Reference gene selection in Carnobacterium maltaromaticum, Lactobacillus curvatus, and Listeria innocua subjected to temperature and salt stress.

Eight putative consistently expressed genes in Carnobacterium maltaromaticum and Lactobacillus curvatus, and nine in Listeria innocua, were examined for their potential as references for the normalization of gene expression. Expression stability of candidate reference genes was evaluated under growth conditions of low (5 °C) and moderately high (40-42.5 °C) temperatures, and high salt (≥3 % NaCl) using the geNormplus and NormFinder algorithms. Under temperature stress, both algorithms ranked elongation factor Tu (Tuf) as the most stably expressed gene in C. maltaromaticum. In L. curvatus, at similar conditions, geNormplus identified Tuf and 6-phosphogluconate dehydrogenase (6PGDH) as suitable for normalization, while NormFinder identified phenylalanyl-tRNA synthase and recombinase A as the best pair. In L. innocua grown under the same temperatures, geNormplus ranked 6PGDH, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Tuf as the top three most stable references, whereas NormFinder identified GAPDH and 6PGDH as suitable for normalization, with Tuf ranked as number six. There was less consistency between algorithms in the salt stress experiment. No gene was identified that exhibited such a constant level of expression as to outperform the other candidates under both experimental conditions. This study underlines the need for normalizing bacterial gene expression using multiple carefully selected references.