3. RESULTS

3.1. DBP degrading ability ofArthrobacter sp. ZJUTW

Strain ZJUTW was capable of rapidly degrading low concentrations of DBP (Figure 2). When DBP concentration in BSM increased to 1000 mg/L, strain ZJUTW could still grow rapidly and degrade more than 90% of DBP within 18 h. The DBP degradation rate of strain ZJUTW could reach 50 mg/L/h. Other reported strains that can efficiently degrade DBP such as,Rhodococcus sp. JDC-11 could completely degrade 1 g/L DBP within 24 h with a degradation rate of 21.33 mg/L/h, Bacillus sp. (NCIM 5220) could degrade 2.783 g/L of DBP within 72 h with a degradation rate of 38.61 mg/L/h, and Gordonia sp. JDC2 could degrade 96% of 400 mg/L DBP within 18 h with a degradation rate of 21.33 mg/L/h. Thus, the ZJUTW strain exhibited the highest degradation rate among all reported DBP-degrading strains (Table 1).

3.2 Genomic analysis of Arthrobactersp. ZJUTW

The sequencing results showed that the Arthrobacter sp. ZJUTW genome contained a chromosome and a plasmid pQL1 (Fig. S1). Its genomic nucleotide sequences were submitted to the GenBank databases under accession number CP043624 (chromosome) and CP043625 (plasmid). Genome features for Arthrobacter sp. ZJUTW are provided in Table 2. Using the IslandViewer for analysis, a total of seven gene islands including 125 hypothetical proteins, nine transposase genes, and some other functional proteins were predicted. Using Pre-phage prediction of PHAST, a total of 45 genes were predicted, including 36 putative proteins, one esterase gene and 8 other functional proteins. The samples were analyzed by Minced software, and a total of six CRISPR-Cas structures with a total of 32 sequences were predicted.

3.3 Comparative genomics analysis between ZJUTW and other 25 Arthrobacterstrains

Twenty-five Arthrobacter strains were selected for whole-genome comparison with the strain ZJUTW (Fig. S2). A total of 218 specific genes, accounting for 5.96% of all genes, were only found inArthrobacte sp. ZJUTW. Among the 218 specific genes, 118 gene functions were undetermined and the others genes were transposase genes, molybdenum absorption and transformation related genes (moaEmoaAmodAmodBmoeA ), benzoic acid metabolism-related genes (xylX , xylY ,xylZ , xylL ), catechol metabolism-related genes (catR ,catB , catC , catA ), and other functional genes. These specific genes suggest Arthrobacter sp. ZJUTW is capable of degrading benzoic acid, catechol and is capable of absorbing molybdate.
The phylogenetic tree analysis results of the whole-genome sequence of 26 strains (Figure 3A) showed that Arthrobacter sp. LS16,Arthrobacter sp. YC-RL1, and Arthrobacter sp. 7749 are evolutionally closer to ZJUTW than the other 22 strains. LS16 is capable of metabolism of phenolic compounds (Hassan et al., 2016), YC-RL1 can efficiently degrade p-xylene, naphthalene, phenanthrene, biphenyl, p-nitrophenol, and bisphenol (Ren et al., 2018). Arthrobacter sp. 7749 can oxidize phenylethanol derivatives (Sastre, Santos, Kagohara, & Andrade, 2017). The protein sequences of LS16, YC-RL1, and 7749 were obtained from NCBI and were subjected to pairwise alignment using local blast-2.4.0+ software (E-value < 10-5). A Venn diagram (Figure 3B) shows 2167 homologous genes in the fourArthrobacter strains and 558 specific genes were found only in stain ZJUTW and not found in other 3 Arthrobacter strains. However, 400 of the 558 specific genes are pseudogenes, the remaining 158 genes are functionally annotated as 1 esterase, 7 ABC transporters,11 MFS transporters, 9 transposases, transcriptional regulators (GntR, PaaX, HxlR, MerR), and other functional enzymes. The related data are presented in Supplementary Table S1 and Table S2.

