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Ecotoxicology and Human Environmental Health
Water disinfection byproducts increase natural transformation rates of environmental DNA in Acinetobacter baylyi ADP1 David Mantilla-Calderon, Michael J Plewa, Gregoire Michoud, Stelios Fodelianakis, Daniele Daffonchio, and Peiying Hong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00692 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 4, 2019
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Environmental Science & Technology
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Water disinfection byproducts increase natural transformation rates of
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environmental DNA in Acinetobacter baylyi ADP1
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David Mantilla-Calderon 1, Michael J. Plewa 2, 3, Grégoire Michoud 4, Stelios Fodelianakis 4,
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Daniele Daffonchio 4 and Pei-Ying Hong 1*.
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1. King
Abdullah University of Science and Technology (KAUST), Water Desalination
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and Reuse Center, Division of Biological and Environmental Science and Engineering,
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Thuwal 239455-6900, Saudi Arabia
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2. Department
of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana,
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IL 61801, United States
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3. Safe
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61801, United States
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4. King
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Biological and Environmental Science and Engineering, Thuwal 239455-6900, Saudi
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Arabia
Global Water Institute, University of Illinois at Urbana-Champaign, Urbana, IL
Abdullah University of Science and Technology (KAUST), Division of
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* Corresponding author:
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Pei-Ying Hong
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Email:
[email protected] 23
Phone: +966-12-8082218 1 ACS Paragon Plus Environment
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Abstract
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The process of natural transformation allows for the stable uptake, integration and
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functional expression of extracellular DNA. This mechanism of horizontal gene transfer has
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been widely linked to the acquisition of antibiotic resistance and virulence factors. Here, we
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demonstrate that bromoacetic acid (BAA) – a regulated drinking water disinfection
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byproduct (DBP) – can stimulate natural transformation rates in the model organism
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Acinetobacter baylyi ADP1. We demonstrate that transformation stimulation in response to
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BAA is concentration-dependent and is linked to the ability of this compound to generate
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DNA damage via oxidative stress. In presence of BAA, transcription of recA was upregulated
34
20-40% compared to the non-treated controls, indicating that this component of the DNA
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damage response could be associated with the increase in transformation. Other genes
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associated with DNA-translocation across the cytoplasmic membrane (i.e. pilX, comA) did not
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exhibited increased transcription in presence of BAA, indicating that the enhancement of
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transformation is not associated with increased translocation rates of environmental DNA.
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Overall, these results lead us to speculate that elevated recA transcription levels could lead
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to increased integration rates of foreign DNA within the recipient cell during DNA repair.
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Lastly, we show that an artificial DBP cocktail simulating the environmental concentrations
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of five water DBP classes stimulates natural transformation by almost 2-fold. The results of
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this study suggest that mutagens like DBPs may play an important role in enhancing the
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fixation rates of extracellular DNA in the environmental metagenome.
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1. Introduction
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The emergence and rapid spread of antimicrobial resistance (AMR) among bacterial
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pathogens is a serious public health concern1. Traditionally, clinical settings were considered
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the main site for the evolution of AMR. However, over the last decade, ARGs conferring
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resistance to last resort antibiotics have been detected in multiple environmental
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compartments, including agricultural soils2, sediments3,
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Consequently, AMR is no longer considered to be a phenomenon exclusive to the clinical
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settings2, 7, but rather, one that is also prevalent in the environment.
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and surface waters4-6.
56 57
Wastewater discharges constitute a key source of environmental resistance as they
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carry high concentrations of emerging microbial pollutants (e.g. ARGs and antibiotic resistant
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bacteria, ARB)3, 8, 9. To mitigate this concern, most countries treat wastewater through a
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series of physical, biological and chemical processes, including a final disinfection step, to
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decrease the nutrient and bacterial counts to permissible levels. The processing of municipal
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and agricultural wastewaters to kill pathogens is important to protect public health and the
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environment. However, it had been demonstrated earlier that different disinfectants or
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disinfection processes alter the toxicological characteristics of treated wastewaters10-14. Even
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with disinfection, studies reported increases in the occurrence of ARB among various types
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of bacterial taxa (e.g. Enterobacteriaceae, Aeromonas spp., Acinetobacter spp.) in downstream
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receiving waters15-17. Besides direct deposition of microbial contaminants, horizontal flow of
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ARGs between wastewater-borne bacteria and the native microbial communities could
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potentially explain the increased prevalence of ARB in wastewater-impacted environments16, 3 ACS Paragon Plus Environment
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18.
The contribution of horizontal gene transfer (HGT) to the ARB enrichment phenomenon
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is inferred from: i) the observation that the resistome fraction comprising ARGs of clinical
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relevance – in wastewater and the environment – are predominantly associated with mobile
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genetic elements2, 19, and ii) the capacity of sub-lethal doses of disinfectants (e.g. chlorine) to
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stimulate conjugation rates by 2-5 fold20. To the best of our knowledge, environmental
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studies had focused exclusively on the contribution of conjugation in the spread of AMR21.
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However, this mechanism of HGT does not present a comprehensive picture of the
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environmental fate and persistence of ARGs, especially those that exist as free extracellular
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DNA.
79 80
In addition to the effect of chlorine on conjugation, many disinfection byproducts
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(DBPs), which are an unintended consequence of water disinfection, are genotoxins and
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mutagens in a number of biological systems22, 23. In bacteria, exposure to mutagens lead to
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the transcriptional activation of the recA gene as part of DNA damage response pathways24,
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25.
85
fundamental process for both repair of DNA double strand breaks26 and chromosomal
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integration of foreign DNA during natural transformation27.
The product of recA is involved in the initiation of homologous recombination, a
87 88
Based on the previous observations, we hypothesized that environmental stressors
89
such as DBPs arising from the final disinfection step of most wastewater treatment plants
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can trigger DNA repair in naturally competent bacteria. This would facilitate the integration
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process of foreign DNA during natural transformation, and hence allow bacterium to gain
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new functional traits. The objective of this study was to determine whether exposure to such 4 ACS Paragon Plus Environment
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environmental mutagens could enhance the process of transformation in naturally
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competent bacteria. As DBPs are particularly abundant in treated wastewater effluents28, 29,
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we tested our hypothesis using the interaction between bromoacetic acid (BAA), a mutagenic
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and genotoxic, regulated DBP30, 31 and the ubiquitous and naturally competent bacterium
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Acinetobacter baylyi ADP1.
98 99 100
2. Material and Methods
101 102
2.1. Bacterial strains and culture conditions
103
A reporter strain of Acinetobacter baylyi ADP1 (BD413) was used in this study32. After a
104
successful natural transformation event, the reporter strain expresses spectinomycin
105
resistance and GFP+ phenotype32. A. baylyi was routinely cultivated at 37°C in LB Miller broth
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and agar, supplemented with kanamycin (50 μg mL-1) and rifampicin (50 μg mL-1). Salmonella
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enterica serovar Typhimurium TA100 was propagated in LB Miller broth supplemented with
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ampicillin at a final concentration of 25 μg mL-1.
