Transcriptome analysis of Pedobacter lusitanus NL19 grown in TSB and PC culture media

Published: 26 July 2021| Version 3 | DOI: 10.17632/t5ryx5mk2k.3
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Description

We report the application of RNA-seq metodology to analyze the differentially expressed genes (DEGs) of Pedobacter lusitanus NL19 grown in two different culture media, TSB and PC, characterized to contain high and low concentration of peptone from casein (PC), respectively. Broth with higher concentrations of PC has been previously associated with the abolishment of antibiotic production by P. lusitanus NL19. RNA-seq results showed that P. lusitanus NL19 grown in TSB and in PC generated 22 783 626 and 43 625 972 sequence reads, respectively. For gene expression analysis, 96,67% and 96,53% of the total fragments obtained for TSB and PC, respectively, were successfully mapped against P. lusitanus NL19 reference genome which allowed the identification of a total of 261 DEGs. More specifically, 108 genes were upregulated when NL19 was grown in TSB compared with its growth in PC, whereas 153 were downregulated. The biological function of most of these DEGs (53,7%) could not be predicted. For the others, genes involved in molecular functions and encoding cellular components were mostly upregulated and genes encoding biological processes were mostly downregulated. In general this study provided some insights on the main cellular pathways that are shaped in the presence of different concentrations of PC in P. lusitanus NL19, showing that high concentrations of PC strongly downregulates the de novo biosynthesis of biotin and the transcription of genes required for pedopeptin’s production, including a L-Dap biosynthetic operon. On the other hand, it increases the transcription of MFS efflux pumps and iron-uptake systems.

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Growth protocol: Pedobacter lusitanus NL19 was grown, overnight, in 5 mL de TSB (peptone from casein 15 g/L, peptone of soy and NaCl 5 g/L) that was used to inoculate 50 mL of TSB and PC (peptone from casein 3.75 g/L, peptone of soy and NaCl 5 g/L) in a 1:100 proportion using 250 mL flasks [23]. Cultures were grown at 26 ◦C, with aeration at 180 rpm, until an OD600nm of 0.6 was reached. At this stage, 1 mL of culture was collected centrifuged at 4 °C for 5 minutes at 8000 rpm and the pelleted cells were used for RNA extraction. Extract protocol: Total RNA was extracted with the PureLinkTM Mini Kit (Invitrogen, USA), isolated on column with PureLinkTM DNase (Invitrogen, USA) and quantified using NanodropTM ND-1000 spectrometer (ThermoFisher Scientific, USA). RNA quality was ensured by 1% agarose gel electrophoresis containing 10% of bleach. Library construction protocol: Total RNA from one replicate grown in each condition (TSB and PC) was sequenced at Stabvida (Caparica, Portugal) since the RNA concentration and integrity number (RIN) were within suitable parameters (amount of total RNA > 1µg; RIN > 7). cDNA library was obtained with Kapa Stranded Total RNA Library Preparation Kit with Ribo-Zero rRNA depletion and the generated DNA fragments (DNA libraries) were sequenced in the lllumina Hiseq 4000 platform, using 150bp paired-end sequencing reads. The obtained sequences were mapped against the reference genome of P. lusitanus NL19 (NZ_JXRA00000000.1). Data processing step: For gene transcription quantification, the RPKM were calculated and the values were used for comparison of genes transcription rates normalized within this experimental setup. Differential expressed genes (DEGs) between the two conditions (TSB vs. PC) were evaluated using the Bioconductor package EdgeR with multi-factorial statistical analysis tool based on a negative binomial model; Data processing steps: a) The sample fold change was calculated from the Generalized Linear Model (GLM), which corrects for differences in library size between the samples and the effects of confounding factors; b) Gene quantification and analysis of the generated sequence raw data was carried out using CLC Genomics Workbench 10.1.1.; c) Functional annotation and Gene Ontology (GO) identification of DEGs were obtained using the InterproScan Processed data files format and content: The differentially expressed genes were filtered using standard conditions, fold change (≥ 2 or ≤ − 2) and FDR p-value ≤0.05.