3.4 Genes and gene clusters involved in DBP metabolism

Based on the Arthrobacter sp. ZJUTW genome annotation results, there are 25 hydrolase genes, 9 esterase genes, and four dioxygenase genes involved in PAE metabolism. Some gene clusters related closely to biodegradation of DBP, including pehA ,phtcluster, and pca gene cluster, were identified in this study. ThepehA encodes α/β hydrolase, which can convert DBP to PA. Thepht cluster encodes phthalic acid catabolic enzymes that catalyze the conversion of PA to PCA. The pca gene cluster encodes the enzymes catalyzing the transformation of PCA into acetyl-CoA
3.4.1 Characteristics of pht gene cluster on ZJUTW plasmid pQL1
In the genome of ZJUTW, a pht gene cluster was found located on plasmid pQL1. The pht gene clusters have been also found in the genomes of other strains including A. keyseri 12B (Eaton, 2001),Gordonia s p. YC-JH1 (Fan et al., 2018), Gordonia sp. HS-NH1 (Li et al., 2016), Arthrobcter sp. 68b, Terrabactersp. DBF63 (Habe et al., 2003), and Mycobacterium vanbaaleniiPYR-1 (Stingley, 2004). However, the gene architecture of the phtcluster in Arthrobacter sp. ZJUTW is different from that inGordonia sp. YC-JH1 and A. keyseri 12B. Each gene of thepht gene cluster is adjacent to one another and is aggregated inA. keyseri 12B plasmid pRE1 and Gordonia sp. YC-JH1. There are phtAa , phtAb , phtAc , phtAd encoded for 3, 4-phthalate dioxygenase on the ZJUTW plasmid pQL1, the phtAb ,phtAc , phtAd have two copies. The positions of gene cluster phtAb1Ac1Ad1 and phtAaAb2Ac2Ad2 on the plasmid are far away from one another, 12113bp, and both of these have the same transcription direction. The phtB and phtC genes are transcribed in the same direction, at a distance of 25325bp. The transcriptional orientation of gene phtB and phtC is opposite to that of the two 3, 4-phthalate dioxygenase genes mentioned above. The amino acid sequence of the pht cluster of Arthrobacter sp. ZJUTW was done homology alignment analysis with known reports and the identities they share with each other are shown in (Figure 4A). The gene clusterphtAaAbAdBC found in ZJUTW was significantly different from thepht gene clusters present in other bacteria.
The genes for the initial hydrolysis of DBP are indispensable on plasmid pQL1 (52 kb) of ZJUTW. For example, the putative phthalate (PA) degrading genes encode the necessary enzymes for the conversion of phthalic acid to protocatechuic acid, are found on pQL1. PA degradation genes also found on other plasmids including pASPHE302 (94 kb) ofArthrobacter phenanthrenivorans Sphe3 (Vandera, Samiotaki, Parapouli, Panayotou, & Koukkou, 2015), pJ30-114 (98 kb) of Arthrobacter sp. J3-40A, p2MP (112 kb) of Arthrobacter sp. 68b (Stanislauskienė et al., 2011), and plasmid 2 (115 kb) of Arthrobacter sp. FB24. A synteny comparison analysis between the above-mentioned four plasmids and the plasmid pQL1 showed that pQL1 is significantly different from the other five plasmids. It is the smallest one among the five plasmids and poorly correlated with other plasmids (Figure 4B).
In addition, according to the annotation results of plasmid nucleic acid sequences, we found that there are 12 sequences related to gene transfer on the plasmid, ten of which are annotated as transposase genes and the other two are resolvase, some transposases are very close to the genes in the pht gene cluster (Figure 4A). These many mobile genetic components are most likely to participate in the shift ingression of the PA decomposition metabolic module, resulting in gene rearrangement, and in complex mosaic gene structures.