109 110
2.2. Mutagenicity test
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Bromoacetic acid (BAA)’s mutagenic potential was evaluated by a modified Ames
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preincubation reversion assay in the tester strain Salmonella enterica serovar Typhimurium
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TA100, as previously described
114
used for mutagenicity tests as this assay is considered the gold standard for assessing
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mutagenicity in bacterial systems34. Briefly, a standardized suspension of 2 × 109 cells mL-1
33.
Salmonella enterica serovar Typhimurium TA100 was
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of the tester strain was incubated at 37°C with 0-600 μM BAA for one hour. Subsequently, 5
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× 108 cells were plated in Vogel-Bonner (VB) minimal medium, and histidine revertant
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colonies were enumerated after 72 h of incubation at 37°C. BAA and glutathione (GSH) assays
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were set to contain an equimolar GSH concentration relative to BAA. Incubation and sample
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processing were done according to standard methods.
121 122
2.3. Dose-response transformation stimulation assays
123 124
A. baylyi was inoculated in 50 mL of LB Miller broth supplemented with kanamycin
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(50 μg mL-1) and rifampicin (50 μg mL-1), and incubated overnight at 37°C × 200 rpm.
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Subsequently, A. baylyi cell cultures were centrifuged (4° C × 6500 rpm × 10 min) and washed
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twice with cold phosphate buffer (PPB). Cell pellets were suspended in 1 mL of PPB and
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adjusted to a standardized suspension containing 2 × 1010 cells mL-1. One hundred μL of
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standardized suspension were added to fresh LB Miller broth (without antibiotics)
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containing 2 μg mL-1 of donor DNA and 0-600 μM BAA (initial inoculum of 2 × 109 cells mL-1
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and 1 mL of final volume per assay tube). Since natural transformation events occur at
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extremely low frequencies, ranging from 10-5 to 10-8, it was important to optimize the
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detection limit of our assays by utilizing high concentrations of donor DNA and high densities
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of A. baylyi cells. Assays were incubated for 24 h at 37°C × 200 rpm. To determine baseline
135
mutation rates that could spontaneously lead to the expression of the cassette, a series of
136
controls lacking the donor DNA were setup in parallel in the 0-600 μM BAA range. These
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controls include i) A. baylyi cells alone (no BAA, no donor DNA added) and ii) A. baylyi cells +
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BAA (0-600 μM, no donor DNA added). No transformants were detected in both types of 6 ACS Paragon Plus Environment
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controls in all biological replicates. This suggests that BAA-induced mutations without the
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presence of any donor DNA would not result in any expression of spectinomycin resistance
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and GFP among A. baylyi. Neither would the A. baylyi undergo spontaneous mutation without
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the presence of stressor (i.e., BAA) and donor DNA.
143 144
Donor DNA was generated by PCR using the primer pair 5’-ATT ATT GAA TTC GGT
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AGA GCC GTT TAT GAA-3’ forward and 5’-TTT GCC CAC TAC CTT GGT GAT-3’ reverse32,
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utilizing as template a cloning vector (pCLT) that contains the functional promoter (Prrn) that
147
activates the aadA::GFP transcriptional fusion. The pCLT cloning vector is propagated in
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Escherichia coli K12. This primer set amplifies the rescue cassette rbcL-Prrn-aadA, which
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inserts by homologous recombination upstream the silenced aadA::GFP fusion. The resulting
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2.1 kb amplicon containing the rbcL- Prrn -aadA fragment was purified using the Wizard® SV
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Gel and PCR Clean-Up System (Promega), and purified DNA was quantified with the Qubit™
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DNA BR Assay Kit (Invitrogen). More details on the transformation reporter system were
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described in earlier study by Rizzi et al32.
154 155
After 24 h, total cell counts were determined by platting in LB Miller agar
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supplemented with kanamycin (50 μg mL-1) and rifampicin (50 μg mL-1) and incubated for
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24 h at 37°C. Screening for transformants was first achieved by additionally supplementing
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LB Miller plates with spectinomycin (100 μg mL-1). No colonies resistant to spectinomycin
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were detected at any BAA concentration when donor DNA was not present, suggesting lack
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of spontaneous expression of gene cassettes by A. baylyi in absence of donor DNA. In the
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assays containing the donor DNA, insertion of the donor was further confirmed by colony 7 ACS Paragon Plus Environment
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PCR in 5 randomly selected transformant colonies per plate. Colony PCR was done using the
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primer pair 5’-ATC TTT CTA TTG TTG TCT TGG AT-3’ forward and 5’-GGT CAC CGT AAC CAG
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CAA ATC AA-3’ reverse32. This primer pair anneals downstream and upstream of the deleted
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promoter. In the presence of the promoter, introduced after a successful transformation of
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the donor DNA, the PCR amplicon would be 333 bp, whereas an unsuccessful transformation
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would derive a PCR amplicon of 190 bp. Therefore, the amplicon size would allow
168
differentiation of whether donor DNA insertion was indeed successful or not. All screened
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colonies were tested positive for the donor DNA insertion, confirming that the observed
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phenotype corresponds to the insertion of the donor DNA, and not to spontaneous mutations
171
that could lead to the expression of the cassette.
172 173
Transformation frequency was defined as the ratio of transformants over the total
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number of cells in each assay vial. Fold induction was defined as the ratio of the
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transformation frequency of each treatment, over the average transformation frequency for
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the 0 μM BAA control in the same replicate run. Statistical differences between treatments
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were calculated by ANOVA. Power test was calculated using SigmaPlot v13 (Systat Software
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Inc, San Jose, CA).
179 180
2.4. BAA and glutathione co-incubation assays
181 182
A BAA and glutathione (GSH) co-incubation assay was designed to determine whether
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reactive oxygen species (ROS) are involved in the increased transformation and cytotoxic
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response to BAA exhibited by A. baylyi. First, the ability of glutathione to modulate 8 ACS Paragon Plus Environment
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mutagenicity was tested in Salmonella enterica serovar Typhimurium TA100. For this, the
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procedure described in section 2.2 was followed with a slight modification. Besides the
187
addition of BAA, an equimolar concentration of GSH was added to the same assay tube (i.e.
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400 μM BAA + 400 μM GSH). Assay incubation and quantification of revertants were then
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conducted as described in section 2.2.
190 191
Once it was demonstrated that equimolar concentration of GSH inhibit the mutagenic
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output of BAA in TA100, transformation assays in A. baylyi were repeated in the presence of
193
equimolar concentrations of GSH (i.e. 400 μM BAA + 400 μM GSH). Assay incubation,
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transformation frequencies and cytotoxic response were determined as described in section
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2.3.
