Copper Oxide Nanoparticles Induce Lysogenic Bacteriophage and

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Copper Oxide Nanoparticles Induce Lysogenic Bacteriophage and Metal Resistance Genes in Pseudomonas aeruginosa PAO1 Jianhua Guo, Shu-Hong Gao, Ji Lu, Philip L. Bond, Willy Verstraete, and Zhiguo Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06433 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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ACS Applied Materials & Interfaces

Copper Oxide Nanoparticles Induce Lysogenic Bacteriophage and Metal Resistance Genes in Pseudomonas aeruginosa PAO1 Jianhua Guo1, *, Shu-Hong Gao1, Ji Lu1, Philip L Bond1, Willy Verstraete2, Zhiguo Yuan1

1

Advanced Water Management Centre, The University of Queensland, St. Lucia, Brisbane, QLD

4072, Australia 2

Center for Microbial Ecology and Technology, Ghent University, Coupure Links 653, B-

9000 Ghent, Belgium

*Corresponding author Jianhua Guo, Advanced Water Management Centre, The University of Queensland, Brisbane, Queensland 4072, Australia Phone: +61 7 3346 3215 Fax: +61 7 3365 4726 E-mail: [email protected]

Running title: CuO nanoparticles induce bacteriophage Keywords: nanoparticles; bacteriophage; genome-wide RNA sequencing; emerging contaminations; denitrification

Abstract: The intensive use of metal-based nanoparticles results in their continuous release into the environment, leading to potential risks for human health and microbial ecosystems. Although previous studies have indicated that nanoparticles may be toxic to microorganisms, there is a scarcity of data available to assess the underlying molecular mechanisms of inhibitory and biocidal effects of nanoparticles on microorganisms. This study used physiological experiments, microscopy, Live/Dead staining and the genome-wide RNA sequencing to investigate the multiple responses of ACS Paragon Plus Environment


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Pseudomonas aeruginosa to the exposure of copper oxide nanoparticles (CuO NPs). The results for the first time show that CuO NPs induce lysogenic bacteriophage, which might render defective within a bacterial host. The presence of CuO NPs causes nitrite accumulation and great increases in N2O emissions. Respiration is likely inhibited as denitrification activity is depleted in terms of decreased transcript levels of most denitrification genes. Meanwhile, CuO NPs exposure significantly up-regulated gene expression for those coding for copper resistance, resistancenodulation-division, P-type ATPase efflux, and cation diffusion facilitator transporters. Our findings offer insights into the interaction between environmental bacteria and CuO NPs at the transcriptional level, and thus improve our understanding of potential risks of nanoparticles on microbial ecosystems and public health.

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1. INTRODUCTION Engineered nanomaterials now make up a significant fraction of material flow in the global economy due to their unique characteristics, e.g. high surface-to-volume ratio, enhanced mechanical properties and high electrical conductivity. They are used in the production of various consumer products, such as electronic components, cigarette filters, cosmetics and cleaning products.1 For example, copper oxide nanoparticles (CuO NPs) are widely used in electronics and technology, including semiconductors, electronic chips, heat transfer nanofluids, and catalytic processes. In addition, CuO NPs are antimicrobial agents commonly used in face masks, stain-resistant fabrics and other personal care products to prevent those from bacterial contamination and spoilage.2 The intensive use of nanoparticles has resulted in their continuous release into the environment. It is documented that the production of CuO NPs was 570 tons in 2014 globally, and the predicated production will be up to 1600 tons by 2025.3 As a consequence, the increasing production and consumption of CuO will result in greater environmental release of CuO NPs.4 As CuO NPs are intentionally applied to fight the undesirable growth of microorganisms,2 the pervasiveness of CuO NPs raises serious concerns about their impacts on the environment and public health.5,

6

For

instance, the exposure of CuO NPs could incur exacerbations of pre-existing respiratory diseases or pulmonary diseases.7 Hence, it is imperative to assess the environmental and public health risks associated with these releases.8-11 Previous studies of toxicity of CuO NPs have focused on human health and on the biocidal effects on target microorganisms.2, 12 There are many questions regarding the broader impact of NPs, their longevity and availability, and there are only limited studies investigating the environmental impacts of these nanoparticles.13 CuO NPs in the environment will pose toxicity to diverse organisms, such as on fish, algae, bacteria, fungi and protozoa. The effective concentration (EC50) or minimal inhibitory concentrations (MIC) for these organisms has been determined.2 In addition, the negative impacts of CuO NPs on the operational performance of wastewater treatment systems has been investigated.14, 15 In those systems was seen that CuO NPs caused deterioration of nitrogen



