Adaption of microarray primers for iron transport and homeostasis gene expression in Pseudomonas fluorescens exposed to nano iron

Ann-Marie Fortuna; Sanjivni Sinha; Tonoy K Das; Achintya N Bezbaruah

doi:10.1016/j.mex.2019.04.006

Adaption of microarray primers for iron transport and homeostasis gene expression in Pseudomonas fluorescens exposed to nano iron

MethodsX. 2019 Apr 30:6:1181-1187. doi: 10.1016/j.mex.2019.04.006. eCollection 2019.

Authors

Ann-Marie Fortuna¹, Sanjivni Sinha², Tonoy K Das², Achintya N Bezbaruah²

Affiliations

¹ Grazinglands Research Laboratory, USDA-ARS, United States.
² Nanoenvirology Research Group, Civil and Environmental Engineering, North Dakota State University, United States.

Abstract

Modified protocols were adapted for PCR and culture based methods for the analysis of Pseudomonas fluorescens cells exposed to nanoscale zero-valent iron (NZVI) and iron (Fe) in bacterial growth nutrient media was determined by a modified atomic absorption spectrometric (AAS) analysis method. We adapted sets of microarray primers used to quantify gene expression of pvdS and a bacterioferritin-associated ferredoxin gene for use in real-time quantitative reverse transcription (qRT-PCR) analysis. pvdS is one of a cluster of genes regulating the synthesis of the siderophore pyoverdine that was also measured using chrome azrul S (CAS) plates. •The current protocol provides a detailed qRT-PCR method for quantifying genes involved in the acquisition and utilization of Fe in P. fluorescens cells exposed to NZVI.•The qRT-PCR results were independently corroborated with 2 culture based methods, growth curves and chrome azurol S (CAS) plate.•The modified AAS method was used to measure Fe in Tryptic Soy Broth (TSB) medium where sodium (Na) causes inference in iron measurement.

Keywords: Atomic absorption; Azrul S (CAS); Ferredoxin; Pyoverdine; Real-time quantitative reverse transcription (qRT-PCR) analysis; qRT-PCR.