Transcriptional response of Vibrio parahaemolyticus to short-chain fatty acids

Microbial cultures for RNA sequencing (RNA-seq) analysis were prepared by inoculating Heart Infusion (HI) broth (BD Biosciences, USA) under two conditions: basal medium and medium supplemented with 200-fold dilution of SCFAs, which are composed of sodium acetate (2.4 M; Shanghai Aladdin Biochemical Technology Co., Ltd., China), sodium propionate (0.8 M; Shanghai Aladdin Biochemical Technology Co., Ltd., China), and sodium butyrate (0.8 ML; Shanghai Aladdin Biochemical Technology Co., Ltd., China). Cultures were grown aerobically at 37 celsius with shaking at 200 rpm until the mid-exponential phase. Due to SCFA-induced growth differences, harvesting was performed at an OD600 of 1.5 for the control and 1.0 for the SCFAs-treated group to ensure comparable physiological states at the time of RNA extraction. Bacteria were collected by centrifugation, and total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the standard protocol. RNA integrity was confirmed by denaturing agarose gel electrophoresis. Three independent biological replicates were prepared per condition to ensure statistical reliability. For transcriptomic sequencing, ribosomal RNA was removed using the Ribo-clean rRNA Depletion Kit Mega (Bacteria) (Vazyme Biotech Co., Ltd., China). cDNA libraries were constructed with the VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme) and sequenced on an Illumina NovaSeq X Plus platform (GENEWIZ Biotechnology, China). Reads were aligned to the complete genome of V. parahaemolyticus RIMD2210633 (RefSeq: NC_004603.1, NC_004605.1), which contains 4832 annotated coding sequences. Transcripts with absolute fold change higher than 2 and adjusted p-value less than 0.05 in the SCFAs treated group versus the untreated control were considered differentially expressed genes (DEGs). The functional annotation of DEGs was carried out by leveraging the Gene Ontology (GO), Cluster of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.