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and phosphorus removal and inhibited sludge treatment. These inhibitory effects were seen to be concentration-dependent and condition-specific. It is suspected that the inhibitory and biocidal effects of various nanoparticles are through the generation of reactive oxygen species (ROS), release of toxic metals or by direct disruption of biological membranes.6, 16-18 Moreover, previous studies in this field mostly measure macro-parameters (e.g. oxygen uptake rate and substrate utilization rate) to assess the toxicity of nanoparticles.2 Polymerase chain reaction (PCR) has been used to examine the expression of certain genes in response to NP exposure.19 However, little has been done to improve understanding of the broader impacts of CuO NPs on bacterial gene expression across the genome, and their affects on general metabolic functions and cell physiology. Pseudomonas aeruginosa is common in a variety of environmental ecosystems, including water, soil and human hosts. It is an opportunistic pathogen and infections can cause acute or chronic morbidity and mortality in cystic fibrosis and immunocompromised patients.20 As a facultative anaerobe, P. aeruginosa is well adapted to proliferate under either aerobic or anaerobic conditions and exhibits a number of virulence factors that contribute to its pathogenicity.21 It is also a ubiquitous denitrifier in wastewater-treatment systems and soil ecosystems, where its activities play a role for nitrogen turnover. Therefore, for revealing the interactions between nanoparticles and bacteria, there is great interest to determine the responses of environmentally and medically relevant organisms to the exposure of nanoparticles, such as that by P. aeruginosa. Recently, the toxicity of nanoparticles on the growth and activities of P. aeruginosa were evaluated.22-24 However, the mechanisms of the inhibitory and biocidal effects of CuO NP were not examined at the genome or gene expression levels. Here we combined physiological experiments, microscopy, Live/Dead staining and genomewide RNA sequencing to examine the microbial response to CuO NPs exposure and reveal the inhibitory and biocidal mechanisms on this prevalent opportunistic pathogen and environmentally relevant denitrifier. Our findings provide insight of the broader picture regarding the mechanisms of microbial susceptibility, tolerance and resistance to NPs exposure, as well, the study highlights the ACS Paragon Plus Environment

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potential risks of CuO NPs to the environment.

2. EXPERIMENT SECTION 2.1 Nanoparticle exposure experiments P. aeruginosa PAO1 strain (DSM No: 22644) was cultured in glycerol modified M9 (GLYM9) medium (details can be found in Text 1 of Supporting Information (SI)).25 To examine the potential impacts of CuO NPs, PAO1 was exposed to 0, 1, 10 and 50 mg/L CuO NPs. In this study, CuO NPs were purchased from Sigma-Aldrich (St. Louis, USA), with a particle size of < 50 nm and surface area of 29 m2/g. Before the dosage, the CuO nanoparticles were sonicated to ensure dispersion of those. After 11h inoculation in the GLYM9 medium to reach early exponential growth phase (OD of 0.2~0.3), a volume of a stock CuO nanoparticles solution was added to reach the initial nanoparticle concentration of 0.1, 1, 10 and 50 mg/L. Control cultures, with no nanoparticle addition, had the same volume of GLYM9 medium added. The concentrations of NO3-, NO2- and glycerol were measured at intervals of 2 or 4 h for a total exposure time of 24 h. The concentrations of NO2-, NO3- and glycerol were determined as described previously26 and details can be found in Text 2 (SI). Experiments of the control cultures and nanoparticle treated samples were conducted in triplicates. 2.2 RNA extraction and sequencing The total RNA extraction was conducted after 2 hours nanoparticle exposure. The bacterial suspension of 6 mL from each serum bottle was harvested and centrifuged at 14,000 rpm for 5 minutes at 4 oC. After discarding supernatants, the pellets were used to extract RNA from each sample. Total RNA extraction was conducted using the QIAGEN miRNeasy Mini Kit (Catalog No: 217004) based on the manufacturer’s protocol. The extracted total RNA was delivered to BGI (Shenzhen, China) for purification, strand-specific cDNA library construction and Illumina pairedend sequencing (HiSeq 2000, Illumina Inc., San Diego, CA, USA, as shown in Figure S2). More than 1 Gb of high quality sequence data were obtained for each sample.