196 197
2.5. Biomass collection for BAA toxico-genomics and RNA extraction
198 199
BAA cytotoxicity was detected at concentrations higher than 200 μM (Figure S1).
200
Given that the transformation induction phenotype is linked to BAA’s mutagenic potential
201
and not due to cytotoxicity, transcriptome analysis was restricted to the 0-200 μM
202
concentration range, where no increased cytotoxicity was detected (Supplementary
203
Information 1, Figure S1-S2). This is to avoid capturing the cytotoxic response to BAA which
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is not accounting for the observed increase in natural transformation.
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One hundred μL of an overnight culture of A. baylyi were inoculated in 100 mL of fresh
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LB Miller broth and incubated at 37°C × 200 rpm. After reaching an OD660 of 0.1, cell cultures 9 ACS Paragon Plus Environment
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were spiked with 2 μg mL-1 of donor DNA and 100, 150 and 200 μM BAA, or mock spiked with
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an equal volume of carrier (DMSO) for the 0 μM control. Cell cultures were placed back in
210
incubation, and 10 mL samples were taken at 0, 10, 30 and 60 min. After collection, samples
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were immediately chilled in an ethanol ice bath, and cell pellets collected by centrifugation
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at 7000 rpm for 15 min at 4° C. Cell pellets were subsequently suspended in RNAlater®
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stabilization solution (Thermo Scientific), following manufacturer instructions. Cell pellets
214
were long term stored at -80° C.
215 216
Cell pellets were extracted for total RNA with an automated Maxwell® RSC
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Instrument, operating with a Maxwell® RSC SimplyRNA Cell Kit (Promega). RNA extraction
218
was done following the manufacturer instructions with a minor technical modification. In
219
order to achieve complete DNA digestion during extraction, 7 extra units of DNase I per
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sample were added to the DNA digestion step. RNA integrity was determined using a 2200
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TapeStation System (Agilent), whereas RNA concentration was assessed with the Qubit™
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RNA HS Assay Kit (Invitrogen).
223 224
2.6. RNA-seq and data analysis
225 226
RNA samples were sent to the KAUST Bioscience Core Lab for paired-end sequencing
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on an Illumina HiSeq 4000 platform. Libraries were first constructed using a TruSeq Stranded
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Total RNA Kit. The quality of the reads was assessed using the fastqc software35, the Illumina
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primers were then removed with the trimmomatic program36. The abundances of transcripts
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were quantified with the kallisto software37 and the differential analyses were done with the 10 ACS Paragon Plus Environment
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sleuth program38. Genome of Acinetobacter sp. ADP1 (assembly ASM4684v1) were used as
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reference for the kallisto software. Concentration-response behavior was evaluated by
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testing for differences at the gene expression (transcripts per million – TPM) with increasing
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concentration of BAA, irrespectively of time. For that, we compared a full model (TPM ~
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BAA+ time) to a reduced model (TPM ~ time) in sleuth. All transcriptomic fastq files are
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deposited in European Nucleotide Archive under study accession number PRJEB28698.
237 238
2.7. Reverse Transcription-qPCR (RT-qPCR) quantification
239 240
Transcriptional changes were confirmed by RT-qPCR using the t = 60 min samples
241
from the toxico-genomic experiment. First, cDNA was generated priming with random
242
hexamers using the SuperScript III First Strand cDNA Synthesis Kit (Thermo Fisher
243
Scientific) following manufacturer’s instructions. Copy numbers were determined by
244
absolute quantification as previously described39 using the primers listed in Table S1.
245 246
2.8. DBP-cocktail transformation stimulation assays
247 248
One hundred μL of an overnight culture of A. baylyi were inoculated in 100 mL of fresh
249
LB Miller broth and incubated at 37° C, 200 rpm. After reaching an OD660 of 0.1, aliquots of 1
250
mL were dosed to 20 mL glass vials containing a simplified DBP-cocktail or an equal volume
251
of DMSO, and incubated for 1 h at 37°C, 200 rpm. A detailed description of the DBP-cocktail
252
can be found in the supplementary material section (Table S2). Transformation frequencies
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were determined by following the protocol described for the BAA concentration-response
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transformation stimulation assays.
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2.9. Determination of potential interaction between BAA and donor DNA
257
To confirm that the transformation induction phenotype is not caused by a potential direct
258
BAA-DNA interaction, a separate set of experiments was conducted in which the donor DNA
259
was first pre-exposed to BAA prior to transformation in absence of BAA. First, donor DNA
260
was pre-exposed to BAA (0-600 M) for 1 h, and subsequently purified using ethanol
261
precipitation. A 1 h exposure time was used as previous transformation assays had shown
262
that only 1 h of exposure to BAA was enough to enhance transformation (Figure S1c).
263
Transformation assays were performed as described in section 2.3 but this time BAA was not
264
added in the culture media and pre-exposed DNA was used instead.
265 266
3. Results and Discussion
267 268
3.1. BAA stimulates natural transformation rates in a concentration-response manner
269 270
BAA-induced mutagenicity concentration response curves were first generated using
271
a modified preincubation test of the his⁻ reversion assay in Salmonella enterica serovar
272
Typhimurium strain TA100
273
concentration range of 100-600 μM, with a linear response from 100-250 μM BAA (Figure
274
1a). Subsequently, we determined whether BAA at these mutagenic concentrations enhanced
275
natural transformation by using a recipient-reporter strain derivative of A. baylyi ADP1
30, 40.
TA100 expressed BAA-induced mutagenicity in the
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(BD413)32. This strain carries a marker-reporter rescue cassette (rbcL-ΔP aadA::GFP)
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integrated in the bacterial chromosome between the lipB and lipA gene. The transcriptional
278
fusion (aadA::GFP), encoding for spectinomycin resistance (aadA) and the green fluorescent
279
protein (GFP), lacks a functional promoter and therefore is not expressed in the recipient
280
strain. A donor DNA carrying a constitutive functional promoter (rbcL-Prrn-aadA) is used to
281
restore the expression of aadA::GFP. Upon uptake of the donor DNA, homologous
282
recombination takes place between the flanking rbcL and aadA loci, leading to the insertion
283
of the Prrn promoter upstream the aadA::GFP fusion. The promoter integration activates the
284
transcription of the gene fusion, restoring spectinomycin resistance and the fluorescent
285
phenotype.
286 287
In accordance with the study hypothesis, BAA significantly increased the
288
transformation frequencies of the reporter strain by nearly two-fold at the highest BAA
289
concentration (600 μM). Moreover, transformation stimulation followed a BAA
290
concentration-response pattern (Figure 1b), establishing a direct link between BAA exposure
291
and the increased rates of DNA transformation. It is important to highlight that no
292
transformants were detected in the control assays lacking donor DNA, and that insertion of
293
donor DNA was further confirmed by PCR in the DNA-positive assays. These results
294
demonstrate that the concentration-response relationship do not correspond to increased
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mutagenic rates that could lead to spontaneous expression of the cassette, but instead,
296
results from an actual increase in transformation frequencies in presence of BAA.
