Reduced expression of iron transport and homeostasis genes in pseudomonas fluorescens during iron uptake from nanoscale iron

Authors: SINHA, SANJIVNI - North Dakota State University; DAS, TONOY - North Dakota State University; BEZBARUAH, ACHINTYA - North Dakota State University; Fortuna, Ann Marie

Submitted to: NanoImpact
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2018
Publication Date: 9/6/2018
Citation: Sinha, S., Das, T.K., Bezbaruah, A.N., Fortuna, A. 2018. Reduced expression of iron transport and homeostasis genes in pseudomonas fluorescens during iron uptake from nanoscale iron. NanoImpact. p. 1-37. https://doi:10.1016/j.impact.2018.08.009.
DOI: https://doi.org/10.1016/j.impact.2018.08.009

Interpretive Summary: Nanoparticles (<100 nm) are in use for biomedical, personal care, industrial and environmental remediation applications. The aim of this research was to ascertain whether and how the presence of (NZVI) affects the growth, viability, siderophore production and iron transport in P. fluorescens. Our research confirms that NZVI can have nutritional or biocidal effects on the growth of P. fluorescens. Treatment of bacterial cultures with 1 mg mL-1 NZVI significantly reduced siderophore production, expression of genes associated with Fe acquisition and significantly increased growth of P. fluorescens relative to the untreated bacterial control and micro-Fe treatments containing bacteria. Cell death was the result of initial NZVI additions to P. fluorescens cultures. Although significant cell death occurred initially in cultures treated with 1 mg mL-1 NZVI after a 6 day lag in cell growth, sufficient cells remained and conditions in the culture (Eh, pH and Fe availability) favored increased growth of P. fluorescens. Microscopy Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray spectroscopy (EDS). Our research confirms that NZVI can have nutritional (1 mg mL-1 NZVI) or biocidal (2 and 5 mg mL-1 NZVI) effects on the growth of P. fluorescens. Treatment of bacterial cultures with 1 mg mL-1 NZVI significantly reduced siderophore production, expression of genes associated with Fe acquisition and significantly increased growth of P. fluorescens relative to the untreated bacterial control and micro-Fe treatments containing bacteria. Cell death was the result of initial NZVI additions to P. fluorescens cultures. Although significant cell death occurred in cultures treated with 1 mg mL-1 NZVI after a 6 day lag in cell growth, sufficient cells remained and conditions in the culture (Eh, pH and Fe availability) favored increased growth of P. fluorescens. The TEM and EDS analysis confirmed that the interior of cells in the log phase of growth treated with 1 mg mL-1 NZVI contained higher concentrations of Fe relative to the cell exterior and nutrient broth. This study verifies that Fe species derived from NZVI in aqueous solution are bioavailable and have the potential to effect Eh, pH, Fe species and the growth and iron acquisition of microorganisms within soil-plant-hydrologic atmospheric systems.

Technical Abstract: Plant growth-promoting rhizobacteria (PGPR) have beneficial effects on the host plant that include acquisition of nutrients such as iron (Fe). In this study, the impact of varying concentrations of nanoscale zero-valent iron (NZVI, 1, 2 and 5 g L-1) on a PGPR, Pseudomonas fluorescens’ viability, growth and production of siderophores was investigated. Microscale zero-valent iron (MZVI) particles were also included in this study to determine and compare the effects of particle size on Fe acquisition and use by Pseudomonas fluorescen. Siderophores chelate insoluble forms of Fe that are then rendered bioavailable to P. fluorescens as well as some plants. This research indicates that the Fe species derived from lower concentrations of NZVI additions (1 g L-1) are bioavailable and can be utilized as a nutrient by P. fluorescens without the bacteria producing siderophores, an energy intensive process. Also, NZVI application as a fertilizer has promise for better utilization of Fe by plants. P. fluorescens colony forming units (log10 CFU mL-1) growth in cultures containing 1 g L-1 NZVI were significantly higher exceeded (14 units) than those grown without NZVI additions (13 units). The majority of P. fluorescens cells treated with 2 and 5 gL-1 were nonviable and produced limited colony forming units (4 units). The effects of NZVI on siderophore production by P. fluorescens were assessed using chrome azurol S (CAS) plates and real time quantitative reverse transcription PCR (qRT-PCR). Further, expression of genes involved in Fe utilization [putative pvdS (PFL 4190) and a bacterioferritin-associated ferredoxin gene (PFL 4858)] were targeted using real time quantitative reverse transcription PCR (qRT-PCR). Expression of both genes was below detection limits in cultures exposed to NZVI but measurable in MZVI and control treatments, again indicating potential ease in uptake of Fe by P. fluorescens from NZVI. Transmission electron microscopy (TEM) revealed the presence of NZVI particles on the exterior and interior of cells; energy dispersive X-ray spectroscopy (EDS) verified that the material was derived from NZVI and that Fe concentrations were higher in the interior of cells exposed to 1 gL-1 NZVI compared to the control. The research findings from this study indicate that NZVI has the potential to accumulate in plant growth-promoting rhizobacteria and affect the growth of organisms in environments like the rhizobiome which may affect plant growth positively. This research opens up opportunities for future studies to determine the effects of Fe and other nutrients and micronutrients derived from nanomaterials, particularly in oligotrophic systems.