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2.3 RNA sequencing data processing and bioinformatics The raw sequencing reads were progressively trimmed at the 3’-ends until a quality value ≥20 was reached. Then, ambiguous characters (Ns/Xs) and poly-As/Ts longer than six based at either end of reads were removed. The 3’end residual adapter and primer sequences were removed and then one base from the 5’-end and three the 3’-end were trimmed. The NGS QC Toolkit (v2.3.3)27 was used for all these sequence-modifying processes. The clean reads with longer than 75 bp were utilized for downstream data analyses. The pipeline of bioinformatics is shown in Figure S3. The sequencing quality control results are shown in Figures S4 and S5, and gene coverage statistics of all samples is shown in Figure S6. Differential expression analysis was performed using the cuffdiff command in Cufflinks (version 2.2.1),28 following the procedures as described previously.26, 29 Briefly, the cleaned sequence reads for each sample were aligned to the PAO1 reference genome (NC_002516) using SeqAlto (version 0.5).30 Statistic analyses and visualization were carried out using the cummeRbund package in R. Gene expression was calculated as reads per kilobase of a gene per million mapped reads (RPKM).28 A cut-off of log2 fold-change (LFC) ≥ 2.0 or ≤ -2.0 with false discovery rate (FDR) < 0.001 was adopted to distinguish the differentially expressed genes. Annotation of the differentially expressed genes was based on the online-curated pathway tools genome database PseudoCyc (http://www.pseudomonas.com). The program Circos was used to visualize expression and determine the RPKM values for each gene.31 2.4 Transmission electron microscopy Cells of P. aeruginosa before and after treatment with CuO NPs were observed under TEM to elucidate the direct influence of CuO NPs on the cell membrane. TEM imaging was performed by standard procedures, as briefly described in Text 3 (SI). 2.5 Chemical and physiological assays Samples were collected and filtered (pore size 0.22 µm, Merck Millipore, USA) for chemical and physiological analyses from the replicate cultures during the different levels of CuO NP exposure. ACS Paragon Plus Environment

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To determine the inhibitory and biocidal effects before and after nanoparticles treatment, LIVE/DEAD Staining was conducted using the flow cytometer (FACSAria™ II, BD Biosciences, San Jose, USA) to study effects of CuO NPs on cell viability. 2.6 Statistical analysis and data availability Significant differences were statistically assessed through Analysis of variance (ANOVA) and Independent-sample t test. Data were shown in mean ± standard deviation. All sequencing data have been deposited to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus through the GEO series (accession no. GSE99981).

3. RESULTS AND DISCUSSION 3.1 Activity of P. aeruginosa during exposure to CuO nanoparticles During the exposure experiments P. aeruginosa PAO1 was grown in denitrifying conditions with glycerol and nitrate as the electron donor and acceptor respectively. When the PAO1 cultures were in early exponential phase of growth, eleven hours after inoculation, nanoparticles were added at various concentrations (0.1, 1, 10, and 50 mg/L) for a 24-hour exposure experiment. Hereby, both environmentally relevant concentrations of the CuO NPs (0.1 and 1 mg/L) and those relevant to extreme industrial wastewater discharges (10 and 50 mg/L) were used. The impacts of CuO NPs on culture proliferation, metabolic activity and cell lysis of P. aeruginosa were assessed by monitoring the optical density at 600 nm (OD600), denitrification rate and cell viability (Figure 1).

The concentration-related inhibitory and biocidal effects of nanoparticles were detected. At low NP concentrations (0.1 and 1 CuO NP mg/L) the PAO1 growth rate was similar to the control cells (no added NPs). Conversely, the cell growth was distinctly inhibited under exposure to the higher CuO NPs concentrations of 10 and 50 mg/L (Figure 1A). The denitrification activity by PAO1 was detected through the utilization of glycerol (data not shown) and nitrate (Figure 1B). At low concentrations of added NPs (0.1 and 1 mg/L) the nitrate reduction rates were very similar to that of ACS Paragon Plus Environment


