Background Nutrients Affect the Biotransformation of Tetracycline by

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Background Nutrients Affect the Biotransformation of Tetracycline by Stenotrophomonas maltophilia as revealed by Genomics and Proteomics yifei leng, Jianguo Bao, Dandan Song, Jing Li, Mao Ye, and Xu Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02579 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Background Nutrients Affect the Biotransformation of Tetracycline by

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Stenotrophomonas maltophilia as revealed by Genomics and Proteomics

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Yifei Leng1,2, Jianguo Bao2,*, Dandan Song2, Jing Li2, Mao Ye3 and Xu Li1,*

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Affiliations:

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1. Department of Civil Engineering, University of Nebraska, Lincoln, NE 68588,

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USA 2. School of Environment Studies, China University of Geosciences, Wuhan 430074, P. R. China 3. State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China

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*Corresponding Authors

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Jianguo Bao bjianguo@cug.edu.cn Xu Li xuli@unl.edu

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ABSTRACT

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Certain bacteria are resistant to antibiotics and can even transform antibiotics in the

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environment.

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resistance and biotransformation processes vary under different environmental

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conditions.

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of tetracycline resistance and biotransformation by Stenotrophomonas maltophilia

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strain DT1 under various background nutrient conditions.

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to tetracycline for seven days with four background nutrient conditions: no

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background (NB), peptone (P), peptone plus citrate (PC), and peptone plus glucose

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(PG).

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Genomic analysis showed that strain DT1 contained tet(X1), a gene encoding an

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FAD-binding monooxygenase, and eight peroxidase genes that could be relevant to

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tetracycline biotransformation.

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nodulation protein transported tetracycline outside of cells; hypoxanthine-guanine

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phosphoribosyltransferase facilitated the activation of the ribosomal protection

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proteins to prevent the binding of tetracycline to the ribosome; and superoxide

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dismutase and peroxiredoxin modified tetracycline molecules.

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nutrient conditions showed that the biotransformation rates of tetracycline were

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positively correlated with the expression levels of superoxide dismutase.

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Keywords: Biotransformation, Tetracycline, Genomics, Quantitative proteomics,

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Stenotrophomonas maltophilia

It is unclear how the molecular mechanisms underlying the

The objective of this study is to investigate the molecular mechanisms

Strain DT1 was exposed

The biotransformation rate follows the order of PC>P>PG>NB≈0.

Quantitative proteomic analyses revealed that

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Comparing different

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1. INTRODUCTION Tetracycline, a member of the tetracycline antibiotic family, is a

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broad-spectrum antibiotic that is widely used to treat human and animal diseases.1, 2

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It inhibits protein synthesis by preventing the attachment of aminoacyl-tRNA to the

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ribosomal acceptor site in bacteria.3

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exist.

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tetracycline binding sites and release tetracycline from the ribosome.4

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pump system can pump tetracycline molecules out of a cell.5

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inactivation is also a mechanism that is used by resistant bacteria to detoxify

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tetracycline.

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tetracycline by adding a hydroxyl group to the C11a position of the molecule.6,7

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Like other antibiotics, tetracycline compounds may be introduced to the

Multiple tetracycline resistance mechanisms

Ribosomal protection proteins can cause an allosteric disruption on The efflux

Enzymatic

A flavin-dependent monooxygenase encoded by tet(X) can inactivate

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environment through the disposal of human and livestock wastes.

Multiple

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processes can affect the fate and the biological impacts of tetracycline antibiotics in

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the environment.

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has high affinity to soil (i.e., low aqueous concentrations in runoff).9

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engineering processes, such as chloramination10 and oxidation11, 12, have also been

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reported to transform tetracycline antibiotics.

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biotransformation of antibiotics, particularly biotransformation carried out by

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bacteria, is rather limited.

The literature on antibiotic biotransformation mostly

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focuses on fungal species.

For example, Pleurotus ostreatus mycelium and the

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laccase of the white rot fungus Trametes versicolor can transform oxytetracycline13

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and tetracycline14, respectively.

For example, chlortetracycline is prone to photo-degradation8 and Abiotic

In comparison, knowledge about the

Noticeably, the biotransformation products are 4

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often less toxic to bacteria than the parent compounds and the transformation

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products from abiotic processes.15

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process is important in predicting the environmental fate of antibiotics, and assessing

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the biological impacts of antibiotics on the microbes in the environment16, 17 and the

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emergence of antibiotic resistance.18, 19

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Hence, understanding the biotransformation

Antibiotics co-occur with other organic compounds in the environments, for

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example, manure-borne antibiotics in soils.20

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can vary substantially from soil to soil and from time to time.