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3.2. Natural transformation stimulation is triggered by DNA damage via oxidative
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stress
300 301
Toxicity endpoints of haloacetic acids (HAAs) in TA100 included mutagenic as well as
302
cytotoxic responses. Recent information demonstrated that the toxicity of the
303
monohalogenated HAAs involves thiol/thiolate reactivity to polypeptides and enzymes41.
304
The involvement of the induction of oxidative stress in monohalogenated HAAs genotoxicity
305
was also previously demonstrated using reactive oxygen species (ROS) inhibitors and human
306
toxicogenomic analyses42-44. In addition, the results from these studies showed no direct
307
interaction between monohalogenated HAAs and DNA. To confirm the absence of direct BAA-
308
DNA interaction, transformation assays (no BAA added in culture media) utilizing only donor
309
DNA that was pre-exposed to increasing BAA concentrations (0-600 M) was performed.
310
Pre-exposed donor DNA did not lead to increased natural transformation frequencies (Figure
311
S3), confirming that the observed transformation induction phenotype do not arise from
312
direct BAA-DNA interactions.
313 314
To establish a direct link between ROS-mediated DNA damage and the transformation
315
stimulation phenotype, the mutagenic response was modulated using an ROS scavenger. An
316
experiment with the addition of equimolar concentrations of glutathione (GSH) in the TA100
317
assay achieved a 73 to 91.4% reduction in the reversion frequency compared to that of the
318
BAA treatment alone (Figure 1c). These results confirm the ability of GSH to modulate the
319
ROS-mediated mutagenic response.
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To further test the effect of GSH on the BAA stimulation of transformation rates in A.
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baylyi, we selected a concentration of 400 μM BAA as it exhibited a prominent transformation
323
enhancement and cytotoxic response (Figure 1b). The addition of GSH completely reversed
324
the increase of transformation rates by 400 μM BAA in the reporter strain (Figure 1d) but did
325
not have any effect on its cytotoxic response (Figure 1e). These data were statistically
326
significant as compared to their concurrent controls and demonstrate a direct causation
327
between ROS-mediated DNA damage and the increase of transformation rates in BAA treated
328
assays.
329 330
The results of prior research with A. baylyi suggested that expression of natural
331
transformation could lead to increased survival under stress conditions 45. Potentially, this
332
could result in artefacts in the estimation of natural transformation rates in response to BAA
333
as increased survival of the transformants could lead to overestimation of transformation
334
events in BAA treated assays. However, this possibility could be ruled out based on the fact
335
that enhancement of transformation was also detected in absence of cell death (i.e. non-
336
cytotoxic BAA levels, Figure 1b), suggesting that this response is not linked to cytotoxicity.
337
This observation was further corroborated in the BAA + GSH co-incubation assays. GSH
338
showed no effect on the BAA cytotoxic response (Figure 1e), therefore, if the enhanced
339
transformation phenotype was indeed an artefact resulting from increased survival of the
340
transformants, it would be anticipated that the transformation rates observed for BAA and
341
BAA + GSH to be similar. Instead, the addition of GSH completely reversed the transformation
342
frequencies in BAA co-incubated treatments. These results provide strong evidence that the
343
observed concentration-response enhancement of transformation frequencies was not the 15 ACS Paragon Plus Environment
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result of an artefact arising from increased survival to cytotoxic stress but rather mediated
345
by ROS.
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3.3. A. baylyi transcriptional response to BAA exposure
348 349
A toxicogenomic experiment was conducted to identify genes that were modulated by
350
BAA concentrations (Figures S1 and S2). Sample quality control data can be found in Table
351
S3. Previous studies demonstrated that the monohalogenated HAAs, which include BAA,
352
blocked glycolysis and reduced cellular ATP levels due to the inhibition of glyceraldehyde-3-
353
phosphate dehydrogenase (GAPDH)44,
354
GAPDH inhibition and ROS formation, A. baylyi up-regulated the expression of GAPDH and
355
ROS detoxification response genes. Indeed, the transcriptomic data revealed a concentration-
356
response increase in the transcripts per million (TPM) of both gene traits (Table S4, Figure
357
S4), and provided a molecular mode of action for both the mutagenicity and enhanced
358
transformation mediated by BAA concentration. These data were in agreement with
359
toxicogenomic studies of BAA genotoxicity in human cell systems 43, 47-49 , but were not yet
360
reported for bacterial cell systems.
46.
Similarly, it was observed that in response to
361 362
A comprehensive list of all genes following a concentration-response change in
363
expression is shown in Table S5. To further elucidate how BAA would modulate gene
364
expressions, genes related to natural transformation were emphasized50. Natural
365
transformation occurs via two steps, namely DNA uptake and DNA integration27. Two
366
essential genes of the DNA uptake machinery, pilX and comA50, were observed to be 16 ACS Paragon Plus Environment
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downregulated with exposure to BAA by RNA-seq (Figure 2a and 2b). However, confirmation
368
by RT-qPCR did not show a concentration-response pattern since the slope of fold-change
369
trend line did not significantly differ from that of zero (Figure 2a and 2b). Hence, the increase
370
in natural transformation observed in response to BAA was unlikely due to increase in active
371
DNA uptake.
372
However, ROS generated by BAA may have altered the physical structure of cell
373
membranes. The exact types of ROS generated by BAA are not known, but hydrogen peroxide
374
is likely to be one of the generated ROS. This is inferred because the addition of glutathione
375
(a scavenger for hydrogen peroxide51) was able to revert mutagenicity level (Figure 1c) and
376
restore natural transformation rates to that of baseline level observed at 0 µM BAA (Figure
377
1d). Hydrogen peroxide can accumulate in large amounts in the cell membranes, resulting in
378
increased oxidized lipid fraction which would cause higher membrane permeability52. This
379
may result in a higher pool of donor DNA within the cytoplasm but further studies would
380
have to be conducted to establish if ROS would indeed result in higher DNA permeability.
381
Next, genes related to DNA integration were further examined. ROS-induced DNA
382
damage causes primarily single strand breaks (SSB), while double strand breaks occur at
383
lower frequencies as a consequence of two proximal SSB or following replication passing
384
through ROS-induced lesions53. In toxicogenomic studies, BAA exposure modulated the
385
expression of genes involved in both SSB and DNA double strand break repair47. While SSB
386
are repaired by nucleotide excision, double-strand breaks are primarily repaired via
387
homologous recombination in a RecA-dependent manner26.
