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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,
8 9 10 11 12
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 [email protected] Xu Li [email protected]
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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
37
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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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
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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 × eିౄా ×்
(A)
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ܥୌ = ܥ − ܥ × eିౄ ×்
(B)
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ܥ = ܥୌ − ܥୌ = ܥ × eିౄ×் −ܥ × eିౄా×்
(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
234
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
237
insert).
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density in the batch reactor, which was 1.0 × 109 CFU/mL for all background
239
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
247
presence of bacterial cells slowed down tetracycline hydrolysis.
One possible
248
explanation was that bacterial cells took up soluble phosphorus from the MM
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medium, resulting in lower phosphorus concentrations (Fig. S3).
250
noticed that reduced phosphorus concentrations resulted in slower tetracycline
251
hydrolysis: when the soluble phosphorus was reduced from 1000 mg L-1 to 500 mg
252
L-1, the hydrolysis rate constant (kH) was reduced from 0.0723 ± 0.0067 day-1 to
253
0.0324 ± 0.0022 day-1.
254
negligible when peptone was not in the background (i.e., the NB condition in Fig.
255
1B).29
256
nutrients were present, suggesting that tetracycline may be transformed through
257
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
260 261 262
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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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
273
slowest under NB.
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Interestingly, the order of the
The
The transformation products of tetracycline from abiotic processes such as
275
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.
278
explain the lack of positive correlation between antibiotic residues, which are often
279
measured as parent compounds, and antibiotic resistance genes in environmental
280
systems.49 In addition, certain background nutrients can enhance the
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biotransformation of toxic compounds by increasing the expression of enzymes
282
critical to the degradation of target compounds. For example, Dai et al. found that
283
sucrose could promote the hydroxylation of imidacloprid by S. maltophilia CGMCC
284
1.1788, likely by increasing the expression of cytochrome P450.50
In contrast, the
These changes in the antimicrobial potency may partially
285 286 287
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
290
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
295
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
297
proteins related to resistance to antibiotics, such as aminoglycoside, fluoroquinolone,
298
and beta-lactam.
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twelve encode beta-lactamase (Table S1).
300
cation/metal efflux pumps for heavy metals such as cobalt, zinc, cadmium, copper
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and arsenic (Table S2).
302
Among them, forty ORFs encode multidrug efflux pumps and Twelve other ORFs encode parts of
Twenty one ORFs encode oxygenases, including eight ORFs encoding
303
monooxygenases, eleven encoding dioxygenases and two encoding oxygenases
304
(Table S3).
305
monohydroxylation and tandem oxidations.54
306
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
308
the tetracycline resistance gene tet(X), FAD-binding monooxygenase encoded by
309
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.
313
from white rot fungi could degrade tetracycline effectively by activating hydrogen
314
peroxide.55
Previous reports showed that peroxidase
315 316
3.4. Proteomic Response to Tetracycline Exposure
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The proteomic response of DT1 to tetracycline was investigated by
318
comparing the proteome of DT1 cells in MM solution containing tetracycline and
319
peptone with the proteome of DT1 cells in MM solution containing only peptone
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(i.e., Comparison #1, Fig. 3A).
321
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).
324
DT1 cells downregulated the glycolysis and TCA cycle while upregulated the purine
325
and the amino acid pathway (Fig. S7).
326
resistance are described in the following sections.
327
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
328
folds in the presence of tetracycline.
This protein can regulate the transcription of
329
genes involved in the nodulation process, and belongs to the
330
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,
334
fusA) were up-regulated by 1.7 and 1.5 folds, respectively, after 24-hour exposure to
335
tetracycline.
336
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
342
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
344
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
347
encoded by tet(34) is classified as a tetracycline deactivation enzyme,58 although
348
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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Tetracycline in hepatocyte63 and plant cell64 can induce the generation of superoxide
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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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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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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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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
36
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Article
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
3 4
Yifei Leng1,2, Jianguo Bao2,*, Dandan Song2, Jing Li2, Mao Ye3 and Xu Li1,*
5 6
Affiliations:
7
1. Department of Civil Engineering, University of Nebraska, Lincoln, NE 68588,
8 9 10 11 12
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
15 16 17 18 19 20
Jianguo Bao [email protected] Xu Li [email protected]
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ABSTRACT
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Certain bacteria are resistant to antibiotics and can even transform antibiotics in the
25
environment.