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quantity of the organic compounds in the background can greatly affect the

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metabolisms of antibiotic resistant bacteria, including their ability to transform

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antibiotics.21, 22 In an environment where the biotransformation of antibiotics is

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enhanced, antibiotic parent compounds would be converted to less toxic

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transformation products15 and consequently the level of selective pressure would

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decrease for the other bacteria in the local microbial community.

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The background organic compounds The composition and

Although the effects of background nutrients on antibiotic biotransformation

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have not been extensively reported, evidence of such effects on the

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biotransformation of other organic contaminants have been documented.

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degradation rates of tetrabromobisphenol A (TBBPA) in activated sludge amended

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with glucose, sucrose, and fructose were 9.75, 5.44, 6.38 times, respectively, of that

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in the absence of these background nutrients.23 Similarly, the release of organic

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acids from plant roots could promote the degradation of polycyclic aromatic

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hydrocarbon (PAH) by Sphingomonas yanoikuyae JAR02 in soil.24 With the recent

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The

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observations on antibiotic biotransformation (e.g., sulfonamides25, 26 and

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tetracycline15, 27), the wide occurrence of this process in the environment is being

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recognized.

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this biotransformation process at the molecular level.

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It is important to understand how background nutrients may impact

In our previous study, we isolated a Stenotrophomonas maltophilia strain that

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was capable of transforming tetracycline by co-metabolism with some background

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nutrient conditions.27

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mechanisms of tetracycline resistance and biotransformation by the S. maltophilia

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strain under various background nutrient conditions.

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sequenced and analyzed.

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under different background nutrient conditions were investigated using quantitative

The objective of this study is to understand the molecular

The genome of the strain was

The global protein expression profile of S. maltophilia

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proteomics.

This study illustrates how biotransformation and other resistance

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mechanisms work in the tetracycline resistant S. maltophilia strain at the molecular

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level and how background nutrient conditions affected the biotransformation of

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tetracycline.

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2. MATERIALS AND METHODS

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2.1. Bacteria, Chemicals and Solutions

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S. maltophilia strain DT1 was isolated at the China University of

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Geosciences (Wuhan, China) and deposited at the China Center for Type Culture

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Collection (CCTCC) with an accession number M2014244.

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ATCC 25922 was purchased from the American Type Culture Collection (ATCC) for

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disc diffusion tests to measure the antimicrobial potency of the tetracycline 6 ACS Paragon Plus Environment

Escherichia coli

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biotransformation products.

All chemicals were purchased from Fisher Scientifics,

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including Mueller-Hinton agar, analytical grade tetracycline, high performance

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liquid chromatography (HPLC) grade methanol, acetonitrile, and 0.1% formic acid

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in water.

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yeast extract, and 5 g L-1 NaCl.

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K2HPO4, 0.5 g L-1 KH2PO4, 1.0 g L-1 NaCl, and 0.2 g L-1 MgSO4·7H2O at pH 7.0.28

Luria-Bertani (LB) medium was prepared using 10 g L-1 tryptone, 5 g L-1 Mineral medium (MM) was made using 1.5 g L-1

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2.2. Exposure Experiment Exposure experiments were conducted in batch reactors.

Strain DT1 was

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grown in LB medium at 30oC on a shaker set at 120 rpm, harvested at early

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stationary phase, washed twice in MM, and transferred to 50 mL MM solutions

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containing 1g L-1 peptone (P), 1g L-1 peptone plus 1g L-1 sodium citrate (PC), 1g L-1

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peptone plus 1g L-1 glucose (PG), or no background nutrient (NB) at final

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concentration of OD600 nm = 1.00.

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co-metabolism of tetracycline by strain DT1.29 Citrate and glucose were used as

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additional nutrients in this study, because they are among the nutrients commonly

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found in soil,30, 31 are used as model nutrients in similar studies,32-34 and exhibited

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different impacts on tetracycline biotransformation kinetics in our study (see below).

