ARTICLE pubs.acs.org/est Cellular and Transcriptional Response of Pseudomonas stutzeri to Quantum Dots under Aerobic and Denitrifying Conditions † ‡ ‡ † Yu Yang, Huiguang Zhu, Vicki L. Colvin, and Pedro J. Alvarez*, †Department of Civil and Environmental Engineering, Rice University, Houston, Texas 77005, United States ‡Department of Chemistry, Rice University, Houston, Texas 77005, United States bS Supporting Information ABSTRACT: Pseudomonas stutzeri was exposed to quantum dots (QDs) with three different surface coatings (anionic polymaleic anhydride-alt-1-octadecene (PMAO), cationic polyethylenimine (PEI), and carboxyl QDs) under both aerobic and anaerobic (denitrifying) conditions. Under aerobic conditions, toxicity (assessed per growth inhibition) increased from PMAO to carboxyl to PEI QDs. The positive charge of PEI facilitated direct contact with negatively charged bacteria, which was verified by TEM analysis. Both PMAO and PEI QDs hindered energy transduction (indicated by a decrease in cell membrane potential), and this effect was most pronounced with PEI QDs under denitrifying conditions. Up- regulation of denitrification genes (i.e., nitrate reductase narG, periplasmic nitrate reductase napB, nitrite reductase nirH, and NO reductase norB) occurred upon exposure to subinhibitory PEI QD concentrations (1 nM). Accordingly, denitri- fication activity (assessed per respiratory nitrate consumption in the presence of ammonia) increased during sublethal PEI QD exposure. However, cell viability (including denitrification) was hindered at 10 nM or higher PEI QD concentra- tions. Efflux pump genes czcB and czcC were induced by PEI QDs under denitrifying conditions, even though Cd and Se dissolution from QDs did not reach toxic levels (exposure was at pH 7 to minimize hydrolysis of QD coatings and the associated release of metal constituents). Up- regulation of the superoxide dismutase (stress) gene sodB occurred only under aerobic conditions, likely due to intracellular production of reactive oxygen species (ROS). The absence of ROS under denitrifying conditions suggests that the antibacterial activity of QDs was not due to ROS production alone. Overall, this work forewarns about unintended potential impacts to denitrification as a result of disposal and incidental releases of QDs, especially those with positively charged coatings (e.g., PEI QDs). ’ INTRODUCTION water pollutants) coupled with the production of dinitrogen gas 12À14 More than one-half century has passed since Richard Feynman (N2) through intermediate nitrogen oxides (NO and N2O). presented the perspective of nanomaterial manipulation and Consequently, denitrification results in a loss of nitrogen 1 (a common limiting nutrient) from aquatic and soil systems with nanotechnology application. Nanotechnology is now leading a 15 concomitant production of a greenhouse gas (N2O). Pseudo- creative and epochal revolution covering various subjects, such as 16,17 pharmaceutics, electronics, physics, chemistry, and biology.2À4 monas stutzeri is one of the most studied denitrifying bacteria, and its denitrification genes have been discerned and sequenced With the increasing worldwide usage of manufactured nano- 17 materials (MNMs), their accidental or incidental releases to (Figure 1). Thus, P. stutzeri is a convenient model bacterium to the environmental seem inevitable. Accordingly, the potential investigate the potential impact of MNMs to denitrification adverse effect of MNMs to different types of biological molecules, processes and associated bacterial response, which has not yet cells, and organisms has received significant recent attention.5À11 been addressed in the literature. However, considerable uncertainty remains about the potential Quantum dots (QDs) offer valuable functionality for bioimag- impacts to microbial ecosystem services (e.g., nutrient cycling) ing, solar cells, electronics, and drug (or gene) delivery, since these semiconductor nanocrystals possess uniform sizes and and the associated risks to ecosystem health. 2,18À20 The nitrogen cycle is one of the fundamental biogeochemical distinct optical and electrical properties. Some QDs can processes on the earth and denitrification is a key component that is critically implicated in water quality, agricultural productivity, Received: December 20, 2010 and climate change. This form of anaerobic respiration (which is Accepted: April 15, 2011 performed by a wide range of Bacteria and Archaea) involves the Revised: April 11, 2011 À À reduction of nitrate (NO3 ) or nitrite (NO2 ) (both common Published: April 28, 2011 r 2011 American Chemical Society 4988 dx.doi.org/10.1021/es1042673 | Environ. Sci. Technol. 