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the control culture with no added NPs. At the NPs exposure of 10 mg/L some nitrate reduction was detected, but nitrite accumulation occurred (Figure 1C). In contrast, denitrification activities were severely lowered at the highest NPs exposure of 50 mg/L. Only 3 mg N/L of nitrate was converted into nitrite at the initial 2 h (Figure 1B) and following that neither glycerol utilization (data not shown) nor nitrate reduction was detected in the presence of 50 mg/L CuO NPs. Meanwhile, different N2O productions were observed during exposure to different CuO NPs concentrations. The average N2O concentrations were very low (~10.5 mg N/L) in the control and low NP exposure conditions (0.1 and 1 mg/L). In contrast, those concentrations were 13.5 and 22.5 mg N/L in the presence of 10 and 50 mg /L CuO NPs after 2 h exposure. The average N2O concentrations after 24h exposure were decreased, possibly because a part of N2O would be further utilized by the PAO1 culture (Figure 1D). Our results are similar with a previous study,32 which reported extracellular copper concentration caused elevated levels of N2O release of P. denitrificans. The PAO1 cell viability was detected at the different CuO NPs exposure levels (Figure 1E). For both the control and the CuO NPs concentration of 0.1 mg /L, the live cell fraction was consistently around 80 % viable cells after 24 h exposure. The live cell percentages in the presence of 1 mg/L CuO NPs were slightly lower than that of the control, but were not significantly different (p > 0.05). There were substantial decreases in the proportions of live cells after 2 h CuO NPs exposure at 10 and 50 mg/L (p < 0.05) and at 50 mg/L exposure for 24 h the P. aeruginosa cells were almost completely killed (Figure 1E and F). This is consistent with the strong biocidal impact of CuO NPs at the same dosage level as indicated from the proliferation profile (Figure 1A). TEM images indicate that the CuO NPs damaged the cell membrane structure. For the control culture, the bacterial membranes were distinct and the cytoplasm was compact (Figure 1G). However, for the bacteria that were treated with added CuO NPs the cell membranes were dissolved or damaged (Figure 1H), and the damage was more extensive with increasing concentrations of CuO NPs (in particular for 50 mg/L). The above result that CuO NPs exhibited dose-dependent inhibitory and biocidal effects is consistent with previous studies12, 33. The antibacterial properties of NPs are mainly attributed to the


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production of ROS, which might result in DNA lesions, altered gene regulation and eventual disintegration of the cell membrane33. However, the bacterial-nanoparticle interactions at the DNA/RNA level remain unclear. 3.2 Global transcript profiles and differential gene expression As bacterial growth and metabolism is linked to changes in transcription, gene expression by whole genome RNA sequencing was conducted to examine the response of PAO1 to CuO NPs exposure. Gene expression was compared when the cells were exposed to 1, 10 and 50 mg/L of CuO NPs for 2 hours to when they were not exposed to added CuO NPs. Following the processing and filtering of the raw sequences more than 1.1 x 107 high-quality reads were obtained for all the CuO NP exposure levels and the control. After mapping these clean reads to the reference genome of P. aeruginosa, more than 96.1% were aligned for each sample to the PAO1 reference genome (Table S1, Supporting Information). A genome overview of the PAO1 gene expression differences between the control and the CuO NPs exposed cells is summarised (Figure 2). Genes showing large changes of expression in terms of log2 fold change (LFC) included the up-regulated PA0717PA0728 and pcoA/B. These genes encode for the bacteriophage Pf1 gene cluster and for the copper resistance protein A/B precursor, and these changes imply a stress response of PAO1 to the CuO NPs exposure. Moreover, the genes coding ATP synthase (atpABCDEFGH) were down regulated under exposure of CuO NPs. These changes in PAO1 in response to CuO NPs exposure are described and discussed in more detail in the sections below. Genes that were significantly differentially transcribed (with a |LFC|> 2.0 and FDR < 0.01) between the control and CuO NPs-exposed cultures were determined. We detected 286, 550 and 456 differentially transcribed genes in response to the exposures of 1, 10 and 50 mg/L CuO NPs respectively. Among them, 106, 191 and 158 genes showed increased transcripts, while 180, 359 and 298 were down regulated in response to those respective exposures (Figure 2B). When arranged into functional categories of orthologous groups (COG) it is seen there are large differences in the number of up or down regulated genes in the class of “metabolism” (Figure 2C). The subcategory