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For all four groups, (1) the initial tetracycline concentration was 50 mg L-1; (2) all

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batch reactors were performed in triplicates; (3) each group included a no-cell

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control; (4) Group P included a autoclaved-cell control, (5) all flasks were covered in

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aluminum foil to prevent photo degradation of tetracycline; and (6) liquid samples

Peptone was included, as it was required for the

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were collected daily for 7 days and the concentration of the parent compound was

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measured using HPLC.29

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2.3. Modeling The decrease in tetracycline concentration in the exposure experiment was

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attributed to both hydrolysis and biotransformation, while the decrease in no-cell

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controls was attributed only to hydrolysis.

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presence of strain DT1 (hydrolysis plus biotransformation, Equation A) and in the

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absence of strain DT1 (hydrolysis only, Equation B) can be described using

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first-order kinetics.

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difference between the two equations (Equation C).

The decrease of tetracycline in the

Tetracycline biotransformation can be described using the

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‫ܥ‬ୌ୆ = ‫ܥ‬଴ − ‫ܥ‬଴ × eି௞ౄా ×்

(A)

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‫ܥ‬ୌ = ‫ܥ‬଴ − ‫ܥ‬଴ × eି௞ౄ ×்

(B)

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‫ܥ‬୆ = ‫ܥ‬ୌ୆ − ‫ܥ‬ୌ = ‫ܥ‬଴ × eି௞ౄ×் −‫ܥ‬଴ × eି௞ౄా×்

(C)

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C0 is the initial concentration of tetracycline (mg L-1), kHB is the first-order reaction

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rate constant of the overall degradation reaction including hydrolysis and

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biotransformation (day-1), kH is the first-order reaction rate constant of hydrolysis

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(day-1), T is the time (day), CHB is the concentration change of tetracycline from the

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overall degradation (i.e., CHB was measured from the reactors containing strain DT1),

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CH is the concentration change of tetracycline due to hydrolysis only (i.e., CH was

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measured from the no-cell control reactors), CB is the concentration change of

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tetracycline due to biotransformation (mg L-1).

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After taking the derivative on both sides of Equation C over time, biotransformation rate (vB, mg L-1 d-1) can be calculated as

‫ݒ‬஻ =

ௗ஼ಳ ௗ௧

= ‫ܥ‬଴ × (݇ୌ × ݁ ି௞ౄ×் − ݇ୌ୆ × ݁ ି௞ౄా ×் )

(D)

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2.4. Antimicrobial Potency Measurements

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The antibacterial activities of tetracycline and its transformation products

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were measured using disk susceptibility tests.35 E. coli ATCC 25922 cells were

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prepared by transferring colonies from an 18- to 24-hour agar plate to a saline

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solution and adjusting the suspension to achieve a turbidity equivalent to a 0.5

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McFarland standard.

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then was used to spread cells on the surface of Mueller-Hinton agar plates.

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process was repeated two more times after rotating the plate approximately 60° each

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time to achieve an even distribution of cells on plate surface.

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a 6 mm diameter (Whatman, USA) were each loaded with 0.02 mL liquid from the

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exposure experiments and placed onto the surface of the agar plates.

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incubated at 30°C for 16-18 hr.

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measured in mm (including disk diameter).35

A sterile cotton swab was dipped into the E. coli solution, and The

Membrane discs with

Plates were

Inhibition zones around the membrane disks were

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2.5. Quantification of Tetracycline using HPLC To measure tetracycline, 1 mL solution from each batch reactor was

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centrifuged at 13,796 × g at 4oC for 3 min.

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0.22-µm PTFE syringe filters (Restek Corp., USA) and preserved at -80°C until

The supernatant was filtered through

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being loaded onto a Waters 2695 HPLC.

A C18 reversed-phase column (4.6×150

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mm, 5 µm, Agilent Technologies) was operated at 40°C, with a 1 mL min-1 mobile

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phase consisting of 67% (v/v) 0.1% formic acid in water, 22% (v/v) acetonitrile, and

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11% (v/v) methanol.36 The injection volume was 20 µL, and the column isocratic

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elution was monitored using a UV detector at 355 nm.

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3.6.Genomic Analysis The genome of S. maltophilia DT1 was sequenced at Genewiz Inc. (SuZhou,

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China) using Illumina HiSeq X Ten, which generated paired-end reads of 150 bp.

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The de novo assembly for the sequences was conducted using Velvet (version 1.2.10)

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and additional scaffolding was performed using SSPACE Basic 2.0.

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was conducted using the Rapid Annotation using Subsystem Technology (RAST)

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annotation server.37 Comparative genomic characterization of DT1 with other S.

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maltophilia strains were conducted using GCview Server.38

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DT1 were deposited at GenBank under the accession numbers MLJK00000000.

