Aerobic Degradation of Sulfadiazine by Arthrobacter spp.: Kinetics

Subscriber access provided by Northern Illinois University

Article

Aerobic Degradation of Sulfadiazine by Arthrobacter spp.: Kinetics, Pathways and Genomic Characterization Yu Deng, Yanping Mao, Bing Li, Chao Yang, and Tong Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02231 • Publication Date (Web): 30 Jul 2016 Downloaded from http://pubs.acs.org on July 31, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Environmental Science & Technology

Aerobic Degradation of Sulfadiazine by Arthrobacter spp.: Kinetics, Pathways and Genomic Characterization

Yu Deng†, Yanping Mao†, Bing Li†, Chao Yang† and Tong Zhang†

Environmental Biotechnology Laboratory, Department of Civil Engineering, The

University of Hong Kong, Pokfulam Road, Hong Kong Corresponding author, +852-28578551; fax:+852-25595337; [email protected]

ACS Paragon Plus Environment


ABSTRACT

Two aerobic sulfadiazine (SDZ)-degrading bacterial strains, D2 and D4, affiliated with the genus Arthrobacter, were isolated from SDZ-enriched activated sludge. The degradation of SDZ by the two isolates followed the first-order decay kinetics. Half-life time of complete SDZ degradation was 11.3 h for strain D2 and 46.4 h for strain D4. Degradation kinetic changed from non-growth to growth-linked when glucose was

introduced as the co-substrate, and accelerated biodegradation rate was observed after the adaption period. Both isolates could degrade SDZ into 12 biodegradation products via three parallel pathways, of which 2-amino-4-hydroxypyrimidine was detected as the principal intermediate product toward the pyrimidine ring cleavage. Compared with five Arthrobacter strains reported previously, D2 and D4 were the only Arthrobacter strains which could degrade SDZ as the sole carbon source. The draft genomes of D2 and D4, with the same completeness of 99.7%, were compared to other genomes of related species. Overall, these two isolates shared high genomic similarities with the s-triazine-degrading Arthrobacter sp. AK-YN10 and the sulfonamide-degrading bacteria Microbacterium sp. C448. In addition, the two genomes contained a few significant regions of difference which may carry the functional genes involved in sulfonamides degradation.


Page 2 of 40

Page 3 of 40


INTRODUCTION

The benefits of global antibiotics use come at the costs of the development and spread of antibiotic resistance.1 As the first systemically used antibiotics group, sulfonamides are still widely used in veterinary medicine due to their broad inhibition ability for bacteria.2 Sulfadiazine (SDZ) is reported as a ‘high priority’ sulfonamide because of its extensive utilization and high potential to enter the environment3 via animal manure4-6 and wastewater discharge.7-9 SDZ was frequently detected in soil and water resources,10 demonstrating its persistent and insufficient removal in natural environments. Residues of sulfonamides pose environmental concerns with regard to both the development and transmission of antibiotic resistance in environmental microbiota,11 which was identified as a global challenge for the health security of human in the twenty-first century.12 It is essential to understand what manages sulfonamides’ environmental fate, and especially their biodegradation, which has been reported as an important role in sulfonamides removal in soil13 and wastewater treatment plants.14

The majority of researches studied the removal efficiency and mineralization rate of



SDZ in soil15,

16

and activated sludge,17,18 however, reports on the sulfonamide

biodegradation by pure cultures are limited although several pure cultures have been isolated from different highly enriched environments. SDZ-degrading Microbacterium lacus strain SDZm4 was isolated from topsoil layer which had been fertilized for 3 years using manure from SDZ medicated pigs.19 A sulfamethoxazole-degrading Microbacterium strain BR1 was isolated from a membrane bioreactor treating a mixture of pharmaceuticals for 10 months.20 Another sulfamethazine-degrading Microbacterium sp. C448. was isolated from the soil after long-term exposure to three veterinary antibiotics.21 However, little information is available regarding to the kinetics, products and pathways of SDZ degradation by these pure cultures. In addition, studies including genomic characteristics of these sulfonamide-degrading strains have been conducted sporadically. Until now, the draft genome of Microbacterium sp. C448 was the only sequenced genome for sulfonamide-degrading bacterium.22

In this study, two SDZ-degrading microbial consortia were enriched and characterized by PCR-cloning approach, and then two Arthrobacter strains that could completely degrade SDZ were isolated from these two consortia. Kinetics, products and pathways of SDZ degradation

by these two isolates were further studied using


Page 4 of 40

Page 5 of 40


ultra-performance liquid chromatography-tandem mass spectrometry. The genomic characteristics of these two isolates were also explored based on their draft genomes obtained from high-throughput sequencing.

MATERIALS AND METHODS

Information about the chemicals, pure cultures, enrichment process, and sample preparation for Scanning Electron Microscope are summarized in the Supporting Information (SI).

Culture enrichment and isolation. SDZ-degrading consortium was enriched in two reactors (SDZ Enriched 1 and SDZ Enriched 2) using activated sludge from a local municipal wastewater treatment plant as inoculum and the defined mineral salts medium (MSM, SI Table S1). After 10 months enrichment, the enriched cultures were serially diluted with sterile phosphate buffered saline (50 mM PBS, pH 7) and plated on agar media comprising 20 mL defined MSM, 50 mg/L SDZ and 1.5% agar. After incubation at 37 ℃ for one week, 10 colonies grown on the agar plates corresponding to the most diluted fraction (10-7) of the enriched cultures were picked and individually transferred for further purification. Finally, two isolates from the



reactor SDZ Enriched 1, D2 and D4, were able to degrade SDZ completely.

16S rRNA gene cloning and sequencing. Total DNA of the enriched cultures and isolates were extracted using FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH). 16S rRNA genes were amplified by polymerase chain reaction (PCR) using primers 27F/1492R,23 and the PCR products were sent to BGI (Shenzhen, China) for sequencing. 40 clones were sequenced from each enriched consortia. After the manual check for low-quality and chimeric sequences, 34 clones (under GenBank accession KU350525 to KU350558) of SDZ Enriched 1 and 31 clones (under GenBank accession KU350559 to KU350589) of SDZ Enriched 2 were finally obtained. 16S rRNA genes of the D2 and D4 were deposited in GenBank under the accession numbers of KU324467 and KU324468, respectively.

Genome sequencing, assembly and analysis. The genomes of D2 and D4 were sequenced using Illumina HiSeq 4000 which generated paired-end reads of 150 bp. The de novo assembly for the sequences was conducted using CLC Genomics Workbench version 6.0.4 (CLC bio, Denmark) by applying the following parameters: word size of 64, a minimum contig length of 500 bp and similarity fraction of 0.9 over 80% read length. Annotation was conducted using NCBI’s Prokaryotic Genome


Page 6 of 40

Page 7 of 40


Annotation Pipeline through the BioProject at GenBank (accession number PRJNA314012 with ID 1776178 for D2 and PRJNA314014 with ID 421054 for D4). Phylogenetic analyses used MUSCLE for sequences alignment and MEGA 6.06 for the trees computation. The completeness and contamination were estimated by CheckM.24 The lineage-specific markers for whole genome phylogenetic analyses were inferred CheckM (SI). Whole genome nucleotide alignments were conducted by BRIG based on BLAST.25 Pairwise comparisons of average amino acid identity were based on the methods of Rodriguez-R and Konstantinidis.26 The draft genomes of D2 and D4 were deposited at GenBank under the accession numbers LUKB00000000 and LUKC00000000, respectively.