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“Amino acid transport and metabolism” contained the largest number of genes that were down regulated in PAO1, this was followed by the numbers of genes assigned to the categories “Energy production and conversion” and “Secondary metabolites biosynthesis, transport and catabolism”. In contrast, the subcategory “Inorganic ion transport and metabolism” contained the largest number of genes that were up regulated in PAO1, followed by “Cell wall/membrane/envelope biogenesis” (Figure S1). Hence, these results indicated that the exposure of CuO NPs is causing distinct effects on metabolism and cellular regulation of PAO1. Additionally, it is worth noting that a high percentage of the up-regulated genes or down-regulated genes after CuO NPs exposure were hypothetical proteins (Table S2). These genes are of interest since they are likely significant for bacterial survival during CuO NPs exposure, but no obvious functions are yet assigned to them. Consequently, this overview suggests that CuO NPs severely affected the protein synthesis process, energy production pathways as well as the iron transport process. More detailed findings of the effects of CuO NPs on particular cell functions are revealed in the following sections. 3.3 Expression genes coding for key metabolic activities It is evident that the presence of CuO NPs affects the metabolism of carbon and inhibits the gene expression coding for key denitrifying enzymes. In PAO1 the complete denitrification is mediated by four essential and dependent denitrifying enzymes, including nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (NOS). These are encoded by the gene clusters of narGHJI (encoding respiratory nitrate reductase), napADE (encoding a second nitrate reductase that is periplasmic), nirSECF (encoding nitrite reductase), norCBQDEF (encoding nitrous oxide reductase), and nosRZD (encoding nitric oxide reductase). A number of transcripts coding for the enzymes involved in denitrification were down regulated during exposure to CuO NPs, this was particularly for the higher concentrations of 10 and 50 mg/L (Figure 3). This included the genes narGHJI and napABD for nitrate reductase, nirS for nitrite reductase, norBC for nitric oxide reductase and nosZL for nitrous oxide reductase (Figure 3). During exposure of 10 mg/L NPs, the down-regulated fold changes for narG, napB, nirC, nirS, ACS Paragon Plus Environment

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norB, nosZ and nosF were 2.4, 3.0, 3.1, 2.1, 1.5, 2.6 and 3.1, respectively. The down-regulated fold change for the nir operon was relatively greater than that for the nor operon, which might explain the observed nitrite accumulation under the exposure of 10 mg/L CuO NPs. Under exposure of 50 mg/L CuO, the down-regulated fold changes for narG, napB, nirC, nirS, norB, nosZ and nosF were 7.8, 4.4, 4.1, 2.3, 2.2, 2.7 and 4.2, respectively. In addition to the denitrification genes mentioned above, genes encoding the nitrite extrusion protein (nirK1 and nirK2) were highly down regulated in the presence of CuO NPs at 10 mg/L (2.9 and 1.9 fold changes) and 50 mg/L (14.5 and 9.1 fold changes), respectively. These two transmembrane proteins (NarK1 and NarK2) play a significant physiological role in the nitrate/nitrite transport by P. aeruginosa.34 Taken together with distinct nitrite accumulation detected in the presence of 10 mg/L NP and substantial levels of nitrate remaining when exposed to 10 and 50 mg/L CuO NP (Figure 1B), it is clear that CuO NPs significantly inhibited the denitrification activity. Also, CuO NPs were documented to reduce the denitrification efficiency from 98.3% to 62.1% with the increase of CuO NPs from 0.05 to 0.25 mg/L,35 where the expressions and catalytic activities of nitrate reductase and nitrite reductase were significantly inhibited. In this study, the glycerol was the electron donor for the denitrification by PAO1. In this metabolism, glycerol would firstly be converted into glyceraldehyde-3-phosphate and then to pyruvate, via the Embden-Meyerhof-Parnas (EMP) pathway, before being utilised in the TCA cycle for energy generation. After the dosage of CuO NPs, most of the genes coding for enzymes of the EMP pathway had decreased transcript expression levels, this included the genes for phosphoglycerate mutase (pgm), phosphoglycerate kinase (pgk), enolase (eno), and pyruvate kinase (pykA). In addition, a number of genes involved in the Entner-Duoforoff (ED) pathway showed slightly decreased transcript levels during exposure to CuO NPs, e.g. PA3181, pgi (encoding glucose-6-phosphate isomerase), zwf (encoding glucose-6-phosphate 1-dehydrogenase), and edd (encoding phosphogluconate dehydratase). As well, the genes encoding enzymes in the TCA cycle (sdhABCD, sucABCD and glcDEF) and genes coding for the ATP synthase (atpABCDFGHI) were