26
resistance and biotransformation processes vary under different environmental
27
conditions.
28
of tetracycline resistance and biotransformation by Stenotrophomonas maltophilia
29
strain DT1 under various background nutrient conditions.
30
to tetracycline for seven days with four background nutrient conditions: no
31
background (NB), peptone (P), peptone plus citrate (PC), and peptone plus glucose
32
(PG).
33
Genomic analysis showed that strain DT1 contained tet(X1), a gene encoding an
34
FAD-binding monooxygenase, and eight peroxidase genes that could be relevant to
35
tetracycline biotransformation.
36
nodulation protein transported tetracycline outside of cells; hypoxanthine-guanine
37
phosphoribosyltransferase facilitated the activation of the ribosomal protection
38
proteins to prevent the binding of tetracycline to the ribosome; and superoxide
39
dismutase and peroxiredoxin modified tetracycline molecules.
40
nutrient conditions showed that the biotransformation rates of tetracycline were
41
positively correlated with the expression levels of superoxide dismutase.
42
Keywords: Biotransformation, Tetracycline, Genomics, Quantitative proteomics,
43
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
45 46
broad-spectrum antibiotic that is widely used to treat human and animal diseases.1, 2
47
It inhibits protein synthesis by preventing the attachment of aminoacyl-tRNA to the
48
ribosomal acceptor site in bacteria.3
49
exist.
50
tetracycline binding sites and release tetracycline from the ribosome.4
51
pump system can pump tetracycline molecules out of a cell.5
52
inactivation is also a mechanism that is used by resistant bacteria to detoxify
53
tetracycline.
54
tetracycline by adding a hydroxyl group to the C11a position of the molecule.6,7
55
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
56
environment through the disposal of human and livestock wastes.
Multiple
57
processes can affect the fate and the biological impacts of tetracycline antibiotics in
58
the environment.
59
has high affinity to soil (i.e., low aqueous concentrations in runoff).9
60
engineering processes, such as chloramination10 and oxidation11, 12, have also been
61
reported to transform tetracycline antibiotics.
62
biotransformation of antibiotics, particularly biotransformation carried out by
63
bacteria, is rather limited.
The literature on antibiotic biotransformation mostly
64
focuses on fungal species.
For example, Pleurotus ostreatus mycelium and the
65
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
71
emergence of antibiotic resistance.18, 19
72
Hence, understanding the biotransformation
Antibiotics co-occur with other organic compounds in the environments, for
73
example, manure-borne antibiotics in soils.20
74
can vary substantially from soil to soil and from time to time.
75
quantity of the organic compounds in the background can greatly affect the
76
metabolisms of antibiotic resistant bacteria, including their ability to transform
77
antibiotics.21, 22 In an environment where the biotransformation of antibiotics is
78
enhanced, antibiotic parent compounds would be converted to less toxic
79
transformation products15 and consequently the level of selective pressure would
80
decrease for the other bacteria in the local microbial community.
81
The background organic compounds The composition and
Although the effects of background nutrients on antibiotic biotransformation
82
have not been extensively reported, evidence of such effects on the
83
biotransformation of other organic contaminants have been documented.
84
degradation rates of tetrabromobisphenol A (TBBPA) in activated sludge amended
85
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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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.
93
It is important to understand how background nutrients may impact
In our previous study, we isolated a Stenotrophomonas maltophilia strain that
94
was capable of transforming tetracycline by co-metabolism with some background
95
nutrient conditions.27
96
mechanisms of tetracycline resistance and biotransformation by the S. maltophilia
97
strain under various background nutrient conditions.
98
sequenced and analyzed.
99
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
100
proteomics.
This study illustrates how biotransformation and other resistance
101
mechanisms work in the tetracycline resistant S. maltophilia strain at the molecular
102
level and how background nutrient conditions affected the biotransformation of
103
tetracycline.
