4. DISCUSSION

4.1 Specific genetic architecture may contribute to efficiently degradation of DBP by ZJUTW

A large number of DBP-degrading strains have been isolated from the natural environment. For example, Delftia tsuruhatensis TBKNP-05 can tolerate and completely degraded 2783 mg/L DBP in 120 h (Patil, Kundapur, Shouche, & Karegoudar, 2006), Bacillus sp. NCIM:5220 entrapped in alginate gels can completely degrade 2783 mg/L DBP in 72 h (Patil & Karegoudar, 2005), and Gordonia sp. JDC2,Gordonia sp. JDC13, and Gordonia sp. JDC33 were obtained from activated sludge and showed a good ability to degrade DBP. JDC2 could degrade 96% of 400 mg/L DBP in 18 h. JDC13 could degrade 98% in 30 h and JDC33 could degrade 78% in 48 h (X. Wu, Wang, Dai, Liang, & Jin, 2011). Pseudomonas sp. V21b, isolated from soil, could degrade 57% of when the initial concentration of DBP was 1997 mg/L DBP, within 192 h (Kumar, Sharma, & Maitra, 2017). When the ZJUTW strain was cultured in BSM containing 1000 mg/L DBP, it could degrade more than 90% of DBP within 18 h. The DBP degrading rate of the ZJUTW strain was the highest among the reported DBP degrading strains (Table 1).
The highly efficient DBP degradation ability of Arthrobacter sp. ZJUTW may be related to the specific genetic architecture of its genome. According to the results of genome sequencing, we found that DBP degradation related gene pehA , and gene clusters pht andpca exhibit a favorable coexisting pattern. As shown in Figure 4A and 4D, the pehA is close to the pht gene cluster. In addition, a series of double copy genes were found both in thepht and pca gene clusters. To our knowledge, this is first reported that there are double copy genes in the pht gene cluster. The above two aspects of genetic architecture may play a role in the efficient degradation of DBP by ZJUTW.
There is only one report of a complete metabolic pathway of DBP in genusArthrobacter for A. keysery 12B (Eaton, 2001). However, its corresponding gene clusters are pht and pcm. The pht gene cluster in A. keysery 12B is homologous to that in strain ZJUTW, while the pcm gene cluster in strain 12B is not homologous to the pca gene cluster in the ZJUTW strain. Thus, the DBP metabolic pathway for ZJUTW is distinctly different from that found in A. keysery 12B.

4.2 Synergistic effect of the pht andpca gene clusters may also contribute to efficient degradation of DBP

Arthrobacter sp. ZJUTW exhibited highly efficient degradation of DBP. This may be closely related to the activity of enzymes encoding bypht and pca gene cluster, the number of copies of key genes, and the location of the two gene clusters in the genome. Some PAEs or PA degrading strains exhibit a different distribution ofpht and pca gene clusters in their genomes. For example, in A. keyseri 12B, both of the pht gene cluster and gene cluster pcmDECABF responsible for PCA metabolism, are located on plasmid pRE1 (Eaton, 2001). In Mycobacterium vanbaalenii PYR-1, both the phtRAaAbAcAd gene cluster and thepcaHGBLIJ gene cluster are located on the chromosome (Stingley, 2004). In Terrabacter sp. strain DBF63, the pht gene cluster is located on its chromosome while the pca gene cluster is unmentioned (Habe et al., 2003). In Rhodococcus sp. RHA1, the PA degradation gene clusters are located two plasmids pRHL1 and pRHL2. (Hara, Stewart, & Mohn, 2010), while its PCA degradation gene cluster is located on the chromosome (Hara et al., 2010). In Gordoniasp.YC-JH1, both of the gene cluster pcaRGHBLIJF andphtRAaAbAcAdBC are located on the chromosome (Fan et al., 2018). In A. phenanthrenivorans Sphe3, two clusters that possibly constitute a phthalic acid operon and share an 87% identity with each other, are found on the two plasmids pASPHE301(190 kb) and pASPHE302 (94 kb) (Vandera et al., 2015). Through these comparisons, we show that the distribution of pht and pca gene cluster on the strain ZJUTW genome is specific to these PAE-degrading strains and each has its own particularity. However, the phenomenon of the two gene clusters, constituting complete metabolic pathway of a substance, distributed on a chromosome and a plasmid is not a special case in aromatic-degrading strains.
Based on the results of the transcriptome, we compared the transcription level between the genes of thepht gene cluster located on the plasmid pQL1 and the genes of thepca gene cluster located on the chromosome (Figure 7). The genes related to DBP degradation detected in the transcriptome show different degrees of up-regulation. The expression levels of the genes in thepht gene cluster were up-regulated range from 2.66- to 5.02-fold, and the expression levels of the genes in thepca gene cluster were up-regulated range from 2.34- to 6.17-fold. The related data are shown in Supplementary Table S3. Overall, the expression level of the pht gene cluster is higher than thepca gene cluster. We speculate that a series of DBP degradation related enzymes encoded by the pht gene cluster and genepehA have high enzyme activity and that these enzymes can transform the initial substrate and metabolic intermediates rapidly, resulting in the accumulation of protocatechuic acid in the cells. The enzyme activity of protocatechuic acid degradation related enzymes encoded by the pca gene cluster may be lower than the enzymes encoded by the pht gene cluster. Therefore, to transform the accumulated protocatechuic acid in time, more key enzymes are needed to participate in the metabolic reaction, causing a significant increase in the transcription level of genes in the pca gene cluster. For DBP to be efficiently degraded and to reduce the consumption of energy and certain nutrients, as much as possible, there may be some regulatory mechanism in cells to regulate the transcription of key enzyme genes in pht gene cluster andpca gene cluster based on the amount of intracellular substrate and the accumulation of intermediate products.