Incubation experiment and treatments. In this study, unless otherwise specified, experiments on SDZ biodegradation were carried out at 37 ℃ and pH 7.0 under aerobic condition. Biomass was indicated by the optical density at 595 nm (OD595) using a Microplate Reader (Bio-Rad, Model 680, USA). Preparation of induced, non-induced cells, growing cells and resting cells were included in SI. SDZ-degrading abilities of D2 and D4 were tested using SDZ-induced and non-induced cells. Growing cells were used to examine the total organic carbon (TOC) reduction during SDZ biodegradation. SDZ degradation by the members of the genus Arthrobacter was



Page 8 of 40

tested in duplicates in 20 mL MSM with 50 mg/L SDZ as the sole carbon source on a rotary shaker (200 rpm) for 11 days. Resting cells were used for identifying SDZ biodegradation products.

Analytical

method.

Ultra-performance

liquid

chromatography-tandem

mass

spectrometry (Acquity UPLC system, Waters) was used for chemical analysis. The mobile phases and elution gradients were the same as our previous study.14

Identification of biodegradation products. An effective detection procedure for biodegradation products was developed based on the reported analytical method from Helbling et al. (Figure 1).27 Non-target full scan was performed in both positive and negative ionization modes. Following the full-scan MS approach, single mass was manually extracted for each suspected ion whose signal intensity was 20% higher than the background in all samples from the same degradation batch. A list of putative mass to charge ratios (m/z) of biodegradation products was generated from the non-target screening. Selected Ion Recording (SIR) mode was applied subsequently to provide a total chromatographic profile of these putative biodegradtion products. To extract the MS/MS spectra for peaks identified in the last step, collision energy (CE) was individually tuned and optimized within the range of 10-45 eV regarding the


Page 9 of 40


response intensity of the total ion chromatograms (TIC). The fragment formed under the optimized CE with the highest signal response was identified as the primary daughter ion for the corresponding peak. Those plausible m/z ratios whose MS/MS spectrum could not be derived from the Daughter Scan were further excluded from the list. Accurate quantification of plausible biodegradation product was carried out by Multiple Reaction Monitoring (MRM) mode. The quantitative assessment was conducted based on the background filtering with a signal-to-noise (S/N) ratio higher than 10,28 and was used to monitor the biodegradation products formation over the time course of the biodegradation experiment. By examining their S/N ratios, the false positives in the plausible m/z list were rejected as low intensity noise. Time course of chromatographic peaks from a selected ion were manually checked to eliminate those random detection noises. In addition, only the peak appeared in all samples after the SDZ reduction with higher intensity than the samples before the SDZ reduction was considered to represent the plausible m/z of a biodegradation product. The remained ions were considered as the candidate m/z of biodegradation products, followed by further MS/MS spectra interpretations. Structure elucidation was manually conducted on the basis of the interpretations of the biodegradation product m/z, corresponding fragments and fragmentation patterns under different CE values. These interpretations were supported and facilitated by the ChemBioOffice software (CambridgeSoft, USA)



and also previously reported main fragment ions and common fragmentation pathways for sulfonamides.29-32 Further confirmation of proposed structures was conducted with purchased reference standards.

RESULTS AND DISCUSSION

Enrichment of SDZ-degrading Consortia. After 10 months enrichment, the removal of SDZ in the two reactors suggested the efficient enrichment of SDZ-degrading consortia (Figure S1). At the concentration of 100 mg/L, SDZ was completely removed within 27 h for both enriched consortia, achieving an average TOC reduction rate of 50.1%, which was increased to 82.5% ~ 85.1% at 72 h. The maximum likelihood phylogenetic analysis of the 16S rRNA gene sequences indicated that SDZ Enriched 1 and 2 had similar microbial communities at phylum-level (Figure S2), having only 4 phyla: Proteobacteria, Actinobacteria, Bacteroidetes and Acidobacteria, although the abundance of each phylum exhibited minor differences in two communities. Interestingly, the genus Arthrobacter was detected to be the sole member of the Actinobacteria in SDZ Enriched 1, where the two isolates D2 and D4 formed a small clade distinct from the reference Arthrobacter ureafaciens strain NC and other strains in this genus (Figure S3). Based on the BLAST results for the 16S


Page 10 of 40

Page 11 of 40


rRNA gene between the two enriched consortia and the isolates, there are 9 sequences in SDZ Enriched 1 and 12 sequences in SDZ Enriched 2 showed the similarity higher than 97% with D2 and D4 (Table S2).

Degradation of SDZ by Isolates D2 and D4. To determine whether these two isolates are distinct in the SDZ degradation capability, we compared the SDZ-degrading ability of these two isolates and other 5 closely related Arthrobacter strains (Table 1). Results showed that only D2 and D4 were capable of utilizing SDZ as the sole carbon source with the accumulation of 2-aminopyrimidine (2-AP) which was later confirmed as the predominate product in the SDZ biodegradation. Moreover, the SDZ-degrading ability in D2 and D4 were examined with their cells grown in the presence (induced) and absence (non-induced) of SDZ using resting cells assays. Degradation of SDZ was inhibited when the non-induced cells of D4 were used compared with its induced-cells. (Table S4). However, D2 exhibited comparable SDZ degradation ability under both induced and non-induced conditions. Both isolates showed high TOC mineralization percentage in the biodegradation experiments. At the SDZ concentration of 50 mg/L, 82.5% of TOC was eliminated by D2 within 7 days of incubation. This was far more than the earlier reported mineralization rate of 1%-32% by isolates from manured soil.19,

33, 34

As for D4, a relatively smaller



mineralization percentage of TOC (34.4%) was observed during the same experiment period.

Kinetic characterization of D2 and D4 on utilizing SDZ as sole carbon source revealed that the SDZ biodegradation followed the first-order decay kinetics with high correlation coefficients (R2) of 0.99 and 0.98. The biodegradation rate constant (k1) and half-life time (t1/2) derived from the fitting model are shown in Table 2. The first-order decay rate constant and half-life time for degradation of 50 mg/L SDZ by D2 were 0.06 h-1 and 11.3 h. SDZ biodegradation at 50 mg/L was not observed for D4 under the same condition. It is well documented that no degradation process happens if the concentration is below the threshold value.35, 36 Insufficient induction of the catabolic genes is often considered as one of the reasons for this threshold value.37, 38 And this could be indicative of the low SDZ biodegradation potential for D4 compared with D2. Thus kinetic experiments for D4 was thus carried out with an increased biomass (OD595 =0.26). D4 exhibited a lower degradation rate constant of 0.02 h-1 corresponding to a longer half-life time of 46.4 h.

When glucose was added as the co-substrate for SDZ biodegradation, the non-growth kinetics was changed to growth-linked kinetics (Figure 2a and c). Since cell growth


Page 12 of 40

Page 13 of 40


was closely correlated with the amount of substrates utilized, the corresponding SDZ biodegradation can be quantitatively characterized on the basis of bacterial growth using a modified Gompertz model.39At an initial SDZ concentration of 50 mg/L together with 2 g/L glucose, accelerating biodegradation was described well by the modified Gompertz model, with R2 of 0.99 for D2 and 0.96 for D4. Degradation of SDZ by D2 required an adaptation with lag phase of 7.4 h and the maximum biodegradation rate was 37.1 mg L-1h-1 after adaptation. For comparison, D4 showed a much shorter lag phase (0.9 h) and a lower maximum biodegradation rate (0.2 mg L-1h-1). The differences in lag phase and maximum biodegradation rate between the two isolates were likely to be the results of different biodegradation abilities associated with the effectiveness of enzymes.40 It is interesting to note that SDZ degradation by the two isolates was enhanced by the presence of glucose. In fact, the actual contaminated environments often contain a mixture of organic carbons. To evaluate the SDZ biodegradation under specific environmental conditions, further studies should provide insights into how organic co-substrates and their concentrations affect the SDZ biodegradation.

