Comparative Transcriptomic Analysis Reveals The Regulatory Mechanisms Of Tetragenococcus Halophilus CICC 10469 In Response To Salt Stress

Tetragenococcus halophilus, a moderately halophilic Gram-positive lactic acid bacterium, was isolated from Chinese style soy sauce, functions as a starter culture to promote the formation of the desired taste and flavor of soy sauce. T. halophilus possesses strong osmotic adjustment capacity to protect itself against deleterious hyperosmotic injury, so the study will help understand the regulatory mechanism of salt stress and promote the salt tolerance capacity of microorganism. We treated T. halophilus in the logarithmic phase with 0 M, 1 M and 3.5 M Na Cl for 5 hours, then obtained high quality RNA samples after extraction, purification and enrichment; and labeled them T1, T2, and T3, respectively. Constructed RNA-seq library by Illumina c DNA library construction methods, then sequenced T. halophilus samples using the Illumina Hi SeqTM 2500 high-throughput sequencing platform. In total, 4.51 Gb high quality clean data were generated, were aligned to the reference genome using Top Hat. The functional annotations of genetic variants were generated using ANNOVAR. With the program cuffdiff and FPKM（Fragments Per Kilobase of transcript per Million mapped reads） method which is a general standardization method of gene expression level, corrected P-values ≤ 0.05 and the value of |log2（fold change）| 3 1.5 was set as the threshold to judge the significance of differences in gene expression between the treatment and control（comparison T1 vs.T2, T3 vs.T2, T1 vs.T3）. Comparative transcriptomic analyses of these significantly differently expressed genes（SDEGs） were performed using GO and KEGG. According to the expression of the SDEGs, analyzed and predicted the regulatory mechanisms of T. halophilus in response to salt stress. In addition, reliability and accuracy validation of RNA-seq data was performed by real-time quantitative PCR（q-PCR）. The study found that: when T. halophilus under low salinity stress, T. halophilus mainly by up-regulating the genes which related to transcription, translation, membrane system and cell division to improve the capacity of salinity resistance, these genes encode ribosomal proteins, structural constituents of ribosomes or were involved in ribosome biogenesis, ribokinase activity, t RNA dihydrouridine synthesis, glycerolipid metabolism or lipid A and fatty acid biosynthesis; when T. halophilus under high salinity stress, T. halophilus mainly by up-regulating the genes which related toheat shock proteins（HSPs） and ABC transport system, such as the genes grp E, gro ES, clp E, hsl V, hsl U, hrc A, dna K, gro EL, rpo D, TEH₀5170, TEH₀5190 and TEH₁7310. Meanwhile, we found that the genes without functional information（i.e., putative and hypothetical genes） occupied a large proportion among the SDEGs. The data and conclusion derive from the study will provide a crucial resource to investigate the regulatory mechanism of T. halophilus and even other halophilic microorganisms in responses to salt stress.