388
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389
As RecA is the molecular link between DNA repair and natural transformation in our
390
hypothesis, we expected overexpression of the recA gene in response to BAA treatment. In
391
accordance to this hypothesis, transcripts for recA were more abundant in BAA treatments
392
than in controls, however, contrary to other SOS-controlled genes such as himA54 (Table S4);
393
the recA induction profile did not follow a clear BAA concentration-response behavior at t =
394
60 min (Figure 2c). To exemplify, recA exhibited more downregulation in the absence of BAA
395
than with 100 to 200 µM BAA at t = 10 min. The gene then experienced an increase in TMP
396
abundance at 30 min, and that the upregulation was higher in presence of BAA than control.
397
There was however an outlier trend at t = 60 min (Figure 2c). RT-qPCR was used to further
398
verify RNA-Seq data at this time point, and revealed a clear linear increase in recA copy
399
numbers in response to BAA at t = 60 min. We cannot explain the discrepancy between the
400
results deriving from RNA-Seq and RT-qPCR at t = 60 min, however, RNA-Seq is a global gene
401
expression profiling tool and may not exactly reflect the expression status of all genes. On the other
402
hand, RT-qPCR is a highly targeted approach, which makes single gene expression data less
403
sensitive to background variations.
404 405
Other components of the DNA repair machinery, such as uvrB, an essential constituent
406
of the nucleotide excision and repair complex, was transcribed in a BAA concentration-
407
dependent manner in both, the RNA-Seq and RT-qPCR data (Figure 2d). These results are
408
consistent with previous studies describing the mutagenic mode of action of BAA47,
409
indicating that DNA damage response to the BAA-induced mutagenicity was predominantly
410
SSB repair.
411 18 ACS Paragon Plus Environment
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412
When examining the expression patterns of genes associated with natural
413
transformation, only recA showed consistent transcriptional modulation in response to BAA
414
in both data sets, RNA-Seq and RT-qPCR data (Figure 2c). The participation of RecA in DNA
415
repair and DNA integration during natural transformation, led us to consider recA
416
overexpression as the mechanism potentially responsible for the increase of natural
417
transformation rates.
418 419
3.4. Environmental concentrations of DBPs increase natural transformation rates
420 421
A DBP cocktail that contained environmental concentrations of 5 classes of DBPs
422
including those regulated by the U.S. Environmental Protection Agency55 (Table S2) was
423
further used to test the hypothesis that enhanced transformation would be associated with
424
DBPs. A. baylyi cell cultures were exposed to the DBP cocktail during early exponential phase
425
as we found that at this point, A. baylyi cell cultures expressed peak transformation
426
frequencies (Figure 3a). Under these conditions, the DBP mixture stimulated donor DNA
427
transformation by nearly 2-fold (Figure 3b). These results demonstrate the environmental
428
relevance of the phenomenon herein described in this study.
429 430 431
4. Implications
432 433
This study reveals that water disinfection byproducts significantly increase the
434
transformation rates of extracellular DNA in the bacterial model A. baylyi ADP1. The 19 ACS Paragon Plus Environment
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435
stimulation phenotype is linked to the ability of BAA to cause DNA damage via oxidative
436
stress, which triggered increased transcription of recA, a key recombinase involved in both
437
DNA damage/repair and foreign DNA integration. The higher cytoplasmic levels of RecA
438
could subsequently increase the chances of foreign DNA to be integrated in the bacterial
439
chromosome and hence resulted in increase in natural transformation. It is important to
440
highlight that DNA damage is the ultimate factor driving the stimulation of transformation,
441
and therefore, exposure to other environmental conditions (e.g. chlorination per se, solar
442
irradiance, heavy metals etc.) that generate mutagenic damage, could potentially result in a
443
similar and/or synergic stimulation response.
444 445
Given that chlorine is a common disinfection strategy in most wastewater treatment
446
plants and that the formation of DBPs is an inevitable process, the implications of DBP-
447
enhanced natural transformation rates are broad. Previous studies have shown that DBPs
448
and other chemical pollutants present in wastewater increase conjugation rates among
449
diverse linages of bacteria
450
over other mechanisms of HGT (i.e. natural transformation) has not been described yet.
451
Unlike conjugation, natural transformation allows for the exchange of information among
452
phylogenetically more distant microorganisms27. Therefore, environmental compartments
453
with mutagenic potential - such as water bodies impacted by wastewater discharges - could
454
facilitate the mobilization of novel ARGs from soil organisms to microorganisms of clinical
455
relevance.
56-59.
However, to the best of our knowledge, the effect of DBPs
456
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457
Even though conjugation and natural transformation are independent mechanisms of
458
HGT, there appear to be several shared elements between the DBP-mediated enhancement
459
of transformation and previous studies on stimulation of conjugation by disinfectants and
460
other environmental pollutants 57, 59. These elements include i) the generation of ROS and ii)
461
the subsequent activation of stress-response. While previous studies showed a correlation
462
between the intracellular ROS production and increased conjugation rates, here, we
463
established a direct link between ROS-mediated DNA damage and the enhancement of
464
transformation. Although A. baylyi does not possess a prototypical S.O.S response60, in
465
agreement with previous research57, 59, we were able to detect downstream activation of
466
stress-response pathways, as evidenced by the increased transcription of ROS-detoxification
467
and DNA repair genes (i.e. recA and uvrB).
468 469
While conjugation might be the predominant factor influencing the environmental
470
spread of ARGs, we speculate that the volumetric magnitude of wastewater discharges, along
471
with the DBPs, could significantly contribute to the persistence of ARGs that exist as naked
472
extracellular DNA. In our opinion, the results from this work help us to elaborate a more
473
comprehensive view of ARG dissemination in the environmental metagenome, introducing
474
extra layers of information concerning i) the ultimate fate of naked extracellular DNA, as well
475
as ii) improved genetic exchange beyond the host range of mobile genetic elements and
476
bacteriophages27. Collectively, these findings raise concerns over our current reliance on
477
conventional chlorine-based wastewater disinfection, and suggest that disinfection
478
strategies should be evaluated not only based on their inactivation efficiencies but also, their
479
potential in stimulating horizontal gene transfer. 21 ACS Paragon Plus Environment
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480
Acknowledgements
481
This study is supported by KAUST baseline funding BAS/1/1033-01-01 awarded to P.-Y.
482
Hong.
483 484
Supplementary Information
485
Methods: Biomass collection for BAA toxico-genomics and RNA extraction. Tables: List of
486
primers for RT-qPCR, composition of the simplified disinfection byproduct (DBP) cocktail,
487
quality control of transcriptome samples, selected differentially expressed genes following a
488
dose-response behavior relative to BAA concentration. Figures: BAA exposure time selection
489
and BAA cytotoxicity for toxico-genomic experiment, toxico-genomic experimental design,
490
verification of transcriptomic data using housekeeping gene with known response to BAA.