Degradation Products and Pathways in Pure Culture. Total chromatograms of 28 putative biodegradation products from SIR mode were included in Figure S4. These



ions were subsequently filtered to exclude false positive noises based on the quantitative assessment from MRM mode (Table S5). 12 out of 28 ions were proposed as candidate biodegradation products after the screening. Four of the 12 candidate products were confirmed with reference standards, and the probable structures for other 8 candidates were proposed. The peak appeared at the retention time of 0.51 min was detected to have the highest signal intensity, containing a sole ion at m/z 96. 2-aminopyrimidine (2-AP) was chosen to be the reference standard since it was reported as the principal biodegradation products by pure cultures belonging to Microbacterium genus.19,

41

Further confirmations on the retention time of

chromatographic peak and the matching MS/MS fragments proved that the detected ion at m/z 96 was 2-AP (Figure S5).

Three biodegradation products were found in one peak near the 2-AP peak, with a retention time of 0.47 min and the observed base ions at m/z of 112, 128 and 130. As an example for confirming the product structure, analytical data of the ion at m/z 112 is presented in Figure 3. The extracted ion chromatogram at m/z 112 showed two peaks, of which the peak at 0.98 min was identified as the noises because the ions have comparable signal intensity than that of samples before the SDZ reduction (Table S5). For the peak at 0.45 min, fragment ions were detected at m/z 70 and 43.


Page 14 of 40

Page 15 of 40


The mass of 112 indicated that this ion could be tentatively assigned as a single hydroxylated product of 2-AP. According to Lamshöft et al.’s study, this hydroxylation could happen at the C4 or C5 position of the pyrimidine ring.42 Although both peaks from the reference standards of 2-amino-4-hydroxypyrimidine (4-OH-2-AP) and 2-amino-5-hydroxpyrimidine (5-OH-2-AP) showed similar retention time (0.45 min for 4-OH-2-AP and 0.47 min for 5-OH-2-AP) with ion at m/z 112, only 4-OH-2-AP was found to have the same MS/MS spectra matching to the detected product. Besides, the prominent ion generated from 5-OH-2-AP fragmentation was at m/z 85 (Figure S6). 4-OH-2-AP was once reported as a biodegradation product by Microbacterium lacus strain SDZm4.31 Ion at m/z 128 showed a 16 Da mas shift compared with 2-AP and thus was proposed as the N-oxide or hydroxylated product of 4-OH-2-AP (Figure S7). Based on its significant fragment ion at m/z 70, the position of the hydroxyl group (OH) at the NH2- group or at the C4 location of the pyrimidine-ring was more likely to be possible. While the characteristic fragment ion observed for reference standard of 2-amino-4, 6-dihydroxypyrimidine (ADHP) was at m/z 60, making the ion at m/z 128 as a result of an OH addition to the NH2- group of 4-OH-2-AP. Similarly, the ion at m/z 130 was likely to be formed by the ring cleavage of the 4-OH-2-AP.



The peak at 0.67 min contained two ions at m/z of 87 and 173. Since sulfanilamide was reported as a common degradation product during photochemical treatment of sulfonamides43 which has the mass of 173 in positive mode, Daughter Scan was then performed on the 250 ppb sulfanilamide standard, biodegradation sample and biodegradation sample containing 125 ppb spiked sulfanilamide standard (Figure S8). It is interesting to find that two peaks all appeared in these three samples at the retention times of 0.51 min and 0.67 min with corresponding predominant ions at m/z 156 and 109, respectively. In order to accurately identify the compounds eluting in these two peaks, MRM detection of characteristic fragment ions at m/z 109 and 156 were further applied to these three samples. Based on the results, it should be further noted that the compound at m/z 173 in biodegradation sample was most likely to be a mixture of 3 isomers which includes sulfanilamide and two other biodegradation products with m/z 109 as their characteristic fragment ion. For peaks at 2.45 and 3.51min along with the other candidate, analytical data for tentative identification on structures including extracted ion chromatograms, MS/MS spectra and proposed MS/MS fragments structures were provided in SI (Figure S9-12).

Based on the identified biodegradation products and their formation patterns in time series (Figure S13), three degradation pathways were proposed in parallel for both


Page 16 of 40

Page 17 of 40


isolates (Figure 4). Apparently, SDZ was predominantly degraded via pathway involving 2-AP (M I-1). Biodegradation experiments showed that around 87.6% and 82.7% SDZ was transformed into M I-1 by induced and non-induced cells of D2 during the complete biodegradation of SDZ. Cleavage of the sulfonamide bond is the initial step in this pathway, with the hydroxylation of the pyrimidine ring at C4 position as the following step. For this major degradation pathway, apart from the sulfanilic acid, 2-AP and 4-OH-2-AP (M I-2) were both detected in large amount as principle products toward the cleavage of pyrimidine ring. M I-2 could be further converted into M I-3 or M I-4 via the addition of an OH to the NH2- group or the hydrolysis of the pyrimidine ring. Apart from this major pathway, the other two parallel pathways which were rarely reported in microbial degradation were detected. The second pathway happened at the bond between the N of the amide moiety and the C2 of the pyrimidine ring. Sulfanilamide and 2-hydroxypyrimidine are expected to be the direct biodegradation products arise from this pathway. In fact, only the concentration of sulfanilamide (M II-2) increased simultaneously to the dissipation of SDZ. While for 2-hydroxypyrimidine, only its further transformation product (M II-1) formed via ring cleavage was detected. Sulfanilamide was then transformed into M II-3 via the binding of a formyl and an acetyl group to two amino groups, and subsequently into M III-2. This could be supported by previous studies, where



Page 18 of 40

formylation and acetylation were considered as the main biotransformation reactions for other sulfonamides and their biodegradation products.30,

44

SDZ could be

transformed into aniline and pyrimidin-2ylsulfamic acid (M III-1) via the third pathway, where the aniline was proposed to be transformed into M III-2 by acetylation, followed by the glucosidation at the N position. García-Galán et al. reported the formation of pyridine-2-ylsulfamic acid during the photolysis of another sulfonamide named sulfapyridine via similar bond cleavage between the sulfonic group and the aniline ring.45

Genomic Characterization of the Isolates. The genome of D2 is composed of 111 chromosome contigs and one circular plasmid, which are 4.63 Mbp and 42,274 bp, respectively. The chromosome and plasmid of D2 genome contain 4651 open reading frames (ORFs), of which 4334 (93.2%) was identified as putative coding sequences (CDS). 59 RNA genes including singe copy of 5S, 16s and 23S rRNA genes, 53 tRNA genes and 3 ncRNA genes were found in these CDS. The genome of D4 only contains 110 chromosome contigs. Compared with D2, except for the plasmid, D4 was found to have similar genome size (4.59 Mbp), number of ORFS (4564), CDS (4293) and RNA genes (56 RNA genes including singe copy of 5S, 16s and 23S rRNA genes, 50 tRNA genes and 3 ncRNA genes ). Consistent with the prevalence of high GC content


Page 19 of 40


in genus Arthrobacter, both isolates have the same GC content of 59.6%. The same completeness (99.71%) and comparable contamination (1.95% and 1.66%) were estimated by CheckM for the genomes of D2 and D4. The inferred placement of isolates genome within the reference genome tree from Checkm revealed their highest similarity to the atrazine-utilizing Arthrobacter sp. AK-YN10.