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down-regulated in the presence the NPs. Typically the activity of respiratory denitrification would contribute to formation of the cellular proton motive force and facilitate ATP generation in P. aeruginosa through the membrane-bound ATPase. Collectively, these events of decreased utilization of nitrate and glycerol, decreased expression of denitrification, carbon utilization, and ATP synthase genes, all strongly support the notion that CuO NPs exhibited an impairing effect on the denitrification pathway and respiration of PAO1. 3.4 CuO NPs exposure results in the upregulation of copper resistance genes In P. aeruginosa it was seen that CuO NPs caused the upregulation of genes involved in copper resistance, this included the genes copR, czcR and irlR. During the exposure to NPs the genes encoding enzymes involved in the cooper resistance protein precursor (pcoAB) were highly up regulated, in the range of 130.0-634.3 fold changes. With the increase of CuO NP concentrations from 1 to 50 mg/L, the upregulation fold changes decreased (Table S3), suggesting exposure to lower CuO NPs concentrations would induce the stronger copper resistance ability. In addition, the important metal efflux transporters, PA1297, PA3920 and czcABC were distinctly up-regulated, and this increased with increasing CuO NPs concentrations from 1 mg/L to 10 mg/L. In contrast, P. aeruginosa significantly changed expression of genes involved in passive transport functions, and incurred down-regulation of several porins belonging to the Opr family, including OprDPBC. In addition, the two-component regulatory regulators copR (PA2809), PA2523 and PA2524 were all up-regulated under exposure of CuO NPs. Collectively, these results indicate that P. aeruginosa regulated their cooper resistance genes and enhanced its transport capacity in order to detoxify CuO NPs. Even under the low level concentrations of CuO NPs (1 mg/L), the upregulation of cooper resistance genes was observed. Previous studies mainly reported the long-term exposure of cooper ions could result in acquiring of Cu resistance genes,36, 37 while very limited studies documented short-term exposure induced copper resistance genes.38 A recent study documented sublethal exposure of quantum dots not only resulted in the up-regulation of metal efflux transporters, but also induced antibiotic resistance genes in P. aeruginosa culture.24 ACS Paragon Plus Environment

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3.5 Defence to CuO NPs involves the resistance-nodulation-division (RND) transporters RND family transporters are a category of bacterial efflux pumps, that form complexes spanning the inner and outer cell membranes, and function in the active transport of heavy metals to remove them from cytoplasm. It is reported that the chromosome of P. aeruginosa carries twelve loci harboring genes homologous to the RND family.39, 40 In this study, we found a number of potential RND genes were significantly up-regulated in response to the CuO NPs exposure (e.g. PA1435, PA1436, PA2520 and PA3522) (Table S4). At the low level of CuO NPs exposure (1 mg/L), the most significantly up-regulated locus was PA1435 and PA1436. Under medium level CuO NPs exposure (10 mg/L), the most distinctly up-regulated genes were PA2520~PA2524, which encode the czrCBARS transport system, and is proven to be linked to the tolerance of the metals Cd, Zn, and Co.41, 42 However, the most prominently up-regulated locus, PA3521 to PA3523, was detected at the highest CuO NPs exposure of 50 mg/L. Other results also indicated that the RND transporters are induced under exposure of increased concentrations of the cooper ion.38 For P. aeruginosa, the RND transporters, the P-type ATPase efflux complexes, and the cation diffusion facilitator (CDF) transporters can form a combined network for adjusting metal concentrations in the periplasm and cytoplasm.38 In this study, the gene PA3920 was up-regulated during the exposure and the fold changes increased with increasing CuO NPs concentrations (8.3, 114.4 and 320.7 fold changes under exposures of 1, 10 and 50 mg/L, respectively). Moreover, the P. aeruginosa chromosome has three genes belonging to the CDF family, including PA0397, PA3693 and PA1297. The gene PA1297 was up-regulated in P. aeruginosa treated with CuO NPs, although, the other two genes did not show any significant changed expression. Consequently, the changes in expression patterns implicate that PAO1 could be using this integrated network for extrusion of CuO NPs from the cell.

