An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

Abstract

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Improved microbiological diagnostic due to utilization of a high-throughput homogenizer for routine tissue processing

“…In addition, the tissue cells could mechanically protect bacteria or fungi from disruption, since specific homogenizing protocols for lysis of bacteria use them in pure suspensions (Verollet, 2008). Finally, employing ceramic beads for tissue disruption could be beneficial for bacterial survival since conventional protocols for preparation of bacterial molecules include the use of glass beads (Verollet, 2008;Holmes et al, 2014).…”

“…The negligible effect on microbial viability could be due to the short and gentle homogenization process used in this study. For comparison, in an E. coli messenger RNA extraction protocol utilizing a bead-based tissue homogenizer, the step of homogenization is repeated several times and thereby is combined with extreme physical and chemical stress (Holmes et al, 2014). In addition, the tissue cells could mechanically protect bacteria or fungi from disruption, since specific homogenizing protocols for lysis of bacteria use them in pure suspensions (Verollet, 2008).…”

The virulence of Streptococcus pneumoniae partially depends on dprA

“…Total RNA was obtained using the hot acid phenol method as previously described 15 . Levels of mRNA for pneumolysin ( ply ), psaA , cbpA , and cps2A genes were quantified by one-step real-time RT-PCR with the Takara SYBR RT-PCR system (Takara Bio).…”

Gluconacetobacter diazotrophicus Changes The Molecular Mechanisms of Root Development in Oryza sativa L. Growing Under Water Stress

“…The samples were frozen in liquid nitrogen, sliced, and then transferred to 1.5 mL microtubes containing a mixture of~250 mg glass and silica beads (both with 1 mm diameter) (Thermo Fisher Scientific; Waltham, MA, USA). Total RNA was extracted according to the bead/SDS/phenol method described by Holmes et al [37]. The concentration, and integrity and purity of the sample were determined with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific; Waltham, MA, USA) and an L-Quant spectrophotometer (Loccus, São Paulo, SP, Brazil), respectively.…”