Phylogenetic evaluation of two isolates based on the 16S rRNA genes was conducted incorporating all publicly available finished genomes of family Micrococcaceae from NCBI and IMG (Figure S14). In addition with Arthrobacter sp. AK-YN10, these two isolates and Arthrobacter sp. ATCC21022 were most closely related. A concatenated protein phylogenetic tree using 148 lineage-specific markers conserved in genus Arthrobacter demonstrated a congruent topology, placing D2 and D4 with Arthrobacter sp. AK-YN10 and Arthrobacter sp. ATCC21022 as a monophyletic sister group to the group of Arthrobacter aurescens TC1 and Arthrobacter sp. Rue61a (Figure 5a). Whole genome nucleotide alignments of relative strains showed the overall conservation of synteny among genes of these two isolates and Arthrobacter sp. AK-YN10 (Figure 5b), with the average amino acid identity (AAI) above 98.18% (Figure 5c). Strikingly, though Microbacterium sp. C448 is more distantly related with these two isolates based on their 16S rRNA genes, the draft genomes of both



isolates were also organized in a way similar to this sulfonamide-degrading draft genome of C448. Whereas, for other eight phylogenetically related finished genomes from Arthrobacter strains including FB24, Hiyo8, Rue61a, TC1, A6, Ar51, Sphe3 and ATCC 21022, a few of genomic regions of difference (RODs) were observed. Pairwise comparisons of AAI between these close relatives resolved that these two isolates were grouped into the same species with Arthrobacter sp. AK-YN10 and Arthrobacter sp. ATCC21022 based on the AAI criterion for species delineation (95%-96%).46

Although gene content conservation between genomes of two isolates and other Arthrobacter strains was high, D2 and D4 were distinct at the genomic RODs. Of these divergences, the circular plasmid of D2 is of great interest since the diverse metabolic abilities in Arthrobacter sp. was considered to be associated with the plasmid. Previous studies have reported the catabolic genes in Arthrobacter sp. for phthalate,47 nicotine,48 atrazine,49 and quinaldine50 were located on plasmids. The 42-kb D2 plasmid contains 40 CDSs and a GC content of 60.4% which is slightly greater than that of the chromosome. Similar to other Arthrobacter plasmid, D2 plasmid also had a putative parA gene (locus tag: A5N17_17360), which is required in the active segregation of plasmids during cell division, especially for plasmids with


Page 20 of 40

Page 21 of 40


low-copy number.51 The parA was the only shared gene between the D2 chromosome and plasmid among the total 40 CDSs. Besides this chromosome partitioning protein, proteins related to replication are plasmid portioning protein (A5N17_17395) and DNA adenine methyltransferase (A5N17_17410). The other plasmid-borne genes encoded proteins with functions involved in nutrients and energy metabolism, transcriptional regulators and transposition, along with 22 proteins of unknown functions (Table S6). Interestingly, one of oxidoreductase-coding gene that were shared by D2 plasmid (Locus tag: A5N17_17290) and D4 chromosome (Locus tag: A5N13_13935) displayed its highest amino acid similarity (73%) to the gene carried by another sulfonamide-degrading Microbacterium sp. C448, which could degrade sulfamethazine to 2-amino-4, 6-dimethyplyrimidine, the aminated heteroaromatic moiety of the sulfonamides. In fact, the sulfadiazine catabolism gene has not yet been fully characterized.

Though

Ricken et al.

proposed a

NADH-dependent

monooxygenase in the sulfonamide-degrading Microbacterrium sp. strain BR1 as the sulfamethoxazole catabolism gene based on the ipso-hydroxylation initiated degradation pathway,41 the expression mode of this gene and hydroxylation activity of the enzyme remain to be further elucidated. The hydroxylation of sulfamethoxazole at ipso-position in BR1, which leads to the release of 3-amino-5-methylisoxazole, benzoquinone-imine and sulfite, presented us an underlying degradation mechanism



Page 22 of 40

for sulfonamide antibiotics in bacteria.52, 53 Laccases,54 peroxidases,55 dioxygenases,56, 57

cytochrome

P450-dependent

monooxygenases58,

59

and

flavoprotein

monooxygenases60, 61 are known enzymes involved in the aromatic ipso-substitutions. Possession of these enzymes in bacteria may decrease their susceptibility to sulfonamide antibiotics through this less obvious degradation mechanism. Coupling with the results of degradation experiments by induced and non-induced resting cells of two isolates, it can be assumed that the catabolism gene initiated the major pathway were constitutively expressed in D2, while in D4, induction of another chromosomal gene is needed to degrade sulfadiazine into innocuous compounds. Overall, the availability of the draft genomes of D2 and D4 will facilitate the comparative analysis on the sulfonamide-degrading ability among the members of Arthrobacter genus, and further mining and refinement for functional genes involved in sulfonamides biodegradation.

Environmental Implications. Members of the genus Arthrobacter were commonly reported as a predominant group in soil microorganisms, and some isolates from this genus are phosphate-solubilizing

62, 63

and nitrogen-fixing64 rhizobacteria. Based on

our results on the sulfonamide-degrading capabilities and the reported plant growth-promoting capacities of Arthrobacter species, the isolates D2 and D4 are


Page 23 of 40


promising candidates to be used as the bacterial inoculant for the stimulation of the bioremediation of SDZ-contaminated soil sites. In addition, the genus Arthrobacter also contains bacteria capable of degrading environmental pollutions like wastewater chlorination byproduct 4-chlorophenol65, industrial byproduct polychlorinated biphenyls66, agricultural herbicides atrazine67-69 and pesticides isocarbophos.70 Therefore, these metabolically diverse Arthrobacter strains deserve particular attention in environmental bioremediation regarding their versatility in degrading a wide range of organic pollutants. The experimental results about the biodegradation kinetics and products of SDZ could provide insights in its environmental fate and potential ecological impacts. As global pollutants, the questions about the efficiency and pathways of antibiotics biodegradation in the presence of co-substrates and also other natural conditions like low-nutrient soil or groundwater will draw more attentions from the researchers.

ASSOCIATED CONTENT

Supporting information Additional results and discussion on the morphologic characterization of the isolates and the identification of detected metabolites were included.



AUTHOR INFORMATION

Corresponding Author Phone: +852-28578551; Fax:+852-25595337; E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was funded by Hong Kong General Research Funds (HKU17209914). Yu Deng would like to thank The University of Hong Kong for the Postgraduate Studentship. Dr. Yanping Mao and Dr. Bing Li would like to thank The University of Hong Kong for the Postdoctoral Fellowship. Dr. Chao Yang wishes to thanks Hong Kong Scholars Program for the financial support of his Postdoctoral Fellowship at The University of Hong Kong.

REFERENCES 1.