Moreover, the pehA is also located on the plasmid pQL1, and it is close to phtR2 . The PehA has been successfully expressed exogenously in BL21 and the enzymatic properties have been determined. It can hydrolyze monoesters and diesters and is a bifunctional enzyme. The gene pehA serves an indispensable role in DBP-degrading.
In summary, the whole pht gene cluster present in the plasmid pQL1 is very different from all other reported pht gene clusters. Elimination of plasmid was performed by exposing the grown culture to sodium dodecyl sulfate (SDS), to obtain a mutant strain of ZJUTW without plasmid to further confirm the critical role of the pehA andpht gene cluster from the strain ZJUTW plasmid pQL1 during DBP degradation. The DBP degradation ability and cell growth of wild-type and mutant strains was evaluated from its growth curve (Figure 4C). The plasmid-eliminated strain was kept in a stagnant state where DBP was its sole carbon source. Wild type grows more rapidly with an OD600 reaching 0.4 in 12 h. This suggests that this strain ZJUTW could no longer degrade DBP after plasmid elimination. In addition, the GC content of the strain ZJUTW chromosome was 61.86%, and that of plasmid pQL1 was 57.23% (Table 2). The plasmid might result from a possible horizontal gene transfer.
3.4.2 Characteristics of the pca gene cluster on ZJUTW chromosome
The gene cluster pcaHGBCDIJF, which is involved in the protocatechuic acid (PCA) branch of the 3-ketoadipate pathway, is located on the chromosome of the ZJUTW strain. Specifically, genes pcaI , pcaJ , and pcaF in this cluster have two copies (i.e. pcaI1 and pcaI2,pcaJ1 and pcaJ2, pcaF1 and pcaF2 ) that encode the enzymes catalyzing the transformation of protocatechuic acid into acetyl-CoA. Some aromatic compound degrading strains, for example,Streptomyces sp. 2065 (Iwagami, Yang, & Davies, 2000),Gordonia sp. YC-JH1 (Fan et al., 2018), A. keyseri12B, Arthrobacter sp.YC-RL1 (Ren et al., 2018) andRhodococcus opacus 1CP (Eulberg, Lakner, Golovleva, & Schlömann, 1998), also carry PCA degradation-related genes in their genomes.
The pca gene cluster located on the ZJUTW strain chromosome displays major differences from all other pca gene clusters mentioned above. As shown in Figure 4D, the pca genes form up to three parts in the chromosome of the ZJUTW strain, and the three parts are far apart from each other. Gene cluster pcaHGBL is 289334 bp away from pcaI1J1F1 , and pcaI1J1F1 is 2571339 bp apart from pcaI2J2F2 . The gene cluster pcaI1J1F1 is closer to gene cluster pcaHGBCD compared with the gene clusterpcaI2J2F2 . The amino acid sequence of the pca cluster ofArthrobacter sp. ZJUTW shares an identity of 36%-71% and 38%-62% with that of Gordonia sp. YC-JH1 and Sreptomycesp. 2065, respectively. However, the gene cluster responsible for PCA degradation in A. keyseri 12B genome is called thepcm gene cluster, which has the same function to pca gene cluster, while carrying different genes. The pcm gene cluster harbors five key genes: pcmA (encoding protocatechuic acid 4,5-dioxygenase), pcmB (encoding 2-hydroxy-4-carboxymuconic semialdehyde dehydrogenase), pcmC (encoding 2-pyrone-4,6-dicarboxylate hydrolase), pcmD (encoding 4-oxalomesaconate hydratase) and pcmE (4-oxalocitramalate aldolase). Moreover, there is no homology between the pca gene clusters in the ZJUTW strain and the pcm gene cluster, as analyzed using Blastp.