491
The Supporting Information is available free of charge via the Internet at:
492
http://pubs.acs.org.
493 494 495 496
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497
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Figure legends
498 499
Figure 1. BAA enhances transformation rates in A. baylyi in a ROS-dependent manner. a)
500
Salmonella enterica serovar Typhimurium TA100 reversion assay showing the mutagenicity
501
of BAA. Data were from four independent replicates. b) Transformation frequencies of A.
502
baylyi ADP1 exposed to 0-600 μM BAA. Data is reported as fold change transformation
503
frequency vs. control. Green circles correspond to individual fold change values of
504
independent biological replicates at each BAA concentration. An ANOVA test statistic
505
demonstrated a significant F-test response (F = 74.09; P < 0.001). Responses in all treatment
506
concentrations were highly significant over the 0 μM BAA control (P < 0.001). Power of
507
performed test with α = 0.050: 1.000. NC and percentage above data points at each specified
508
BAA concentration depicts percent of A. baylyi ADP1 cell survival at that particular BAA
509
concentration; NC = non-cytotoxic. c) Mutagenic response in TA100 to BAA + glutathione
510
(GSH) treatments. GSH and BAA were added at equimolar concentrations (n = 4). d) Fold
511
change transformation frequencies of A. baylyi ADP1 exposed to BAA and BAA + GSH (n = 3).
512
e) Percent of A. baylyi ADP1 cell survival in BAA and BAA + GSH treated cell cultures (n = 3).
513
Plots indicate the mean response ± SE. Statistical comparisons in bar graphs were done using
514
ANOVA all pairwise comparisons. * denotes P < 0.001. Power of the performed test with α =
515
0.050: 1.000.
516 517
Figure 2. A. baylyi ADP1 transcriptional changes in response to BAA. Upper panel shows heat
518
maps representing percent changes in transcript per million (TPM) abundance at ti vs. t = 0
519
min for genes associated with DNA translocation machinery: a) pilX and b) comA, and genes 23 ACS Paragon Plus Environment
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Page 24 of 32
520
associated with DNA integration/repair c) recA and d) uvrB. Lower panels correspond to
521
samples at t = 60 min quantified by RT-qPCR to confirm transcriptional changes observed by
522
RNA-Seq. Linear models were fitted to fold changes in copy numbers to stablish
523
concentration-response transcriptional changes. Slopes of linear models were statically
524
tested for deviation from zero using Graph Pad Prism 8 linear regression package with α =
525
0.050.
526 527
Figure 3. Environmental concentrations of DBPs stimulate transformation. a) Growth
528
dependent donor DNA transformation frequencies in A. baylyi. Peak competence and
529
transformation was detected at the very early stages of the growth curve, establishing the
530
experimental window used for the DBP cocktail transformation assays. b) Fold change
531
transformation frequencies of A. baylyi ADP1 exposed to a DBP cocktail simulating the
532
environmental concentrations in treated wastewater effluents of 5 DBP classes (Table S2) vs.
533
a mock-treated control (n = 3). * denotes P < 0.001.
534 535 536 537 538 539 540
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References
542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584
1. Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K.; Wertheim, H. F.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens, H., Antibiotic resistance—the need for global solutions. The Lancet infectious diseases 2013, 13, (12), 1057-1098. 2. Forsberg, K. J.; Reyes, A.; Wang, B.; Selleck, E. M.; Sommer, M. O.; Dantas, G., The shared antibiotic resistome of soil bacteria and human pathogens. science 2012, 337, (6098), 11071111. 3. Czekalski, N.; Gascon Diez, E.; Burgmann, H., Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake. ISME J 2014, 8, (7), 138190. 4. Ahammad, Z. S.; Sreekrishnan, T. R.; Hands, C. L.; Knapp, C. W.; Graham, D. W., Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the upper ganges river. Environ Sci Technol 2014, 48, (5), 3014-20. 5. Luo, Y.; Yang, F.; Mathieu, J.; Mao, D.; Wang, Q.; Alvarez, P. J. J., Proliferation of Multidrug-Resistant New Delhi Metallo-β-lactamase Genes in Municipal Wastewater Treatment Plants in Northern China. Environmental Science & Technology Letters 2014, 1, (1), 26-30. 6. Mantilla-Calderon, D.; Jumat, M. R.; Wang, T.; Ganesan, P.; Al-Jassim, N.; Hong, P.-Y., Isolation and characterization of NDM-positive Escherichia coli from municipal wastewater in Jeddah, Saudi Arabia. Antimicrobial Agents and Chemotherapy 2016, 60, (9), 5223-5231. 7. Wellington, E. M.; Boxall, A. B.; Cross, P.; Feil, E. J.; Gaze, W. H.; Hawkey, P. M.; JohnsonRollings, A. S.; Jones, D. L.; Lee, N. M.; Otten, W., The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. The Lancet infectious diseases 2013, 13, (2), 155-165. 8. Mao, D.; Yu, S.; Rysz, M.; Luo, Y.; Yang, F.; Li, F.; Hou, J.; Mu, Q.; Alvarez, P. J., Prevalence and proliferation of antibiotic resistance genes in two municipal wastewater treatment plants. Water Res 2015, 85, 458-66. 9. Al-Jassim, N.; Ansari, M. I.; Harb, M.; Hong, P. Y., Removal of bacterial contaminants and antibiotic resistance genes by conventional wastewater treatment processes in Saudi Arabia: Is the treated wastewater safe to reuse for agricultural irrigation? Water Res 2015, 73, 277-90. 10. Masalha, N.; Dong, S.; Plewa, M. J.; Borisover, M.; Nguyen, T. H., Spectroscopic indicators for cytotoxicity of chlorinated and ozonated effluents from wastewater stabilization ponds and activated sludge. Environ. Sci. Technol. 2018, 52, 3167−3174. 11. Dong, S.; Masalha, N.; Plewa, M. J.; Nguyen, T. H., The impact of disinfection Ct values on cytotoxicity of agricultural wastewaters: ozonation vs. chlorination. Water Res. 2018, 144, 482-490. 12. Dong, S.; Masalha, N.; Plewa, M. J.; Nguyen, T. H., Toxicity of wastewater with elevated bromide and iodide after chlorination, chloramination, or ozonation disinfection. Environ. Sci. Technol. 2017, 51, 9297−9304. 13. Dong, S.; Nguyen, T. H.; Plewa, M. J., Comparative mammalian cell cytotoxicity of wastewater with elevated bromide and iodide after chlorination, chloramination, or ozonation. J. Environ. Sci. 2017, 58, 296-301. 25 ACS Paragon Plus Environment