Andersson, D. I.; Hughes, D., Antibiotic resistance and its cost: is it possible to

reverse resistance? Nature Reviews Microbiology 2010, 8, (4), 260-271. 2. Zhang, Y.; Marrs, C. F.; Simon, C.; Xi, C., Wastewater treatment contributes to


Page 24 of 40

Page 25 of 40


selective increase of antibiotic resistance among Acinetobacter spp. Science of the Total Environment 2009, 407, (12), 3702-3706. 3. Boxall, A. B.; Fogg, L. A.; Kay, P.; Blackwel, P. A.; Pemberton, E. J.; Croxford, A., Prioritisation of veterinary medicines in the UK environment. Toxicology letters 2003, 142, (3), 207-218. 4. Lamshöft, M.; Sukul, P.; Zühlke, S.; Spiteller, M., Behaviour of 14 C-sulfadiazine and 14 C-difloxacin during manure storage. Science of the Total Environment 2010, 408, (7), 1563-1568. 5. Hou, J.; Wan, W.; Mao, D.; Wang, C.; Mu, Q.; Qin, S.; Luo, Y., Occurrence and distribution of sulfonamides, tetracyclines, quinolones, macrolides, and nitrofurans in livestock manure and amended soils of Northern China. Environmental Science and Pollution Research 2015, 22, (6), 4545-4554. 6. Xian-Gang, H.; Yi, L.; Qi-Xing, Z.; Lin, X., Determination of thirteen antibiotics residues in manure by solid phase extraction and high performance liquid chromatography. Chinese Journal of Analytical Chemistry 2008, 36, (9), 1162-1166. 7. Li, B.; Zhang, T., Biodegradation and adsorption of antibiotics in the activated sludge process. Environmental science & technology 2010, 44, (9), 3468-3473. 8. Göbel, A.; Thomsen, A.; McArdell, C. S.; Joss, A.; Giger, W., Occurrence and sorption behavior of sulfonamides, macrolides, and trimethoprim in activated sludge treatment. Environmental science & technology 2005, 39, (11), 3981-3989. 9. Göbel, A.; McArdell, C. S.; Joss, A.; Siegrist, H.; Giger, W., Fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies. Science of the Total Environment 2007, 372, (2), 361-371. 10. Gothwal, R.; Shashidhar, T., Antibiotic pollution in the Environment: a review. CLEAN–Soil, Air, Water 2015, 43, (4), 479-489. 11. Larcher, S.; Yargeau, V., Biodegradation of sulfamethoxazole: current knowledge and perspectives. Applied microbiology and biotechnology 2012, 96, (2), 309-318. 12. G8 Science Ministers. 12 June 2013. G8 science ministers statement. G8 Centre, London, United Kingdom. http://www.g8.utoronto.ca /science/130613-science.html. 13. Ding, H.; Wu, Y.; Zou, B.; Lou, Q.; Zhong, J.; Zhang, W.; Lu, L.; Dai, G., Simultaneous removal and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption. Journal of Hazardous Materials 2016, 307, 350-358 14. Deng, Y.; Li, B.; Yu, K.; Zhang, T., Biotransformation and adsorption of pharmaceutical and personal care products by activated sludge after correcting matrix effects. Science of The Total Environment 2016, 544, 980-986. 15. Yang, J.-F.; Ying, G.-G.; Yang, L.-H.; Zhao, J.-L.; Liu, F.; Tao, R.; Yu, Z.-Q.; Peng, P. a., Degradation behavior of sulfadiazine in soils under different conditions.



Journal of Environmental Science and Health Part B 2009, 44, (3), 241-248. 16. Selvam, A.; Zhao, Z.; Wong, J. W., Composting of swine manure spiked with sulfadiazine, chlortetracycline and ciprofloxacin. Bioresource technology 2012, 126, 412-417. 17. Joss, A.; Zabczynski, S.; Göbel, A.; Hoffmann, B.; Löffler, D.; McArdell, C. S.; Ternes, T. A.; Thomsen, A.; Siegrist, H., Biological degradation of pharmaceuticals in municipal wastewater treatment: proposing a classification scheme. Water research 2006, 40, (8), 1686-1696. 18. Pérez, S.; Eichhorn, P.; Aga, D. S., Evaluating the biodegradability of sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim at different stages of sewage treatment. Environmental Toxicology and Chemistry 2005, 24, (6), 1361-1367. 19. Tappe, W.; Herbst, M.; Hofmann, D.; Koeppchen, S.; Kummer, S.; Thiele, B.; Groeneweg, J., Degradation of sulfadiazine by Microbacterium lacus strain SDZm4, isolated from lysimeters previously manured with slurry from sulfadiazine-medicated pigs. Applied and environmental microbiology 2013, 79, (8), 2572-2577. 20. Bouju, H.; Ricken, B.; Beffa, T.; Corvini, P. F.-X.; Kolvenbach, B. A., Isolation of bacterial strains capable of sulfamethoxazole mineralization from an acclimated membrane bioreactor. Applied and environmental microbiology 2012, 78, (1), 277-279. 21. Topp, E.; Chapman, R.; Devers-Lamrani, M.; Hartmann, A.; Marti, R.; Martin-Laurent, F.; Sabourin, L.; Scott, A.; Sumarah, M., Accelerated Biodegradation of Veterinary Antibiotics in Agricultural Soil following Long-Term Exposure, and Isolation of a Sulfamethazine-degrading sp. Journal of environmental quality 2013, 42, (1), 173-178. 22. Martin-Laurent, F.; Marti, R.; Waglechner, N.; Wright, G. D.; Topp, E., Draft genome sequence of the sulfonamide antibiotic-degrading Microbacterium sp. strain C448. Genome announcements 2014, 2, (1), e01113-13. 23. Weisburg, W. G.; Barns, S. M.; Pelletier, D. A.; Lane, D. J., 16S ribosomal DNA amplification for phylogenetic study. Journal of bacteriology 1991, 173, (2), 697-703. 24. Parks, D. H.; Imelfort, M.; Skennerton, C. T.; Hugenholtz, P.; Tyson, G. W., CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome research 2015, 25, (7), 1043-1055. 25. Alikhan, N.-F.; Petty, N. K.; Zakour, N. L. B.; Beatson, S. A., BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC genomics 2011, 12, (1), 402. 26. Rodriguez-R, L. M.; Konstantinidis, K. T., Bypassing cultivation to identify bacterial species. Issues 2010, 9 (3), 111-118.


Page 26 of 40

Page 27 of 40


27. Helbling, D. E.; Hollender, J.; Kohler, H.-P. E.; Singer, H.; Fenner, K., High-throughput identification of microbial transformation products of organic micropollutants. Environmental science & technology 2010, 44, (17), 6621-6627. 28. Ahrer, W.; Scherwenk, E.; Buchberger, W., Determination of drug residues in water by the combination of liquid chromatography or capillary electrophoresis with electrospray mass spectrometry. Journal of Chromatography A 2001, 910, (1), 69-78. 29. Klagkou, K.; Pullen, F.; Harrison, M.; Organ, A.; Firth, A.; Langley, G. J., Fragmentation pathways of sulphonamides under electrospray tandem mass spectrometric conditions. Rapid communications in mass spectrometry 2003, 17, (21), 2373-2379. 30. Majewsky, M.; Glauner, T.; Horn, H., Systematic suspect screening and identification of sulfonamide antibiotic transformation products in the aquatic environment. Analytical and bioanalytical chemistry 2015, 407, (19), 5707-5717. 31. Tappe, W.; Hofmann, D.; Disko, U.; Koeppchen, S.; Kummer, S.; Vereecken, H., A novel isolated Terrabacter-like bacterium can mineralize 2-aminopyrimidine, the principal metabolite of microbial sulfadiazine degradation. Biodegradation 2015, 26, (2), 139-150. 32. Wang, J.; Gardinali, P. R., Identification of phase II pharmaceutical metabolites in reclaimed water using high resolution benchtop Orbitrap mass spectrometry. Chemosphere 2014, 107, 65-73. 33. Kreuzig, R.; Höltge, S., Investigations on the fate of sulfadiazine in manured soil: laboratory experiments and test plot studies. Environmental Toxicology and Chemistry 2005, 24, (4), 771-776. 34. Schmidt, B.; Ebert, J.; Lamshöft, M.; Thiede, B.; Schumacher-Buffel, R.; Ji, R.; Corvini, P. F.-X.; Schäffer, A., Fate in soil of 14C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic. Journal of Environmental Science and Health 2008, 43, (1), 8-20. 35. Pahm, M. A.; Alexander, M., Selecting inocula for the biodegradation of organic compounds at low concentrations. Microbial ecology 1993, 25, (3), 275-286. 36. Roch, F.; Alexander, M., Inability of bacteria to degrade low concentrations of toluene in water. Environmental toxicology and chemistry 1997, 16, (7), 1377-1383. 37. Santos, P. M.; Blatny, J. M.; Di Bartolo, I.; Valla, S.; Zennaro, E., Physiological Analysis of the Expression of the Styrene Degradation Gene Cluster in Pseudomonas fluorescensST. Applied and environmental microbiology 2000, 66, (4), 1305-1310. 38. Tros, M. E.; Bosma, T.; Schraa, G.; Zehnder, A., Measurement of minimum substrate concentration (Smin) in a recycling fermentor and its prediction from the kinetic parameters of Pseudomonas strain B13 from batch and chemostat cultures. Applied and environmental microbiology 1996, 62, (10), 3655-3661.