3.5 Differential transcriptional profile of ZJUTW under DBP and glucose

The transcriptional profile of Arthrobacter sp. ZJUTW grown on DBP and glucose was analyzed using RNA-seq. Among total 2908 genes detected (including that on chromosome and plasmid), 677 genes were up-regulated, 416 genes were down-regulated, and 1815 genes did not change significantly (Figure 5A). The gene expression level changed under growth on DBP and glucose carbon sources as shown in Figure 5B. It was also found that most of pcacluster genes fall in the up-regulated fields. Among 677 up-regulated and 416 down-regulated genes, 126 and 126 genes were significantly up-regulated (log2FoldChange 2.0, p-value < 0.05) and down-regulated (log2FoldChange < 2.0, p-value < 0.05), respectively (Supplementary Table S3).
Among the total of 558 specific genes in the Arthrobacter sp. ZJUTW genome, 60 genes were up-regulated (Supplementary Table S4), 23 genes were down-regulated (Supplementary Table S5), and 475 genes did not change significantly (Figure 3B). It is notable that not all specific areas exhibit significant responses to DBP. Only three genes (gene 2924, gene 3435, and gene 3504) out of 126 significantly up-regulated genes and six genes (gene 0928, gene 0929, gene 1081, gene 1082, gene 1083, and gene 3144) out of 126 significantly down-regulated genes, belong to 558 specific genes. Three significantly up-regulated specific genes of the ZJUTW strain, are annotated as hypothetical proteins (gene 2924 and gene 3435) and α-ketoglutarate transporter (gene 3504), respectively. The up-regulation of alpha-ketoglutarate transporter (gene 3504) may be a response of ZJUTW to DBP environment. Gram-positive bacteria have cell walls that contain high levels of peptidoglycans, approximately reaching 90%. WhenArthrobacter sp. ZJUTW is grown in a BSM with high concentration DBP, its cell wall may be damaged. Peptidoglycan synthesis related genes will be induced to express to adapt to environmental pressure of DBP. Peptidoglycan consists of three parts: disaccharide unit, tetrapepitide side chain and peptide interbridge. The tetrapeptide side chain consists of four amino acids, and they are connected to each other by the L-type and D-type alternately. Because α-ketoglutarate is involved in most L-form amino acid transformations, alpha-ketoglutarate transporter (gene 3504) is crucial for the process. Therefore, about 5-fold upregulation of gene 3504 was detected under DBP stress (Supplementary Table S3).
Among the top ten genes that are most up-regulated, three are chaperone protein genes (GrpE, DnaK, and GroEL), two genes encode ClpB protein, one gene encodes ArsR family transcriptional regulator, one gene encodes anti-sigma factor, and the other three genes encode the MFS (major facilitator superfamily) transporter, flavin-dependent oxidoreductase and NADPH-dependent FMN reductase, respectively (Supplementary Table S6). According to previous publications (Thomas, Ayling, & Baneyx, 1997; Arnau, Sorensen, Appel, Vogensen, & Hammer, 1996; Hartke, Frère, Boutibonnes, & Auffray, 1997), it is known that when cells are exposed to extreme conditions, such as extreme temperatures and arsenite, a series of high-level expressions of hot shock proteins (HSPs),including chaperone proteins, such as GrpE, DnaK, GroEL and ClpB, are induced. PAEs are toxic to cells, when the ZJUTW strain is grown in BSM medium at high concentrations DBP, the permeability of the cell membrane will be changed, causing damage to the cells, causing some functional proteins to fail to fold properly. These factors induce the up-regulation of MFS (major facilitator superfamily) transporter and a series of chaperone protein (GrpE, DnaK, GroEL, ClpB). Significantly up-regulated expression of the MFS transporter gene can be correlated with DBP efflux. GrpE, DnaK belongs to the HSP70 protein family, GroEL belongs to the HSP60 protein family, and two ClpB belong to the HSP100 protein family. These chaperone proteins fold the newly synthesized peptide chain correctly, repair of misfolded polypeptides, degrade the inactive protein and enable the cells to grow normally and metabolize. Expression of GrpE, DnaK, GroEL chaperone proteins regulated by the σ32 factor. When intracellular stress response was reduced, the anti-sigma factor binds and sequester σ32, terminating the sustained transcription of these chaperone proteins. This may be the reason for the expression level of the anti-sigma factor is significantly up-regulated. A series of stress responses in the cells is caused by the high concentration of DBP,these biochemical reactions involve many intracellular redox reactions. We conclude that a significant up-regulation of flavin-dependent oxidoreductases and NADPH-dependent FMN reductase genes may be associated with this.