Environmental Science & Technology
585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629
Page 26 of 32
14. Dong, S.; Lu, J.; Plewa, M. J.; Nguyen, T. H., Comparative mammalian cell cytotoxicity of wastewaters for agricultural reuse after ozonation or chlorination. Environ. Sci. Technol. 2016, 50, 11752-11759. 15. Guardabassi, L.; Petersen, A.; Olsen, J. E.; Dalsgaard, A., Antibiotic resistance in Acinetobacterspp. isolated from sewers receiving waste effluent from a hospital and a pharmaceutical plant. Applied and Environmental Microbiology 1998, 64, (9), 3499-3502. 16. Zhang, Y.; Marrs, C. F.; Simon, C.; Xi, C., Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Science of the Total Environment 2009, 407, (12), 3702-3706. 17. Goñi-Urriza, M.; Capdepuy, M.; Arpin, C.; Raymond, N.; Caumette, P.; Quentin, C., Impact of an Urban Effluent on Antibiotic Resistance of Riverine Enterobacteriaceae andAeromonas spp. Applied and environmental Microbiology 2000, 66, (1), 125-132. 18. Rizzo, L..; Manaia, C.; Merlin, C.; Schwartz, T.; Dagot, C.; Ploy, M.C.; Michael, I.; FattaKassinos, D., Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review. Science of the Total Environment 2013, 447, 345-360 19. Li, A.-D.; Li, L.-G.; Zhang, T., Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants. Frontiers in microbiology 2015, 6: 1025. 20. Guo, M.-T.; Yuan, Q.-B.; Yang, J., Distinguishing effects of ultraviolet exposure and chlorination on the horizontal transfer of antibiotic resistance genes in municipal wastewater. Environmental science & technology 2015, 49, (9), 5771-5778. 21. von Wintersdorff, C. J.; Penders, J.; van Niekerk, J. M.; Mills, N. D.; Majumder, S.; van Alphen, L. B.; Savelkoul, P. H.; Wolffs, P. F., Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Frontiers in microbiology 2016, 7: 173. 22. Richardson, S. D.; Plewa, M. J.; Wagner, E. D.; Schoeny, R.; Demarini, D. M., Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research. Mutat Res 2007, 636, (1-3), 178-242. 23. Wagner, E. D.; Plewa, M. J., CHO cell cytotoxicity and genotoxicity analyses of disinfection by-products: an updated review. J. Environ. Sci. 2017, 58, 64-76. 24. Min, J.; Kim, E. J.; LaRossa, R. A.; Gu, M. B., Distinct responses of a recA∷ luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 1999, 442, (2), 61-68. 25. Rauch, P. J.; Palmen, R.; Burds, A. A.; Gregg-Jolly, L. A.; van der Zee, J. R.; Hellingwerf, K. J., The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation. Microbiology 1996, 142, (4), 1025-1032. 26. Hiom, K., DNA repair: common approaches to fixing double-strand breaks. Current Biology 2009, 19, (13), R523-R525. 27. Thomas, C. M.; Nielsen, K. M., Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nature reviews microbiology 2005, 3, (9), 711. 28. Krasner, S. W.; Weinberg, H. S.; Richardson, S. D.; Pastor, S. J.; Chinn, R.; Sclimenti, M. J.; Onstad, G. D.; Thruston, A. D., Occurrence of a new generation of disinfection byproducts. Environmental science & technology 2006, 40, (23), 7175-7185. 26 ACS Paragon Plus Environment
Page 27 of 32
630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673
Environmental Science & Technology
29. Zeng, T.; Plewa, M. J.; Mitch, W. A., Chemical and toxicological assessment of Nnitrosamines and halogenated disinfection byproducts in potable reuse treatment trains. Water Res. 2016, 101, 176-186. 30. Kargalioglu, Y.; McMillan, B. J.; Minear, R. A.; Plewa, M. J., Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium. Teratogen. Carcinogen. Mutagen. 2002, 22, (2), 113-128. 31. Plewa, M. J.; Simmons, J. E.; Richardson, S. D.; Wagner, E. D., Mammalian cell cytotoxicity and genotoxicity of the haloacetic acids, a major class of drinking water disinfection by-products. Environ. Mol. Mutagen. 2010, 51, 871-878. 32. Rizzi, A.; Pontiroli, A.; Brusetti, L.; Borin, S.; Sorlini, C.; Abruzzese, A.; Sacchi, G. A.; Vogel, T. M.; Simonet, P.; Bazzicalupo, M.; Nielsen, K. M.; Monier, J. M.; Daffonchio, D., Strategy for in situ detection of natural transformation-based horizontal gene transfer events. Appl Environ Microbiol 2008, 74, (4), 1250-4. 33. Maron, D. M.; Ames, B. N., Revised methods for the Salmonella mutagenicity test. Mutation Research/Environmental Mutagenesis and Related Subjects 1983, 113, (3-4), 173215. 34. Hengstler, J. G.; Oesch, F., In Encyclopedia of Gentics Academic Press: 2001; pp 51-54. 35. Andrews, S., FastQC: a quality control tool for high throughput sequence data. 2014, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. 36. Bolger, A. M.; Lohse, M.; Usadel, B., Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, (15), 2114-2120. 37. Bray, N. L.; Pimentel, H.; Melsted, P.; Pachter, L., Near-optimal probabilistic RNA-seq quantification. Nature biotechnology 2016, 34, (5), 525-527. 38. Pimentel, H.; Bray, N. L.; Puente, S.; Melsted, P.; Pachter, L., Differential analysis of RNA-seq incorporating quantification uncertainty. Nature methods 2017, 14, (7), 687-690. 39. Al-Jassim, N.; Mantilla-Calderon, D.; Wang, T.; Hong, P., Inactivation of a virulent wastewater Escherichia coli and non-virulent commensal Escherichia coli DSM1103 strains and their gene expression upon solar irradiation. Environmental Science & Technology 2017, 51, (7), 3649-3659. 40. Maron, D. M.; Ames, B. N., Revised methods for the Salmonella mutagenicity test. Mutat.Res. 1983, 113, (3-4), 173-215. 41. Pals, J. A.; Wagner, E. D.; Plewa, M. J., Energy of the lowest unoccupied molecular orbital, thiol reactivity, and toxicity of three monobrominated water disinfection byproducts. Environ. Sci. Technol. 2016, 50, (6), 3215-3221. 42. Cemeli, E.; Wagner, E. D.; Anderson, D.; Richardson, S. D.; Plewa, M. J., Modulation of the cytotoxicity and genotoxicity of the drinking water disinfection byproduct iodoacetic acid by suppressors of oxidative stress. Environmental science & technology 2006, 40, (6), 18781883. 43. Pals, J.; Attene-Ramos, M. S.; Xia, M.; Wagner, E. D.; Plewa, M. J., Human cell toxicogenomic analysis linking reactive oxygen species to the toxicity of monohaloacetic acid drinking water disinfection byproducts. Environ. Sci. Technol. 2013, 47, (21), 12514-12523. 44. Pals, J. A.; Ang, J. K.; Wagner, E. D.; Plewa, M. J., Biological mechanism for the toxicity of haloacetic acid drinking water disinfection byproducts. Environ Sci Technol 2011, 45, (13), 5791-5797.