39. Fang, H. H.; Liang, D.; Zhang, T., Aerobic degradation of diethyl phthalate by Sphingomonas sp. Bioresource technology 2007, 98, (3), 717-720. 40. Li, J.; Gu, J.-D.; Pan, L., Transformation of dimethyl phthalate, dimethyl isophthalate and dimethyl terephthalate by Rhodococcus rubber Sa and modeling the processes using the modified Gompertz model. International Biodeterioration & Biodegradation 2005, 55, (3), 223-232. 41. Ricken, B.; Corvini, P. F.; Cichocka, D.; Parisi, M.; Lenz, M.; Wyss, D.; Martínez-Lavanchy, P. M.; Müller, J. A.; Shahgaldian, P.; Tulli, L. G., ipso-Hydroxylation and subsequent fragmentation: a novel microbial strategy to eliminate sulfonamide antibiotics. Applied and environmental microbiology 2013, 79, (18), 5550-5558. 42. Lamshöft, M.; Sukul, P.; Zühlke, S.; Spiteller, M., Metabolism of 14C-labelled and non-labelled sulfadiazine after administration to pigs. Analytical and bioanalytical chemistry 2007, 388, (8), 1733-1745. 43. Boreen, A. L.; Arnold, W. A.; McNeill, K., Photochemical fate of sulfa drugs in the aquatic environment: sulfa drugs containing five-membered heterocyclic groups. Environmental science & technology 2004, 38, (14), 3933-3940. 44. García-Galán, M. J.; Rodríguez-Rodríguez, C. E.; Vicent, T.; Caminal, G.; Díaz-Cruz, M. S.; Barceló, D., Biodegradation of sulfamethazine by Trametes versicolor: Removal from sewage sludge and identification of intermediate products by UPLC–QqTOF-MS. Science of the total environment 2011, 409, (24), 5505-5512. 45. García-Galán, M. J.; Díaz-Cruz, M. S.; Barceló, D., Kinetic studies and characterization of photolytic products of sulfamethazine, sulfapyridine and their acetylated metabolites in water under simulated solar irradiation. Water research 2012, 46, (3), 711-722. 46. Konstantinidis, K. T.; Tiedje, J. M., Towards a genome-based taxonomy for prokaryotes. Journal of bacteriology 2005, 187, (18), 6258-6264. 47. Eaton, R. W., Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B. Journal of Bacteriology 2001, 183, (12), 3689-3703. 48. Igloi, G. L.; Brandsch, R., Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system. Journal of bacteriology 2003, 185, (6), 1976-1986. 49. Sajjaphan, K.; Shapir, N.; Wackett, L. P.; Palmer, M.; Blackmon, B.; Tomkins, J.; Sadowsky, M. J., Arthrobacter aurescens TC1 atrazine catabolism genes trzN, atzB, and atzC are linked on a 160-kilobase region and are functional in Escherichia coli. Applied and environmental microbiology 2004, 70, (7), 4402-4407. 50. Overhage, J.; Sielker, S.; Homburg, S.; Parschat, K.; Fetzner, S., Identification of large linear plasmids in Arthrobacter spp. encoding the degradation of quinaldine to


Page 28 of 40

Page 29 of 40


anthranilate. Microbiology 2005, 151, (2), 491-500. 51. Jerke, K.; Nakatsu, C. H.; Beasley, F.; Konopka, A., Comparative analysis of eight Arthrobacter plasmids. Plasmid 2008, 59, (2), 73-85. 52. Ricken, B.; Kolvenbach, B. A.; Corvini, P. F., Ipso-substitution—the hidden gate to xenobiotic degradation pathways. Current opinion in biotechnology 2015, 33, 220-227. 53. Kolvenbach, B. A.; Helbling, D. E.; Kohler, H.-P. E.; Corvini, P. F., Emerging chemicals and the evolution of biodegradation capacities and pathways in bacteria. Current opinion in biotechnology 2014, 27, 8-14. 54. Chivukula, M.; Renganathan, V., Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Applied and Environmental Microbiology 1995, 61, (12), 4374-4377. 55. Davila-Vazquez, G.; Tinoco, R.; Pickard, M. A.; Vazquez-Duhalt, R., Transformation of halogenated pesticides by versatile peroxidase from Bjerkandera adusta. Enzyme and Microbial Technology 2005, 36, (2), 223-231. 56. Locher, H. H.; Leisinger, T.; Cook, A. M., 4-Sulphobenzoate 3, 4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2. Biochemical journal 1991, 274, (3), 833-842. 57. Bünz, P. V.; Cook, A. M., Dibenzofuran 4, 4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system. Journal of bacteriology 1993, 175, (20), 6467-6475. 58. Ohe, T.; Mashino, T.; Hirobe, M., Substituent Elimination Fromp-Substituted Phenols by Cytochrome P450 ipso-Substitution by the Oxygen Atom of the Active Species. Drug metabolism and disposition 1997, 25, (1), 116-122. 59. Nakamura, S.; Tezuka, Y.; Ushiyama, A.; Kawashima, C.; Kitagawara, Y.; Takahashi, K.; Ohta, S.; Mashino, T., Ipso substitution of bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of estrogenic activity. Toxicology letters 2011, 203, (1), 92-95. 60. Tang, H.; Yao, Y.; Zhang, D.; Meng, X.; Wang, L.; Yu, H.; Ma, L.; Xu, P., A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida. Journal of Biological Chemistry 2011, 286, (45), 39179-39187. 61. Yadid, I.; Rudolph, J.; Hlouchova, K.; Copley, S. D., Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol. Proceedings of the National Academy of Sciences 2013, 110, (24), E2182-E2190. 62. Banerjee, S.; Palit, R.; Sengupta, C.; Standing, D., Stress Induced Phosphate Solubilization by'Arthrobacter'Sp. and'Bacillus' Sp. Isolated from Tomato Rhizosphere. 2010, 4 (6), 378.