Annotation

The draft genomes of

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2.7. Proteomic Analyses Following the triplicate exposure experiments, a fourth experiment was

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conducted to collect biomass samples for proteomic analyses.

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were included to cover five experimental conditions in triplicate: MM solution

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containing 50 mg L-1 tetracycline under four background nutrient conditions (i.e., P,

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PC, PG, and NB) at 24 hr, cells grown in MM solution containing 1g L-1 peptone but

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no tetracycline at 24 hr.

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initial biotransformation rates, during Day 0 and approximately Day 2, were the

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highest (see below).

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extracted, analysed, identified and quantified following a published protocol39 at the

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UNL Proteomics and Metabolomics Core Facility.

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Supplemental Information.

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they exhibited at least 1.5 fold change in abundance between treatment and reference

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proteomes40 and the change was statistically significant (p < 0.05) according to

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Fisher's exact test on results from the triplicate protein extracts for each condition.

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24 hr was chosen for the proteomic study because the

Harvested cells were washed twice with MM.

Proteins were

More details can be found in

Proteins were considered differentially expressed when

Identified proteins were further analyzed based on QuickGO for Gene

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Ontology (GO) annotation analysis database at the EBI.41

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into metabolic pathways using Kyoto Encyclopedia of Genes and Genomes

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(KEGG).42

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predicted using the STRING database.43

Proteins were mapped

Protein–protein interactions between co-expressed proteins were

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2.8. Inhibition on the N-demethylation of Tetracycline The inhibition of tetracycline N-demethylation activity was tested using

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p-aminobenzoic acid,44 aminopyrine,45 and methimazole.46

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nutrient condition, P, was used in this experiment.

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added to the exposure experiment, and samples were collected for tetracycline

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measurements at the end of the experiment.

Only one background

1 mmol L-1 of each inhibitor was

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3. RESULTS AND DISCUSSION

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3.1. Biotransformation of Tetracycline under Different Background Nutrient

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Conditions In the no-cell controls, the hydrolysis of tetracycline followed first order

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The hydrolysis rate constant, kH, was measured to be 0.0819 ±

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kinetics (Fig. 1A).

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0.0032, 0.0679 ± 0.0050, 0.0756 ± 0.0023, and 0.0681 ± 0.0052 day-1 when peptone

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(P), peptone plus sodium citrate (PC), peptone plus glucose (PG), or no background

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nutrient (NB) was in the MM solution, respectively (Fig. 1A insert).

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Adsorption of tetracycline to bacterial cells was negligible (Fig. S1).

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biotransformation of tetracycline in the exposure experiment increased initially and

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then plateaued (Fig. 1B).

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and used to compare biotransformation kinetics under various experimental

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conditions (Equation D).

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(-0.59 ± 0.19 mg L-1d-1), the biotransformation rate increased to 9.23 ± 0.30, 18.76 ±

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0.68, and 4.25 ± 0.24 mg L-1d-1 in P, PC and PG, respectively (p < 0.05, Fig. 1B

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insert).

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density in the batch reactor, which was 1.0 × 109 CFU/mL for all background

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nutrient conditions tested.

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background nutrient conditions tested during the course of the 7-day experiments

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(Fig. S2).

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Erlenmeyer flasks in the exposure experiment.

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The

Initial biotransformation rates (i.e., t=0) were calculated

Compared to biotransformation rate of the NB control

The biotransformation rate reported above are rates based on the initial cell

The amount of suspended biomass decreased under all

It is noticed that some biofilm formed on the inner wall of the

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trending down (Fig. 1B) and consequently had a slightly negative mean for the initial

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biotransformation rate (Fig. 1B insert).

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no-cell control overestimated the hydrolysis in the NB reactors.

In other words, the

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presence of bacterial cells slowed down tetracycline hydrolysis.

One possible

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explanation was that bacterial cells took up soluble phosphorus from the MM

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medium, resulting in lower phosphorus concentrations (Fig. S3).

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noticed that reduced phosphorus concentrations resulted in slower tetracycline

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hydrolysis: when the soluble phosphorus was reduced from 1000 mg L-1 to 500 mg

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L-1, the hydrolysis rate constant (kH) was reduced from 0.0723 ± 0.0067 day-1 to

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0.0324 ± 0.0022 day-1.

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negligible when peptone was not in the background (i.e., the NB condition in Fig.