27 ACS Paragon Plus Environment
Environmental Science & Technology
674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719
Page 28 of 32
45. Hülter, N.; Sørum, V.; Borch-Pedersen, K.; Liljegren, M. M.; Utnes, A. L.; Primicerio, R.; Harms, K.; Johnsen, P. J., Costs and benefits of natural transformation in Acinetobacter baylyi. BMC microbiology 2017, 17, (1), 34. 46. Dad, A.; Jeong, C. H.; Pals, J.; Wagner, E. D.; Plewa, M. J., Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection byproducts. Environ. Mol. Mutagen. 2013, 54, 629-637. 47. Muellner, M. G.; Attene-Ramos, M. S.; Hudson, M. E.; Wagner, E. D.; Plewa, M. J., Human cell toxicogenomic analysis of bromoacetic acid: a regulated drinking water disinfection byproduct. Environ. Mol. Mutagen. 2010, 51, 205-214. 48. Pals, J. A.; Wagner, E. D.; Plewa, M. J.; Xia, M.; Attene-Ramos, M. S., Monohalogenated acetamide-induced cellular stress and genotoxicity are related to electrophilic softness and thiol/thiolate reactivity. J. Environ. Sci. 2017, 58, 224-230. 49. Attene-Ramos, M. S.; Wagner, E. D.; Plewa, M. J., Comparative human cell toxicogenomic analysis of monohaloacetic acid drinking water disinfection byproducts. Environ. Sci. Technol. 2010, 44, (19), 7206-7212. 50. Leong, C. G.; Bloomfield, R. A.; Boyd, C. A.; Dornbusch, A. J.; Lieber, L.; Liu, F.; Owen, A.; Slay, E.; Lang, K. M.; Lostroh, C. P., The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi. PloS one 2017, 12, (8), e0182139. 51. Mishra, S.; Imlay, J., Why do bacteria use so many enzymes to scavenge hydrogen peroxide? Archives of Biochemistry and Biophysics 2012, 525, (2), 145-160. 52. Itri, R.; Junqueira, H. C.; Mertins, O.; Baptista, M. S., Membrane changes under oxidative stress: the impact of oxidized lipids. Biophysical Reviews 2014, 6, (1), 47-61. 53. Lindahl, T., Instability and decay of the primary structure of DNA. nature 1993, 362, (6422), 709-715. 54. Miller, H. I.; Kirk, M.; Echols, H., SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli. Proceedings of the National Academy of Sciences 1981, 78, (11), 6754-6758. 55. U. S. Environmental Protection Agency, National primary drinking water regulations: Stage 2 disinfectants and disinfection byproducts rule. Federal Register 2006, 71, (2), 387493. 56. Li, D.; Zeng, S.; He, M.; Gu, A. Z., Water disinfection byproducts induce antibiotic resistance-role of environmental pollutants in resistance phenomena. Environmental science & technology 2016, 50, (6), 3193-3201. 57. Zhang, Y.; Gu, A. Z.; He, M.; Li, D.; Chen, J., Sub-inhibitory Concentrations of Disinfectants Promote the Horizontal Transfer of Multidrug Resistance Genes within and across Genera. Environmental Science & Technology 2016, 51, (1), 570-580. 58. Lu, J.; Jin, M.; Nguyen, S. H.; Mao, L.; Li, J.; Coin, L. J.; Yuan, Z.; Guo, J., Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation. Environment international 2018, 118, 257-265. 59. Wang, Y.; Lu, J.; Mao, L.; Li, J.; Yuan, Z.; Bond, P. L.; Guo, J., Antiepileptic drug carbamazepine promotes horizontal transfer of plasmid-borne multi-antibiotic resistance genes within and across bacterial genera. The ISME journal 2019, 13, (2), 509-522. 60. Hare, J. M.; Ferrell, J. C.; Witkowski, T. A.; Grice, A. N., Prophage induction and differential RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacter baumannii and Acinetobacter baylyi. PloS one 2014, 9, (4), e93861. 28 ACS Paragon Plus Environment
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Competent bacteria
Baseline transformation
eDNA +
=
Vs. +
Increased transformation
+ MUTAGEN =
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Assays with Acinetobacter baylyi ADP1 reporter strain b
1200
2.00
Transformation frequency fold change vs. control
900 600 300 0 0
200
400
50%
1.50
NC
1.25 1.00
600
0
200
d
1200
Transformation frequency fold change vs. control
900 600 300
*
1.75 1.50 1.25
600
*
e 100
50
1.00
400
600
Figure 1
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Co SH ntro l 4 BA 00 µ A M 4 BA 00 µ M A + G SH
200 BAA (µM) BAA BAA + GSH
G
0
Co SH ntro l 4 BA 00 A µM 4 BA 00 µ M A + G SH
0
G
Revertants per 5 x 10^8 cells plated
400 BAA (µM)
BAA (µM) c
90%
NC
1.75
% survival vs control
Revertants per 5 x 10^8 cells plated
Assays with Salmonella TA 100 a
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DNA uptake machinery
a
RNA-Seq data
+40%
b
pilX
BAA Time (min) (µM) 0 10
30
DNA damage and repair
c
comA
0
60
10
30
d
recA *
0
60
10
30
uvrB*
60
0
0
0
0
100
100
100
100
150
150
150
150
200
200
200
200
1.2
1.10
1.3
1.05
1.2
1.00
1.1
1.2
0.95
1.0
1.0
10
30
60
0%
fold change copy numbers vs. control
-40%
RT-qPCR data (Time: 60 min)
0
1.1 1.0
*
0.9 0.8 0.7
Slope = 0 ?
0.90 0
100
150
200
0
100
150
200
0.9
*
*
0
100
150
*
200
1.6
*
1.4
0.8
0
100
*
150
BAA (µM)
BAA (µM)
BAA (µM)
BAA (µM)
Not significant p-val = 0.107
Not significant p-val = 0.690
Significant p-val = 0.001
Significant p-val = 0.017
Figure 2
ACS Paragon Plus Environment
*
200
Environmental Science & Technology
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Assays with Acinetobacter baylyi Experimental window
1
2
3 Time (h)
Log trans. freq.
4
5
CFU / mL
Figure 3
ACS Paragon Plus Environment
1.50 1.25 1.00 Co nt ro l co ck ta il
1x10 8
-8
1.75
DB P
2x10 8
-7
Transformation frequency fold change vs. control
3x10 8
-6
-9
*
b
-5
CFU / mL
Log transformation fequency
a