63. Yu, X.; Liu, X.; Zhu, T. H.; Liu, G. H.; Mao, C., Isolation and characterization of phosphate-solubilizing bacteria from walnut and their effect on growth and phosphorus mobilization. Biology and Fertility of Soils 2011, 47, (4), 437-446. 64. Upadhyay, S. K.; Singh, D. P.; Saikia, R., Genetic diversity of plant growth promoting rhizobacteria isolated from rhizospheric soil of wheat under saline condition. Current microbiology 2009, 59, (5), 489-496. 65. Westerberg, K.; Elväng, A.; Stackebrandt, E.; Jansson, J. K., Arthrobacter chlorophenolicus sp. nov., a new species capable of degrading high concentrations of 4-chlorophenol. International Journal of Systematic and Evolutionary Microbiology 2000, 50, (6), 2083-2092. 66. Stella, T.; Covino, S.; Burianová, E.; Filipová, A.; Křesinová, Z.; Voříšková, J.; Větrovský, T.; Baldrian, P.; Cajthaml, T., Chemical and microbiological characterization of an aged PCB-contaminated soil. Science of The Total Environment 2015, 533, 177-186. 67. Zhang, Y.; Jiang, Z.; Cao, B.; Hu, M.; Wang, Z.; Dong, X., Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil. International Biodeterioration & Biodegradation 2011, 65, (8), 1140-1144. 68. Wang, Q.; Xie, S., Isolation and characterization of a high-efficiency soil atrazine-degrading Arthrobacter sp. strain. International Biodeterioration & Biodegradation 2012, 71, 61-66. 69. Sagarkar, S.; Bhardwaj, P.; Storck, V.; Devers-Lamrani, M.; Martin-Laurent, F.; Kapley, A., s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil. Applied Microbiology and Biotechnology 2016, 100, (2), 903-913. 70. Li, R.; Guo, X.; Chen, K.; Zhu, J.; Li, S.; Jiang, J., Isolation of an isocarbophos-degrading strain of Arthrobacter sp. scl-2 and identification of the degradation pathway. J Microbiol Biotechnol 2009, 19, (11), 1439-1446. 71. CLARK, F. E., The Designation of Corynebacterium Ureafaciens Krebs and Eggleston as Arthrobacter Ureafaciens (Krebs and Eggleston) Comb. Nov. International Bulletin of Bacteriological Nomenclature and Taxonomy 1955, 5, (3), 111-113. 72. Adams, E., The enzymatic synthesis of histidine from histidinol. Journal of Biological Chemistry 1954, 209, (2), 829-846. 73. Kotoučková, L.; Schumann, P.; Durnová, E.; Spröer, C.; Sedláček, I.; Neča, J.; Zdráhal, Z.; Němec, M., Arthrobacter nitroguajacolicus sp. nov., a novel 4-nitroguaiacol-degrading actinobacterium. International journal of systematic and evolutionary microbiology 2004, 54, (3), 773-777.


Page 30 of 40

Page 31 of 40


74. Phillips, H. C., Characterization of the soil globiforme bacteria. 1951. 75. Zhang, W.-W.; Wen, Y.-Y.; Niu, Z.-L.; Yin, K.; Xu, D.-X.; Chen, L.-X., Isolation and characterization of sulfonamide-degrading bacteria Escherichia sp. HS21 and Acinetobacter sp. HS51. World Journal of Microbiology and Biotechnology 2012, 28, (2), 447-452.



Table and Figure Legends Table 1. Capability of utilizing sulfonamides as the sole carbon source in 7 strains tested in this study and 5 strains from references Table 2. Kinetic parameters of SDZ degradation by isolates D2 and D4 Figure 1.Schematic workflow of the developed procedure. Figure 2. SDZ degradation by two isolates. Kinetic characterizations of D2 and D4 on degradation of SDZ were tested with (red line) and without (blue line) 2 g/L glucose as co-substrate in MSM. Experiments for the bacterial growth coupling SDZ degradation were conducted with MSM containing 50 mg/L together with 2 g/L glucose. (a) Best-fitted curves for SDZ biodegradation by the Gompertz model and first-order decay model for D2; (b) Growth of D2 coupling biodegradation of SDZ; (c) Best-fitted curves for SDZ biodegradation by the Gompertz model and first-order decay model for D4; (d) Growth of D4 coupling biodegradation of SDZ. The biodegradation of SDZ was initially investigated with both isolates which use SDZ as the sole carbon at an initial OD595 of 0.09. Figure 3. Analytical data for detected ion at m/z 112 including (a) chromatogram of the ion at m/z 112; (b) MS/MS spectrum of the ion at m/z 112; (c) chromatogram of the standard 2-amino-4-hydroxypyrimidine (4-OH-2-AP); (d) MS/MS spectrum of the standard 4-OH-2-AP. Figure 4. Proposed pathways for the SDZ degradation by two isolates. Compounds with * were confirmed with reference standards. Compounds in bracket were not detected in this study. Figure 5. Genomic characterization of D2. (a) Maximum-likelihood tree of two isolates genomes in the context of other 21 finished genomes in family Micrococcaceae and one draft genome (AK-YN10) in genus Arthrobacter using 148 concatenated proteins. Genomes selected for further comparative analysis were highlight in blue. Bootstrap values were supported by 1000 replications. The scale bar represents 5% estimated sequence divergence. (b) Comparative genomic characterization of isolate D2. Rings from inside to outside: track1, GC skew; track 2, contig boundaries of isolate D2 (alternating red and blue), the plasmid of isolate D2 was highlighted in yellow; tracks 3-5, nucleotide alignment to reference isolate D2 with draft genomes of isolate D4, Microbacterium sp. C448 and Arthrobacter sp. AK-YN10; tracks 6-13, nucleotide alignment to reference isolate D2 with eight finished genomes of selected Arthrobacter strains including FB24, Hiyo8, Rue61a, TC1, A6, Ar51, Sphe3 and ATCC 21022. (c) Average amino acid and 16S rRNA gene identities. Colors represent values in each cell. Dash line indicates the species-level boundaries based on the 95~96% cutoff value for average amino acid identity recommended by Konstantinidis and Tiedje.


Page 32 of 40

Page 33 of 40

1


Table 1. Capability of utilizing sulfonamides as the sole carbon source in 7 strains tested in this study and 5 strains from references. Sulfonamides biodegradation batch experiment a

Strain characterization Strain Organism

DSMZ Origin

designation

number

16S rRNA identity (%)b

Primary Biomass

Removal

Time metabolite

Arthrobacter sp.

D2

AS c

N/A d

N/A

OD595=0.3

99.8% SDZ

53h

2-AP

Arthrobacter sp.

D4

AS

N/A

99

OD595=0.3

99.0% SDZ

11d

2-AP

Arthrobacter ureafaciens

NC

Soil71

20126

99

OD595=0.3

No removal

11d

No

Arthrobacter histidinolovorans

DSM 20115

Soil72

20115

98

OD595=0.3

No removal

11d

No

Arthrobacter nicotinovorans

DSM 420

N/A

420

98

OD595=0.6

No removal

11d

No

Arthrobacter nitroguajacolicus

G2-1

Soil73

15232

97

OD595=0.6

No removal

11d

No

Arthrobacter aurescens

DSM 20116

Soil74

20116

97

OD595=0.6

No removal

11d

No

Microbacterium lacus

SDZm4

Soil19

26765

90

NA

100% SDZ

21d

2-AP

Microbacterium sp.

C448

Soil21

NA

90

NA

100% SMZ

NA

ADMP e

MBR20

2.5h

3A5MI g

NA

90

OD595=0.541

100% SMX

Microbacterium sp.

BR1 f

100% SDZ

2h

2-AP

2d

No detection

2d

No detection

Escherichia sp.

Acinetobacter sp.