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1B).29

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nutrients were present, suggesting that tetracycline may be transformed through

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co-metabolism.

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was used to subtract from the overall degradation in the NB reactors, the calculated

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biotransformation rate was slightly negative.

This indicates that the hydrolysis in the

In this study, we

In addition, the biotransformation of tetracycline was

Biotransformation of tetracycline appeared to occur only when background

Together, when the hydrolysis measured in the no-cell controls

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3.2. Antibacterial Potency of Tetracycline Degradation Products The antimicrobial potency of the degradation products from hydrolysis and

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biotransformation decreased over the course of seven days (Fig. 2).

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hydrolysis was the only mechanism, the degradation products under different

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background nutrient conditions exhibited similar antibacterial potencies (Fig. 2A). 13 ACS Paragon Plus Environment

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The antimicrobial potency of the degradation products differed among the

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background nutrient conditions tested (Fig. 2B).

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decreases in antimicrobial potency among the four background nutrient conditions

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matched the order of the biotransformation rates (Fig. 2B and Fig. 1B insert).

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diameter of the inhibition zones dropped the fastest under PC (from 13.38 mm to

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8.27 mm) and the slowest under NB (from 13.27 mm to 11.50 mm).

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Correspondingly, the biotransformation rate was the highest under PC and the

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slowest under NB.

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Interestingly, the order of the

The

The transformation products of tetracycline from abiotic processes such as

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photolysis had higher toxicity than the parent compound.47, 48

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biotransformation products of tetracycline by DT1 or fungi14 had lower toxicity than

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the parent compound.

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explain the lack of positive correlation between antibiotic residues, which are often

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measured as parent compounds, and antibiotic resistance genes in environmental

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systems.49 In addition, certain background nutrients can enhance the

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biotransformation of toxic compounds by increasing the expression of enzymes

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critical to the degradation of target compounds. For example, Dai et al. found that

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sucrose could promote the hydroxylation of imidacloprid by S. maltophilia CGMCC

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1.1788, likely by increasing the expression of cytochrome P450.50

In contrast, the

These changes in the antimicrobial potency may partially

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3.3. Genomic Characterization of S. maltophilia DT1 The draft genome of S. maltophilia DT1 consists of 41 scaffolds with

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30,242,004 reads, a mean read length of 148.6 base pairs (bp), and N50 of 206.2 Kb.

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The draft genome included 4,532,597 bp, with an average contig length of 110,551

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bp and a maximum contig length of 476,742 bp.

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to be 66.48%.

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all ORFs, 2,787 (69%) could be functionally annotated using RAST (Fig. S4).

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total of 1,153 hypothetical proteins were predicted for S. maltophilia DT1.

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genomic regions of difference (RODs) were observed between DT1 with the finished

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genomes of other S. maltophilia strains R551-3,51 K297a,52 ISMMS253 (Fig. S5).

The GC content was determined

There were 4,052 predicted open reading frames (ORFs).

Out of A

A few

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Fifty seven ORFs of S. maltophilia DT1 were predicted to encode for

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proteins related to resistance to antibiotics, such as aminoglycoside, fluoroquinolone,

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and beta-lactam.

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twelve encode beta-lactamase (Table S1).

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cation/metal efflux pumps for heavy metals such as cobalt, zinc, cadmium, copper

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and arsenic (Table S2).

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Among them, forty ORFs encode multidrug efflux pumps and Twelve other ORFs encode parts of

Twenty one ORFs encode oxygenases, including eight ORFs encoding

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monooxygenases, eleven encoding dioxygenases and two encoding oxygenases

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(Table S3).

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monohydroxylation and tandem oxidations.54

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FAD-binding monooxygenase encoded by tet(X1) was identified in the draft genome.

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Unlike the better known flavin-dependent monooxygenase, which was encoded by

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the tetracycline resistance gene tet(X), FAD-binding monooxygenase encoded by

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tet(X1) lacks the N-terminal domain 1 and is hence inactive.7, 27

Dioxygenases can oxidize aromatic compounds by dihydroxylation, It is worth noting that an

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Eight peroxidase genes were also identified in the draft genome (Table S4).

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Peroxidases are wildly found in bacteria, fungi, plants and animals, and catalyze

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oxidative reactions by hydrogen peroxide.

