Microbiol Spectr. 2026 Feb 27:e0307725. doi: 10.1128/spectrum.03077-25. Online ahead of print.
ABSTRACT
RNA isolation is a critical first step for gene expression analysis; however, obtaining high-quality RNA from polysaccharide-rich or biofilm-producing microbial samples remains challenging. High polysaccharide content hinders cell lysis, decreases RNA yield, and reduces sample purity, limiting the reliability and accuracy of downstream techniques such as RNA-seq and RT-qPCR. In this study, we evaluated the use of polysaccharide lyase, Smlt1473, as a pre-processing step to improve RNA isolation from Pseudomonas species. Incorporation of Smlt1473 into a commercial RNA extraction kit workflow significantly improved RNA extraction for mucoid clinical and agricultural pseudomonads and did not adversely affect the non-mucoid isolate, PA14. More specifically, RNA-seq analysis demonstrated that pre-processing with Smlt1473 increased the proportion of assigned reads without introducing significant changes in gene expression. Only a limited set of genes-primarily hypothetical proteins and potential phage-associated elements-were differentially expressed, while global transcriptional profiles remained stable. Together, these findings demonstrate that pre-processing with Smlt1473 provides an effective, easily integrated method to enhance RNA isolation from biofilm-forming bacteria and improves RNA-seq data quality without perturbing the underlying biology.IMPORTANCEPseudomonas aeruginosa, along with other clinically relevant pathogens, is notorious for forming complex biofilms. Microbial biofilms can be composed of anywhere from 50% to 90% polysaccharides. This high polysaccharide content of microbial biofilms severely hinders RNA extraction by complicating bacterial cell lysis, causing a decrease in yield and purity. Challenges with isolating RNA from clinically relevant biofilm-forming pathogens limit our ability to study and better understand bacterial pathogenesis. Low quality and quantity of RNA impede the accuracy and reproducibility of downstream analysis and may ultimately obstruct the discovery of novel drug targets and therapeutic interventions. Developing strategies to overcome these barriers, such as enzymatic pre-processing, is therefore critical to improving RNA recovery from biofilm-producing bacteria to enable more accurate transcriptomic studies that advance both basic science and clinical applications.
PMID:41759553 | DOI:10.1128/spectrum.03077-25