104 105
2. MATERIALS AND METHODS
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2.1. Bacteria, Chemicals and Solutions
107
S. maltophilia strain DT1 was isolated at the China University of
108
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
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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
114
liquid chromatography (HPLC) grade methanol, acetonitrile, and 0.1% formic acid
115
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
118 119 120
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
122
stationary phase, washed twice in MM, and transferred to 50 mL MM solutions
123
containing 1g L-1 peptone (P), 1g L-1 peptone plus 1g L-1 sodium citrate (PC), 1g L-1
124
peptone plus 1g L-1 glucose (PG), or no background nutrient (NB) at final
125
concentration of OD600 nm = 1.00.
126
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
128
found in soil,30, 31 are used as model nutrients in similar studies,32-34 and exhibited
129
different impacts on tetracycline biotransformation kinetics in our study (see below).
130
For all four groups, (1) the initial tetracycline concentration was 50 mg L-1; (2) all
131
batch reactors were performed in triplicates; (3) each group included a no-cell
132
control; (4) Group P included a autoclaved-cell control, (5) all flasks were covered in
133
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
136 137 138
2.3. Modeling The decrease in tetracycline concentration in the exposure experiment was
139
attributed to both hydrolysis and biotransformation, while the decrease in no-cell
140
controls was attributed only to hydrolysis.
141
presence of strain DT1 (hydrolysis plus biotransformation, Equation A) and in the
142
absence of strain DT1 (hydrolysis only, Equation B) can be described using
143
first-order kinetics.
144
difference between the two equations (Equation C).
The decrease of tetracycline in the
Tetracycline biotransformation can be described using the
145
ܥୌ = ܥ − ܥ × eିౄా ×்
(A)
146
ܥୌ = ܥ − ܥ × eିౄ ×்
(B)
147
ܥ = ܥୌ − ܥୌ = ܥ × eିౄ×் −ܥ × eିౄా×்
(C)
148
C0 is the initial concentration of tetracycline (mg L-1), kHB is the first-order reaction
149
rate constant of the overall degradation reaction including hydrolysis and
150
biotransformation (day-1), kH is the first-order reaction rate constant of hydrolysis
151
(day-1), T is the time (day), CHB is the concentration change of tetracycline from the
152
overall degradation (i.e., CHB was measured from the reactors containing strain DT1),
153
CH is the concentration change of tetracycline due to hydrolysis only (i.e., CH was
154
measured from the no-cell control reactors), CB is the concentration change of
155
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)
159 160
2.4. Antimicrobial Potency Measurements
161
The antibacterial activities of tetracycline and its transformation products
162
were measured using disk susceptibility tests.35 E. coli ATCC 25922 cells were
163
prepared by transferring colonies from an 18- to 24-hour agar plate to a saline
164
solution and adjusting the suspension to achieve a turbidity equivalent to a 0.5
165
McFarland standard.
166
then was used to spread cells on the surface of Mueller-Hinton agar plates.
167
process was repeated two more times after rotating the plate approximately 60° each
168
time to achieve an even distribution of cells on plate surface.
169
a 6 mm diameter (Whatman, USA) were each loaded with 0.02 mL liquid from the
170
exposure experiments and placed onto the surface of the agar plates.
171
incubated at 30°C for 16-18 hr.
172
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
173 174 175
2.5. Quantification of Tetracycline using HPLC To measure tetracycline, 1 mL solution from each batch reactor was
176
centrifuged at 13,796 × g at 4oC for 3 min.
177
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
180
phase consisting of 67% (v/v) 0.1% formic acid in water, 22% (v/v) acetonitrile, and
181
11% (v/v) methanol.36 The injection volume was 20 µL, and the column isocratic
182
elution was monitored using a UV detector at 355 nm.
183 184 185
3.6.Genomic Analysis The genome of S. maltophilia DT1 was sequenced at Genewiz Inc. (SuZhou,
186
China) using Illumina HiSeq X Ten, which generated paired-end reads of 150 bp.
187
The de novo assembly for the sequences was conducted using Velvet (version 1.2.10)
188
and additional scaffolding was performed using SSPACE Basic 2.0.
189
was conducted using the Rapid Annotation using Subsystem Technology (RAST)
190
annotation server.37 Comparative genomic characterization of DT1 with other S.
191
maltophilia strains were conducted using GCview Server.38
192
DT1 were deposited at GenBank under the accession numbers MLJK00000000.
Annotation
The draft genomes of
193 194 195
2.7. Proteomic Analyses Following the triplicate exposure experiments, a fourth experiment was
196
conducted to collect biomass samples for proteomic analyses.