HS21

Seawater75

NA

83

107 CFU/mL

HS51

Seawater75

NA

82

107 CFU/mL

66%SFP

h

45%SFT i 72%SFP; 67%SFT

2

a

SDZ biodegradation batch experiments by growing cells were carried out in this study in duplicates for 7 strains including D2, D4, NC, DSM



Page 34 of 40

3

20115, DSM 420, G2-1, and DSM 20116, with standard deviations of removal rate within 0.6%. b16S rRNA gene sequences of other strains

4 5

were aligned with strain D2 using a web-based nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All the query cover values were 100%. cAS: activated sludge. dN/A: not available. eADMP,2-amino-4,6-dimethylpyrimidine. fRichen et al. demonstrated the strain BR1 also has the ability to completely degrade other 3 sulfonamides including sulfadimethoxine, sulfamethazine and sulfamethazine. g3A5MI-

6 7 8 9

3-imino-5-methylisoxazole. hSFP, sulfapyridine. iSFT, sulfathiazole. Table 2. Kinetic parameters of SDZ degradation by isolates D2 and D4 Initial SDZ

Isolate

Carbon source

D2

SDZ

50 mg/L

0.09

D4

SDZ

100 mg/L

0.26

Isolate

Carbon source

D2

SDZ + Glucosed

50 mg/L

0.09

D4

SDZ + Glucose

50 mg/L

0.08

concentration

Initial SDZ concentration

OD595

OD600

First-order decay model fitting R2

S0 (mg/L)a

k1 (h-1)b

t1/2 (h)c

t

0.99

49.0

0.06

11.3

t1

0.98

107.0

0.02

46.4

Equation

S = 0 + 1 × exp (− )

Modified Gompertz model fitting Equation μm × e

S = 0 − × exp{−exp[

A

× (λ − t) + 1]}

R2

A (mg/L)e

µm (mg L-1h-1)f

λ (h)g

0.99

50.0

37.1

7.4

0.96

29.4

0.2

0.9

10

a

S0 is the calculated initial SDZ concentration. bk1 is the degradation rate derived from 1/t1. ct1/2 is the half-life derived from t1 × In 2. d 2 g/L glucose

11

(weight/weight) was added as co-substrate in this treatment. eA is the biodegradation potential. fµm is the maximum biodegradation rate. gλ is the lag phase

12

time.


Page 35 of 40


13

14 15

Table of Contents (TOC) Art



16

17 18

Figure 1.Schematic workflow of the developed procedure.


Page 36 of 40

Page 37 of 40


19 20 21

Figure 2.

SDZ degradation by two isolates. Kinetic characterizations of D2 and D4 on degradation of SDZ were tested with (red line) and without (blue line)

22

2 g/L glucose as co-substrate in MSM. Experiments for the bacterial

23

growth coupling SDZ degradation were conducted with MSM containing

24

50 mg/L together with 2 g/L glucose. (a) Best-fitted curves for SDZ

25

biodegradation by the Gompertz model and first-order decay model for

26

D2; (b) Growth of D2 coupling biodegradation of SDZ; (c) Best-fitted

27

curves for SDZ biodegradation by the Gompertz model and first-order

28

decay model for D4; (d) Growth of D4 coupling biodegradation of SDZ.

29

The biodegradation of SDZ was initially investigated with both isolates

30

which use SDZ as the sole carbon at an initial OD595 of 0.09.

31



32 33 34

Figure 3.

Analytical data for detected ion at m/z 112 including (a) chromatogram of the ion at m/z 112; (b) MS/MS spectrum of the ion at m/z 112; (c)

35

chromatogram

of

the

standard

2-amino-4-hydroxypyrimidine

36

(4-OH-2-AP); (d) MS/MS spectrum of the standard 4-OH-2-AP.

37


Page 38 of 40

Page 39 of 40


38 39 40 41

Figure 4.

Proposed pathways for the SDZ degradation by two isolates. Compounds with * were confirmed with reference standards. Compounds in bracket were not detected in this study.

42



Page 40 of 40

100 Arthrobacter sp. D2 (b) 73 Arthrobacter sp. D4 100 Arthrobacter sp. ATCC 21022 100 Arthrobacter sp. AK-YN10 Arthrobacter aurescens TC1 100 100 Arthrobacter sp. Rue61a 100 Arthrobacter sp. Hiyo8 Arthrobacter sp. FB24 Arthrobacter sulfonivorans Ar51 100 Arthrobacter chlorophenolicus A6 100 100 Arthrobacter phenanthrenivorans Sphe3 100 Arthrobacter sp. A3 99 100 Arthrobacter alpinus ERGS4:06 100 Arthrobacter sp. PAMC25486 Arthrobacter sp. ERGS1:01 99 Arthrobacter alpinus R3.8 98 Arthrobacter sp. IHBB 11108 100 Renibacterium salmoninarum ATCC 33209 100 Arthrobacter sp. YC-RL1 Arthrobacter sp. LS16 100 Arthrobacter arilaitensis RE117 100 Arthrobacter arilaitensis KLBMP5180 Micrococcus luteus trpE16 100 Micrococcus luteus NCTC 2665 0.05

(a)

(c)

Figure 5.

97.63 97.63 96.84 96.52 96.98 96.52 97.17 100 100 100

91.3 91.3 91.24 91.12 91.05 91.05 90.8 90.16 90.16 90.16 90.16

TC1 Rue61a Hiyo8 Sphe3 A6 FB24 Ar51 ATCC21022 D2 D4 AK-YN10 C448

50.6 C448

97.63 97.63 96.84 96.52 96.98 96.52 97.17 100 100

98.18 98.23 99.99 98.21 97.25 97.32 50.92 51.83 51.22

43 44 45

97.63 97.63 96.84 96.52 96.98 96.52 97.17 100

AK-YN10

97.63 97.63 96.84 96.52 96.98 96.52 97.17

D4

Ar51

97.63 97.63 97.43 99.01 98.35 98.55

D2

FB24

96.7 96.7 97.04 98.02 97.43

ATCC21022

74.27 74.07 74.17 73.59 50.6

A6

78.6 75.27 74.9 74.92 74.48 51.1

Hiyo8

Rue61a

77.85 74.61 74.45 74.82 74.53 84.08 83.85 83.93 83.8 51.27

TC1

Average amino acid identity (%)

99.17 77.74 74.55 74.61 75.11 75.04 84.28 83.94 84.04 83.93 51.22

98.21 97.56 97.56 98.21 97.56 97.56 97.89 98.02 74.14 98.61 74.59 83.41 76.1 78.49 77.48 74.47 80.01 79.28 76.96 74.7 74.81 76.08 74.67 74.61 76.08 74.67 74.6 75.51 74.27 74.68 51.18 51.84 51.64 Sphe3

16S rRNA gene identity (%) 100

Genomic characterization of D2. (a) Maximum-likelihood tree of two isolates genomes using 148 concatenated proteins. Bootstrap values were

46

supported

by

1000

replications.

(b)

47

characterization of D2. Rings from inside to outside: track1, GC skew;

48

track 2, contig boundaries and plasmid (yellow) of D2; tracks 3-5,

49

nucleotide alignment to D2 with draft genomes of D4, C448 and

50

AK-YN10; tracks 6-13, nucleotide alignment to D2 with eight finished

51

genomes of selected Arthrobacter strains. (c) Average amino acid and 16S

52

rRNA gene identities. Dash line indicates the species-level boundaries

53

(95~96% AAI) recommended by Konstantinidis and Tiedje.

54


Comparative

genomic

Aerobic Degradation of Sulfadiazine by Arthrobacter spp.: Kinetics

Recommend Documents