3.6 CuO NPs induce lysogenic bacteriophage production



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Interestingly in PAO1 the most up regulated genes in response to the CuO NPs exposure were those of the bacteriophage Pf1 gene cluster (PA0717-PA0728). These genes displayed extremely high expression upon exposure to any concentration of CuO NPs, with up-to a 2031 fold change for PA0720 in the presence of 50 mg/L (Figure 4). To our knowledge, this is the first report of CuO NPs promoting such increased expression of bacteriophage genes. During CuO NPs exposure, we tried to detect the release of bacteriophage through TEM and flow cytometry. However, we did not observe any increases of lytic bacteriophage. Also, cell lysis was not observed under low CuO NPs exposure conditions (0.1 and 1 mg/L) in terms of Live/Dead staining, implying the suppression of the lytic cycle by these NPs. It is likely that lysogenic phage, rather than lytic phage, were caused under the exposure of CuO NPs. Through integrating themselves into the host genome, lysogenic phage can still pose risks to the host, either via conferring a selective advantage on the host bacteria, or influencing accumulating mutations that render it defective.43 In previous studies, increased expression of bacteriophage genes are reported in P. aeruginosa biofilms upon exposure to nitric oxide,44, 45 and biofilm dispersal was caused by phage-induced cell lysis. In addition, it is reported that the bacteriophage Pf1 operon (PA0717) is highly expressed in P. aeruginosa biofilms in comparison to planktonic cells.46 Indeed, we observed that planktonic cells would cluster together to form biofilm when dosed with CuO NPs, but this phenomenon was not observed in the control cells (no added NPs) in this study. This might imply CuO NPs stimulated the biofilm development of P. aeruginosa in order to tolerate the toxicity posed by CuO NPs, which is consistent with a previous report about sublethal concentrations of Ag NPs stimulating biofilm development.47 The bacteriophage Pf1 operon is found to be involved in various cell traits that include virulence, biofilm structural integrity and phenotypic variant formation.43 These traits are potentially associated with the increased adaptability of the stressed bacterial population.48

3.7 The complex response of PAO1 to CuO NPs exposure


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In this study we have applied a genome wide RNA-Seq analysis to detect the impacts of CuO NPs on the global transcriptome of P. aeruginosa PAO1. As an opportunistic pathogen, PAO1 also possesses intrinsic resistance to a variety of antimicrobials due to the presence of multidrug efflux transporters, the bacterial outer-membrane barrier and endogenous antimicrobial inactivation.21 We identify critical determinants in the bacteriostatic impacts of CuO NPs on this bacterium and the protective response of PAO1 against CuO NPs stress. Here we propose a model to describe the effects of CuO NPs and the multiple responses of PAO1 against CuO NPs stress (Figure 5). During exposure to bacteriostatic levels of CuO NPs, respiration through denitrification activity was inhibited as decreased transcript levels of most denitrification genes were detected. Meanwhile, all levels of NPs exposure up-regulated genes encoding several active transport functions. In order to cope with CuO NPs stress, active efflux would transport metal cations or NPs out of the cytosol. Our results indicate that two RND efflux systems, a two-component regulator, and a P-type ATPase in P. aeruginosa might paly important roles in the tolerance to CuO NPs. ATP hydrolysis mediates the transportation of CuO NPs or Cu ions from the cytoplasm into the periplasm, then RND transporters, whose complexes span the inner and outer membranes, mediate proton-driven efflux of copper cations or NPs from the periplasm. In addition, bacteriophage genes were activated most highly, which suggested the increased presence of the prophage in the PAO1 population that could result in defective within the host.

3.8 Implications to environment and public health In this work, we for the first time found that genes coding for a Pf1-like filamentous bacteriophage, which exists as a prophage in the genome of PAO1, displayed up-to hundred- or thousand-fold increased gene expression. Even under the environmental relevant concentration (1 mg/L CuO NPs), the genes for bacteriophage were significantly upregulated. The occurrence of bacteriophage is one of the major causes of bacteria death in the environment.49 In addition, bacteriophage play an important role in horizontal gene transfer via the transduction pathway, in which donor DNA could ACS Paragon Plus Environment


