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
(moaE ,moaA ,modA ,modB ,moeA ),
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.