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from white rot fungi could degrade tetracycline effectively by activating hydrogen

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peroxide.55

Previous reports showed that peroxidase

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3.4. Proteomic Response to Tetracycline Exposure

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The proteomic response of DT1 to tetracycline was investigated by

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comparing the proteome of DT1 cells in MM solution containing tetracycline and

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peptone with the proteome of DT1 cells in MM solution containing only peptone

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(i.e., Comparison #1, Fig. 3A).

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and a subset of these proteins were listed in Table 1.

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exposure on various protein functional categories in S. maltophilia DT1 were

323

analyzed using GO annotation (Fig. S6).

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DT1 cells downregulated the glycolysis and TCA cycle while upregulated the purine

325

and the amino acid pathway (Fig. S7).

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resistance are described in the following sections.

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Efflux pump.

A total of 40 proteins were up-regulated (Table S5), Effects of tetracycline

Upon the exposure to tetracycline, overall

Genes closely related to tetracycline

Nodulation protein (nodW, Table 1) was up-regulated by 2.6

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folds in the presence of tetracycline.

This protein can regulate the transcription of

329

genes involved in the nodulation process, and belongs to the

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resistance-nodulation-division (RND) family efflux pumps system.56

331

efflux pumps broadly exist in bacteria and constitute a superfamily of transporter

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proteins that can pump out a broad range of substrates, including antibiotics.52

Ribosomal protection.

Elongation factors Tu (EF-Tu, tuf) and G (EF-G,

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fusA) were up-regulated by 1.7 and 1.5 folds, respectively, after 24-hour exposure to

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tetracycline.

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proteins (RPPs) encoded by tet(M) and tet(O),57 which can interact with the

337

ribosome, cause allosteric disruption of the tetracycline binding sites, and lead to the

338

release of tetracycline from binding sites.58

339

EF-Tu and EF-G are highly homologous to the ribosomal protection

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was

340

up-regulated by 1.5 folds upon exposure to tetracycline (Table 1).

341

catalyzes guanine to form GMP and increases the production of GTP indirectly.5, 59

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The oversupply of GTP can accelerate the binding of aminoacyl-tRNA to

343

EF-Tu•GTP, which attenuates the binding of tetracycline to the ribosome (Fig. 4A).60

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In addition, members of the HGPRT family are closely related to members of the

345

xanthine-guanine phosphoribosyltransferase (XGPRT) family,61 several of which are

346

homologous to the product of the tetracycline resistance gene tet(34).60

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encoded by tet(34) is classified as a tetracycline deactivation enzyme,58 although

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Thaker et al. did not believe the protein could bring about any alteration to

349

tetracycline molecules.5

350

Enzymatic transformation.

HGPRT

The protein

Superoxide dismutase [Cu-Zn]

351

(DF40_007275, Table 1) was up-regulated by 3.6 folds after 24-hour exposure to

352

tetracycline.

353

damaging species such as oxygen (O2) or hydrogen peroxide (H2O2).62

•‐

This enzyme can convert superoxide radicals (O2 ) into less

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354

Tetracycline in hepatocyte63 and plant cell64 can induce the generation of superoxide

355

radicals (O2 )65 due to drug-target binding and resultant common changes in

356

metabolism.

357

•‐

Peroxiredoxin (BurJV3_0701, Table 1) was up-regulated by 1.8 folds upon

358

exposure to tetracycline.

It belongs to a peroxidase family of antioxidant enzymes

359

and shows peroxidase activities.

360

various xenobiotic compounds including antibiotics.55

361

activities of peroxiredoxin can demethylate the N-methyl group from compounds

362

such as N-methyl aryl amines,66 aminopyrine,45 and methylene blue.67

363

the oxygen radical (O2 ) resulting from tetracycline stress was reduced to hydrogen

364

peroxide by superoxide dismutase [Cu-Zn].

365

activator peroxiredoxin removed the N-methyl groups of tetracycline (Fig. 4B).

Peroxidase can participate in the degradation of In particular, the peroxidase

In this study,

•‐

With hydrogen peroxide as an

366 367 368

3.5. Biochemical Evidence of Tetracycline Biotransformation In the biotransformation pathway we previously proposed, S. maltophilia

369

DT1 first removed two methyl groups from the dimethylamino group at the C4

370

position of tetracycline.29

371

peroxiredoxin (Table 1), which can catalyze demethylation reactions, supports our

372

previous finding.

373

tetracycline biotransformation by DT1, p-aminobenzoic acid and aminopyrine, two

374

competitive substrates for peroxidases, were added separately to exposure

375

experiments.