2011, 45, 4988–4994 Environmental Science & Technology ARTICLE Random primers, dNTP set, Superscript II reverse transcriptase, Bacteria Counting Kit, BacLight Bacterial Membrane Potential Kit, and BacLight RedoxSensor Green Vitality Kit. RNAprotect Figure 1. Genes coding for denitrification in Pseudomonas stutzeri. The bacteria reagent, QIAquick PCR Purification Kit, and RNeasy effect of QDs on the genes depicted in bold was assessed by RT-qPCR. Mini Kit were obtained from Qiagen Inc. (Valencia, CA). SYBR Green Master Mix was purchased from Applied Biosystems be toxic to microorganisms and animals, and their toxicity (Carlsbad, CA), and PCR primers were synthesized by Integrated depends on their physiochemical properties such as core com- DNA Technologies, Inc. (San Diego, CA). Lysozyme, ethylene- position, size, charge, and surface coatings,6,8,21À24 which are diamine-tetraacetic acid (EDTA) buffer, Tris-EDTA (TE) buffer, critical determinants of QD stability and biosafety.25,26 Previous phosphate buffered saline (PBS), multielement standard solution, research showed that the chemical composition and stability of and other chemicals were provided by Sigma-Aldrich (St. Louis, the organic coating play a key role in cytotoxicity of QDs, MO). CHEMets colorimetric dissolved oxygen test kit was influencing the release of toxic heavy metal ions and the potential purchased from CheMetrics, Inc. (Calverton, VA). 25,27,28 uptake of QDs. Effect of QDs on Bacterial Growth. P. stutzeri was exposed to In this study we address the effect of three types of QDs on PEI QDs (1À80 nM) or PMAO QDs (50À800 nM) under P. stutzeri. Exposure was conducted in minimal mineral medium either aerobic or anaerobic conditions for 24 h at 37 °C. Higher under neutral pH conditions to minimize extensive weathering concentrations (0.5 and 1 μM) were used for carboxyl QDs, (e.g., hydrolysis) of the QD coating and avoid confounding which were less toxic; these tests were conducted under aerobic effects on bacterial toxicity associated with the release of Cd and conditions. Bacterial growth was monitored using flow cytome- Se ions.26 Bacterial response is assessed at cellular and molecular try, according to the Bacteria Counting Kit instructions, using a levels, under both anaerobic (denitrifying) and aerobic condi- BD LSRFortessa cell analyzer (Franklin Lakes, NJ). The experi- tions. Anaerobic conditions accentuated the susceptibility of ments were performed at least in triplicate and calculation of half- P. stutzeri to the toxic effect of the positively charged PEI QDs, maximal inhibitory concentration (IC50) was described in Sup- indicated by decreases in cell viability, cell membrane potential, porting Information (SI). Experiments were repeated for con- and reductase activity. We also demonstrated the up-regulation firmation purposes. of denitrification genes as a result of sublethal exposure, which is To assess the role of released heavy metal ions in microbial a novel finding with implications on nitrogen cycling. toxicity, the supernatant was collected by ultracentrifugation (35 000 rpm for 3.5 h) and Cd and Zn concentrations were ’ MATERIALS AND METHODS measured by inductively coupled plasma-mass spectrometry (ICP-MS) Elan 9000 (Perkin-Elmer, Waltham, MA). Then, QD Preparation and Characterization. Three different types additional growth inhibition tests were conducted by exposing of QDs were used in this work. CdSe/CdZnS quantum dots were P. stutzeri for 24 h to similar concentrations of heavy metal ions 29 synthesized as described earlier. Briefly, a mixture of purified (without QDs). The heavy metals were added as ZnCl2 and CdSe nanocrystal chloroform solution, octadecylamine (ODA), Cd(NO3)2 salts under both aerobic and anaerobic conditions. zinc/cadmium precursor, and thiourea/ethanol solution was The tested concentrations were 2 ppm Zn2þ and 0.2 ppm Cd2þ, stirred and heated to 150 °C, cooled to 60 °C, and separated 1 ppm Zn2þ and 0.1 ppm Cd2þ, and 0.5 ppm Zn2þ and 0.05 ppm by precipitation. Polyanionic polymaleic anhydride-alt-1-octade- Cd2þ. Growth was monitored by measuring optical density at cene (PMAO, Mn = 30 000À50 000) and polycationic polyethy- 600 nm with a SpectraMax plus spectrometer (Molecular Device, lenimine (PEI, Mn = 10 000) were used to transfer the as- Sunnyvale, CA). synthesized QDs into aqueous phase (Figure S1).30,31 Qdot Assessment of Membrane Potential and Redox Activity. 655 ITK carboxyl QDs were purchased from Invitrogen Inc. P. stutzeri was grown aerobically or anaerobically on Difco (Carlsbad, CA). Their size distribution and zeta-potential were nutrient broth at 37 °C overnight, and diluted to an OD600 of measured in the exposure medium using a Zetasizer Nano 0.002 in PBS buffer.35 The cells were exposed to 0.5 μM PEI or (Malvern Instruments, U.K.), and concentrations were calcu- PMAO QDs for 15 min. The BacLight Bacterial