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.