197
were included to cover five experimental conditions in triplicate: MM solution
198
containing 50 mg L-1 tetracycline under four background nutrient conditions (i.e., P,
199
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.
201
initial biotransformation rates, during Day 0 and approximately Day 2, were the
202
highest (see below).
203
extracted, analysed, identified and quantified following a published protocol39 at the
204
UNL Proteomics and Metabolomics Core Facility.
205
Supplemental Information.
206
they exhibited at least 1.5 fold change in abundance between treatment and reference
207
proteomes40 and the change was statistically significant (p < 0.05) according to
208
Fisher's exact test on results from the triplicate protein extracts for each condition.
209
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
210
Ontology (GO) annotation analysis database at the EBI.41
211
into metabolic pathways using Kyoto Encyclopedia of Genes and Genomes
212
(KEGG).42
213
predicted using the STRING database.43
Proteins were mapped
Protein–protein interactions between co-expressed proteins were
214 215 216
2.8. Inhibition on the N-demethylation of Tetracycline The inhibition of tetracycline N-demethylation activity was tested using
217
p-aminobenzoic acid,44 aminopyrine,45 and methimazole.46
218
nutrient condition, P, was used in this experiment.
219
added to the exposure experiment, and samples were collected for tetracycline
220
measurements at the end of the experiment.
Only one background
1 mmol L-1 of each inhibitor was
221
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3. RESULTS AND DISCUSSION
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3.1. Biotransformation of Tetracycline under Different Background Nutrient
224
Conditions In the no-cell controls, the hydrolysis of tetracycline followed first order
225
The hydrolysis rate constant, kH, was measured to be 0.0819 ±
226
kinetics (Fig. 1A).
227
0.0032, 0.0679 ± 0.0050, 0.0756 ± 0.0023, and 0.0681 ± 0.0052 day-1 when peptone
228
(P), peptone plus sodium citrate (PC), peptone plus glucose (PG), or no background
229
nutrient (NB) was in the MM solution, respectively (Fig. 1A insert).
230
Adsorption of tetracycline to bacterial cells was negligible (Fig. S1).
231
biotransformation of tetracycline in the exposure experiment increased initially and
232
then plateaued (Fig. 1B).
233
and used to compare biotransformation kinetics under various experimental
234
conditions (Equation D).
235
(-0.59 ± 0.19 mg L-1d-1), the biotransformation rate increased to 9.23 ± 0.30, 18.76 ±
236
0.68, and 4.25 ± 0.24 mg L-1d-1 in P, PC and PG, respectively (p < 0.05, Fig. 1B
237
insert).
238
density in the batch reactor, which was 1.0 × 109 CFU/mL for all background
239
nutrient conditions tested.
240
background nutrient conditions tested during the course of the 7-day experiments
241
(Fig. S2).
242
Erlenmeyer flasks in the exposure experiment.
243
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
It is noticed that in the NB reactors the tetracycline biotransformation was 12 ACS Paragon Plus Environment
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trending down (Fig. 1B) and consequently had a slightly negative mean for the initial
245
biotransformation rate (Fig. 1B insert).
246
no-cell control overestimated the hydrolysis in the NB reactors.
In other words, the
247
presence of bacterial cells slowed down tetracycline hydrolysis.
One possible
248
explanation was that bacterial cells took up soluble phosphorus from the MM
249
medium, resulting in lower phosphorus concentrations (Fig. S3).
250
noticed that reduced phosphorus concentrations resulted in slower tetracycline
251
hydrolysis: when the soluble phosphorus was reduced from 1000 mg L-1 to 500 mg
252
L-1, the hydrolysis rate constant (kH) was reduced from 0.0723 ± 0.0067 day-1 to
253
0.0324 ± 0.0022 day-1.
254
negligible when peptone was not in the background (i.e., the NB condition in Fig.
255
1B).29
256
nutrients were present, suggesting that tetracycline may be transformed through
257
co-metabolism.
258
was used to subtract from the overall degradation in the NB reactors, the calculated
259
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
260 261 262
3.2. Antibacterial Potency of Tetracycline Degradation Products The antimicrobial potency of the degradation products from hydrolysis and
263
biotransformation decreased over the course of seven days (Fig. 2).