3.6 Transcription level changes of DBP degrading related genes

The transcription levels of genes located on the pht gene cluster and pca cluster and pehA were measured to obtain a comprehensive understanding of the metabolic process of DBP. As mentioned above, when Arthrobacter sp. ZJUTW was cultured in BSM with DBP as the sole carbon source, a total of 126 genes are significantly up-regulated (Supplementary Table S3).
As shown in Supplementary Table S3, the expression level of thepehA is upregulated by 2.66-fold compared to the control group. Among pht gene cluster, which comprises phtAb1 ,phtAc1 , phtAc1 , phtR1 , phtAa , phtAb2 ,phtAc2 , phtAd2 , phtB , and phtC , the expression level of the phtAa , phtAb1 , phtAb2 ,phtAc2, phtAc1, and phtB were dramatically upregulated with 3.05-, 3.52-, 3.47-, 5.12-, 4.25- and 5.02-fold, respectively. However, the expression level of phtAc1 andphtAd2 did not change significantly, while the transcription level of phtC is unknown. In summary, these results clearly indicate that the pehA and pht gene clusters play a significant role in DBP metabolism.
PcaH (the protocatechuic acid 3,4-dioxygenase β-subunit, gene 0406), and PcaG (the protocatechuic acid 3,4-dioxygenase α-subunit, gene 0407) are protocatechuic acid 3,4-dioxygenases,catalyzing the ring cleavage and the transformation of protocatechuic acid into 3-carboxy-cis, cis-muconate through ortho-cleavage. Compared to the control group, the expression levels of PcaH and PcaG were up-regulated by 6.17-fold, 5.64-fold, respectively. The PcaB (3-carboxy-cis, cis-muconate cycloisomerase, gene0405), catalyzing conversion of 3-carboxy-cis, cis-muconate to 4-carboxymuconolactone, was not detected using RNA-seq in transcriptomic analysis. The PcaC (4-carboxymuconolactone decarboxylase, gene0403) catalyzes the 4-carboxymuconolactone decarboxylation to form 3-oxoadipate enol-lactone. Its expression is increased by 3.31-fold. The PcaD (gene 0404) for the beta-ketoadipate enol-lactone hydrolase can hydrolyze 3-oxoadipate enol-lactone to form the 3-oxoadipate. Its expression level is up-regulated by 3.28-fold. Next, the 3-oxoadipate transformed into acetyl-CoA associated with two key enzymes. The one is 3-oxoadipate CoA-transferase, composed by PcaI1 (3-oxoadipate CoA-transferase subunit A) and PcaJ1 (3-oxoadipate CoA-transferase subunit B), 3-oxoadipate is converted to 3-oxoadipyl-CoA by this enzyme. The other one is PcaF1 (acetyl-CoA acetyltransferase), catalyzing the conversion of 3-oxoadipyl-CoA to acetyl-CoA. The expression of pcaF2 , pcaI12 and pcaJ 2 was not detected in the transcriptome. This result suggests that pcaF1 ,pcaI1 , and pcaJ1 play a leading role in the 3-oxoadipate transformation process.
RT-qPCR analysis on some of the DBP degradation-related genes was performed to confirm the transcriptomic analysis. The RT-qPCR results also show that the expression level of pehA , phtAa ,pcaD , pcaG , pcaF1 and pcaJ1 , were up-regulated, consistent with transcriptome data (Figure 6). Combining the results of genomic analysis on the genes and gene clusters involved in DBP degradation and transcriptomic analysis, a possible complete metabolic pathway of DBP in strain ZJUTW could be proposed (Figure 7). In this specific pathway, two ester bonds of DBP are hydrolyzed by α, β-hydrolase (pehA encoded) to form PA. PA is then converted to PCA by a series of enzymes encoded by the pht gene cluster. Finally, PCA is transferred to acetyl-CoA through related enzymes (pca gene cluster encoded). Thus far, only one complete metabolic pathway for DBP in A. keyseri 12B has been reported among allArthrobacter strains. In A. keyseri 12B,PCA is catalyzed by some enzymes encoded by the gene cluster pcm . The pcaand pcm gene clusters encode completely different enzymes (Eaton, 2001). In addition, some key genes in the pht and pca gene clusters have double copies. Overall, the metabolic pathway of DBP in strain ZJUTW is distinct from the pathway in A. keyseri 12B.