNN matches, 20. Phylogenetic trees were constructed using the neighbour-joining method with the Kimura method.

Supplementary Figure 4. Phylogenetic tree of CJ1 with strains of the genus Burkholderia.
The 16S rRNA sequence of CJ1 was aligned by the Sequence Match application on the RDP website (http://rdp.cme.msu.edu/). Data Set Options were selected as follows: strain, Type; source, Isolates; size, >1200; quality, good; KNN matches, 20. Phylogenetic trees were constructed using the neighbour-joining method with the Kimura method.

Supplementary Figure 5. Detection of BBR metabolites in GBD-1 and CJ1.
HPLC chromatograms, monitored at 280 nm, of culture medium containing BBR at the initial time and after incubation of GBD-1 and CJ1. GBD-1 and CJ1 were grown in LB medium containing 0.5 mM BBR. The sampling times of the cultures are indicated in the chromatograms. The peaks at 15.3 min retention time, indicated by grey arrows, correspond to the retention time of the standard D-BBR. The detection of D-BBR in all BBR-degraders suggests that demethylenation of BBR is an important step of BBR degradation. Absorption at 280 nm 1