In this study the observation of the up-regulation of

To further verify the involvement of peroxidase activities in

The addition of either competitive substrate significantly slowed

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376

down tetracycline biotransformation as shown in the relative biotransformation rates

377

in Fig. 5 (i.e., the biotransformation rate of the control condition was set as 100%).

378

This result provides direct evidence of the involvement of peroxidase activities in

379

tetracycline biotransformation and supports the biotransformation reaction proposed

380

in Fig. 4B.

381

The flavin-dependent monooxygenase encoded by tet(X)68 was not detected

382

in any of the replicate proteome samples, while an ORF of a presumably inactive

383

FAD-binding monooxygenase encoded by tet(X1) was observed in the draft genome

384

(Table S3).

385

monooxygenase competitive inhibitor, methimazole,69 was added to an exposure

386

experiment.

387

tetracycline (Fig. 5), confirming that monooxygenase was not responsible for

388

tetracycline biotransformation.

To rule out the potential involvement of monooxygenase, a

The addition of this compound did not affect the biotransformation of

389 390 391

3.6. .Protein-Protein Interactions Proteins related to tetracycline resistance were selected for protein-protein

392

interaction analyses.

The expression of peroxiredoxin, superoxide dismutase

393

[Cu-Zn], and thioredoxin were correlated (Fig. 6).

394

can transform reactive oxygen species to hydrogen peroxides, which serves as

395

substrate for peroxidase in tetracycline biotransformation. Reduced thioredoxin,

396

which was up-regulated 2.1 folds albeit not statistically significant (p = 0.189), can

397

donate electrons to restore the catalytic activity of peroxiredoxin.70

Superoxide dismutase [Cu-Zn]

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398 399 400

3.7. Protein Expression Under Different Nutrient Conditions The difference in background nutrient condition did not cause substantial

401

difference in protein expression (Table S6).

402

proteome while P, PC and PG were treated as treatment proteomes in Comparison #2,

403

#3, and #4, respectively (Fig. 3B).

404

were focused on in Comparisons #2-4 (Table S6).

405

down-regulated in PC and in PG upon exposure to tetracycline, whereas these

406

proteins were either not differentially expressed or up-regulated in P.

407

nodulation protein and peroxiredoxin were up-regulated in P, but not differentially

408

expressed in PC or PG upon exposure to tetracycline (Table S6).

409

NB was treated as the reference

The key enzymes identified in Comparison #1 HGPRT, EF-Tu and EF-G were

The

In an attempt to match the order of biotransformation rates (PC>P>PG, Fig.

410

1B insert) with that of the expression level of a protein (i.e., searching for

411

up-regulated proteins with the fold changes in the order of PC>P>PG), we found one

412

protein, superoxide dismutase [Cu-Zn] (Fig. 7). Superoxide dismutase [Cu-Zn]

413

was up-regulated with the highest fold change under PC (1.7 folds) and with the

414

lowest fold change under PG (1.2 folds).

415

Consistent with our finding, one study reported that glucose led to

416

down-regulation of superoxide dismutase in E. coli.71

417

monitored the superoxide radical levels in the presence of glucose and in the

418

presence of organic acids.

419

superoxide dismutase than did organic acids, likely because the catabolism of

In that study, the authors

They found that glucose led to lower levels of

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420

glucose produced less superoxide radicals than did the catabolism of organic acids.

421

In contrast, the presence of sodium citrate can cause the formation of reactive

422

oxygen species (ROS) in Cryptococcus laurentii,72 leading to increased expression

423

of enzymes such as superoxide dismutase to mitigate the stressed caused by ROS.

424

Together, these could explain why the biotransformation rate of tetracycline was

425

lower in PG (peptone plus glucose) than in PC (peptone plus sodium citrate).

426

Interestingly, a recent study reported that 41.5% of the resistance genes in estuaries

427

samples belonged to the mechanism of enzymatic deactivation.73

428

occurrence of resistance genes specializing in enzymatic deactivation of antibiotics

429

and the involvement of commonly occurring enzymes in antibiotic biotransformation

430

(i.e., superoxide dismutase and peroxidase) suggest that enzymatic inactivation of

431

antibiotics in bacteria may exist more broadly than we have realized.

432

Nutrients co-occur with antibiotics in the environment.

The broad

In this study we

433

revealed how background nutrients may influence the fate of antibiotics by affecting

434

the enzyme expression of tetracycline degrading bacteria S. maltophilia DT1.