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be packaged into the phage head and injected into other recipient bacteria.50 Potentially, through the increase of phage activity, microbial exposure to NPs could cause increased transduction of genes that are of concern to public health, such as in the dissemination of antibiotic resistance genes. We also revealed the potential risks of CuO NPs to nitrate removal during microbial denitrification. Denitrification is essential in the global nitrogen cycle for returning nitrogen to the atmosphere from terrestrial and aquatic environments. Increasing the exposure of CuO NPs to denitrifiers could cause decreased levels of environmental nitrogen cycling, which, for example could further worsen human-induced water eutrophication. In addition, the incomplete denitrification induced by CuO NPs could cause N2O accumulation, which is a potent greenhouse gas, since it has a 300-fold higher warming potential than CO2.51 Based on our results, it is evident that the responses of bacteria under the exposure of nanoparticles are very complex. A clear and comprehensive understanding of inhibition or toxicity of NPs under environmentally relevant conditions (e.g. at the community or biofilm level) is still lacking. In summary, our findings provide insights on the whole picture regarding the fundamental mechanisms of microbial susceptibility, tolerance and resistance to exposure of CuO NPs, additionally, they highlight some potential risks of CuO NP to the public health and the environment. 4. CONCLUSIONS In summary, using P. aeruginosa PAO1 as a model environmental bacterium, we examined the impacts of CuO NPs on the response of environmental bacteria at the cellular and gene expression levels, with application of a suite of advanced molecular and imaging methods, including flow cytometry, TEM and genome-wide RNA sequencing. Our results indicate the multiple responses of Pseudomonas aeruginosa to the exposure of copper oxide nanoparticles. Increasing release of CuO NPs into the environment would cause lower nitrate removal and higher N2O emission. In particular, the expressions of bacteriophage genes and copper resistance genes were activated during exposure


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to environmentally relevant CuO NPs concentrations, which would pose a secondary danger to the host bacteria. This study advances understanding of interactions between nanoparticles and environmental microorganisms, and will assist in assessing the potential risks of nanoparticles on microbial ecosystems and public health.

Acknowledgements We acknowledge the Australian Research Council for funding support through the project DP170104330. Jianhua Guo acknowledges the support from the Australian Research Council Future Fellowship (FT170100196). We thank Dr. Beatrice Keller, Ms. Jianguang Li and Mr. Nathan Clayton, at The University of Queensland for IC, FIA and HPLC analysis and Dr. Michael Nefedov,  The University of Queensland for assistance with the BD FACSAriaTM II flow cytometer and data analysis.   Conflict of interest The authors declare that they have no conflict of interest.

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Alignment statistics of samples. Table S2. Differentially expressed genes encoding hypothetical proteins by addition of CuO NPs Table S3. Differentially expressed genes encoding copper resistance by addition of CuO NPs Table S4. Differentially expressed genes encoding RND by addition of CuO NPs Figure S1. An overview of differentially expressed genes by PAO1 in response to CuO NPs exposure when assigned to different functional COG categories functional COG categories. Figure S2. Transcriptome resequencing experimental process Figure S3. The bioinformatic pipeline for RNA-sequencing data Figure S4. Base composition analysis of samples (A, control; B, CuO NPs of 1 mg/L; C, CuO NPs of 10 mg/L; D, CuO NPs of 50 mg/L.) ACS Paragon Plus Environment


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Figure S5. Base sequence quality of samples (A, control; B, CuO NPs of 1 mg/L; C, CuO NPs of 10 mg/L; D, CuO NPs of 50 mg/L.) Figure S6. Gene coverage statistics of samples (A, control; B, CuO NPs of 1 mg/L; C, CuO NPs of 10 mg/L; D, CuO NPs of 50 mg/L.)

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Figure Captions Figure 1. Physiological features of P. aeruginosa PAO1 exposed to different levels of CuO NPs. The optical density at 600nm (OD600) profile (A), nitrate levels (B), nitrite levels (C) and N2O accumulation (D), when CuO NPs were added at T=0 h. Cell viability of P. aeruginosa cultures were determined by the Live/Dead BacLight stain (E). Side scatter statistics analysis of cells exposed to CuO NPs after 2h (F). Images of the control cells (no added NPs) (G) and of cells in the presence of 10 mg/L CuO NPs (H) by transmission electron microscopy (TEM), bar = 200 nm. The data in (A-E) are shown as mean value ± standard deviation from the triplicated experiments. In (D and E), Student’s t-test was conducted, compared with the control, *p < 0.05. Figure 2. Global transcript profiles of PAO1 samples under exposure of CuO NPs. (A) A Circos image of global transcript changes in the presence of different levels of CuO NPs. The outmost circle (defined as the 1st circle) represents the full genome of strain PAO1. Moving inwards, the second, third, and fourth circles represent the differentially expressed genes in samples treated with 1, 10, and 50 mg/L CuO NPs, respectively when compared to the no-exposure control culture. Genes were considered differentially regulated if |log2| >2 and false discovery rate (FDR)2 and false discovery rate (FDR)

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