264
hydrolysis was the only mechanism, the degradation products under different
265
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The antimicrobial potency of the degradation products differed among the
267
background nutrient conditions tested (Fig. 2B).
268
decreases in antimicrobial potency among the four background nutrient conditions
269
matched the order of the biotransformation rates (Fig. 2B and Fig. 1B insert).
270
diameter of the inhibition zones dropped the fastest under PC (from 13.38 mm to
271
8.27 mm) and the slowest under NB (from 13.27 mm to 11.50 mm).
272
Correspondingly, the biotransformation rate was the highest under PC and the
273
slowest under NB.
274
Interestingly, the order of the
The
The transformation products of tetracycline from abiotic processes such as
275
photolysis had higher toxicity than the parent compound.47, 48
276
biotransformation products of tetracycline by DT1 or fungi14 had lower toxicity than
277
the parent compound.
278
explain the lack of positive correlation between antibiotic residues, which are often
279
measured as parent compounds, and antibiotic resistance genes in environmental
280
systems.49 In addition, certain background nutrients can enhance the
281
biotransformation of toxic compounds by increasing the expression of enzymes
282
critical to the degradation of target compounds. For example, Dai et al. found that
283
sucrose could promote the hydroxylation of imidacloprid by S. maltophilia CGMCC
284
1.1788, likely by increasing the expression of cytochrome P450.50
In contrast, the
These changes in the antimicrobial potency may partially
285 286 287
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.
289
The draft genome included 4,532,597 bp, with an average contig length of 110,551
290
bp and a maximum contig length of 476,742 bp.
291
to be 66.48%.
292
all ORFs, 2,787 (69%) could be functionally annotated using RAST (Fig. S4).
293
total of 1,153 hypothetical proteins were predicted for S. maltophilia DT1.
294
genomic regions of difference (RODs) were observed between DT1 with the finished
295
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
296
Fifty seven ORFs of S. maltophilia DT1 were predicted to encode for
297
proteins related to resistance to antibiotics, such as aminoglycoside, fluoroquinolone,
298
and beta-lactam.
299
twelve encode beta-lactamase (Table S1).
300
cation/metal efflux pumps for heavy metals such as cobalt, zinc, cadmium, copper
301
and arsenic (Table S2).
302
Among them, forty ORFs encode multidrug efflux pumps and Twelve other ORFs encode parts of
Twenty one ORFs encode oxygenases, including eight ORFs encoding
303
monooxygenases, eleven encoding dioxygenases and two encoding oxygenases
304
(Table S3).
305
monohydroxylation and tandem oxidations.54
306
FAD-binding monooxygenase encoded by tet(X1) was identified in the draft genome.
307
Unlike the better known flavin-dependent monooxygenase, which was encoded by
308
the tetracycline resistance gene tet(X), FAD-binding monooxygenase encoded by
309
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).
311
Peroxidases are wildly found in bacteria, fungi, plants and animals, and catalyze
312
oxidative reactions by hydrogen peroxide.
313
from white rot fungi could degrade tetracycline effectively by activating hydrogen
314
peroxide.55
Previous reports showed that peroxidase
315 316
3.4. Proteomic Response to Tetracycline Exposure
317
The proteomic response of DT1 to tetracycline was investigated by
318
comparing the proteome of DT1 cells in MM solution containing tetracycline and
319
peptone with the proteome of DT1 cells in MM solution containing only peptone
320
(i.e., Comparison #1, Fig. 3A).
321
and a subset of these proteins were listed in Table 1.
322
exposure on various protein functional categories in S. maltophilia DT1 were
323
analyzed using GO annotation (Fig. S6).
324
DT1 cells downregulated the glycolysis and TCA cycle while upregulated the purine
325
and the amino acid pathway (Fig. S7).
326
resistance are described in the following sections.
327
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
328
folds in the presence of tetracycline.
This protein can regulate the transcription of
329
genes involved in the nodulation process, and belongs to the
330
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,
334
fusA) were up-regulated by 1.7 and 1.5 folds, respectively, after 24-hour exposure to
335
tetracycline.
336
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
342
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
344
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
347
encoded by tet(34) is classified as a tetracycline deactivation enzyme,58 although
348
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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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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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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