435

involvement of superoxide dismutase and peroxiredoxin in the co-metabolism of

436

tetracycline have implications on how other environmental factors, which can trigger

437

the up-regulation of the genes encoding these enzymes, may affect the

438

biotransformation of tetracycline.

439 440 441

4. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS

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The

Environmental Science & Technology

442 443

Publications Website: Experimental procedure of protein preparation, proteomic analysis using 2D

444

LC-MS/MS, and protein identification and quantitation, as well as mechanisms of

445

peroxidase.

446

different background nutrient conditions, soluble phosphorus concentrations in the

447

NB reactor, distribution of genes in the S. maltophilia genome, comparative genomic

448

mapping, gene ontology, and proteins that were detected and differentially

449

expressed.

Results on tetracycline adsorption on cells, biomass density under

450 451 452

5. ACKNOWLEDGEMENTS This study was supported by the US National Science Foundation

453

(CBET-1351676), the Natural Science Foundation of China (41373083 and

454

41611130185), and the Hubei Key Laboratory for Mine Environmental Pollution

455

Control and Remediation (2014103).

456

Core Facility was supported by the NIH Grant P30GM103335.

The UNL Proteomics and Metabolomics

457 458

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670

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671

FIGURES

A

672

B

673 674 675 676 677 678 679 680

Figure 1. The temporal change of tetracycline concentration due to hydrolysis (A) and biotransformation (B) under various nutrient background conditions. P: peptone, PC: peptone plus sodium citrate, PG: peptone plus glucose, NB: no background nutrient. The curves describing hydrolyses and biotransformation were simulated using Equations (B) and (C). Error bars are standard deviation from triplicate experiments. The values of R2 are 0.9452-0.9939 in (A) and 0.9285-0.9957 in (B). “*” indicates that the difference was significant at the 0.05 significance level.

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681

A

682

B

683 684 685 686 687 688

Figure 2. The antimicrobial potency measured using disc diffusion tests for degradation products from hydrolysis only (A) and hydrolysis plus biotransformation (B). The diameter of the inhibition zone includes the diameter of the disks, which was 6 mm. The error bars are standard deviations from triplicate experiments. 30 ACS Paragon Plus Environment

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689 690 691 692 693 694 695

Figure 3. Comparison scheme used in the proteomic analyses to illustrate (A) the proteomic responses of DT1 to tetracycline and (B) the impact of background nutrient conditions on tetracycline biotransformation. Arrows originate from reference proteomes and point to treatment proteomes. TC: tetracycline; P: peptone; PC: peptone and sodium citrate; PG: peptone and glucose; and NB: no background.

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696 697 698 699 700

Figure 4. Proposed resistance mechanisms that S. maltophilia DT1 used upon exposure to tetracycline: (A) ribosomal protection and (B) enzymatic biotransformation.

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701

702 703 704 705 706 707

Figure 5. Effects of the inhibitors to peroxidase (p-aminobenzoic and aminopyrine) and the inhibitor to monooxygenase (methimazole) on tetracycline biotransformation in terms of relative biotransformation rate (i.e., the biotransformation rate in the control experiment without inhibitors was set at 100%). “*” and “**” indicate that the differences were significant at p < 0.05 and p < 0.01, respectively.

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708 709 710 711

Figure 6. Specific protein were up regulated in cells with tetracycline (network nodes).

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Environmental Science & Technology

712 713 714

Figure 7. Differentially expressed proteins in S. maltophilia DT1 under various background nutrient conditions.

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TABLE Table.1. Differentially expressed proteins in S. maltophilia DT1 upon exposure to tetracycline. Protein Name Efflux pump Nodulation protein W Ribosomal protection Elongation factor Tu Elongation factor G Hypoxanthine-guanine phosphoribosyltransferase Enzymatic modification Superoxide dismutase [Cu-Zn] Peroxiredoxin

Gene From Uniprot

Accession Number

KO

Unique peptide

Mol weight [kDa]

2

Comparison #1 Fold chng

p

23.4

2.6

0.012

nodW

A0A031HFA2

tuf

A0A0H2QQ41

K02358

1

43

1.7

0.048

J7VNC6

K02355

1

77.9

1.5

0.049

AVW14_18300

A0A0J8PLL7

K00760

2

20.3

1.5

0.012

DF40_007275

A0A064BR80

K04565

1

19

3.6

0.010

BurJV3_0701

G0JYI5

K13279

1

20.7

1.8

0.010

fusA

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