Transcriptomics and Real-Time PCR analysis reveals a cross-modulatory effect between riboflavin and iron and outlines common and specific responses to riboflavin biosynthesis and uptake in Vibrio cholerae. Transcriptomics and Real-Time PCR analysis reveals a cross-modulatory effect between riboflavin and iron and outlines common and specific responses to riboflavin biosynthesis and uptake in Vibrio cholerae

Riboflavin-derived molecules such as flavin mononucleotide and flavin adenine dinucleotide comprise the most important redox coenzymes all across life kingdoms. Vibrio cholerae, a pandemic diarrheagenic bacterium, is able to synthesize riboflavin through the riboflavin biosynthetic pathway (RBP) and to internalize exogenous riboflavin using the RibN importer. This bacterium thrives in different environments such as estuarine waters and the human intestinal tract. The presence of two independent riboflavin supply pathways in this species may be related to the variable riboflavin availability and requirements across different niches. In order to gain insights into the role of the riboflavin provision pathways in the physiology of V. cholerae, we analyzed the bacterial transcriptomics response to extracellular riboflavin and to the deletions of ribD (RBP-deficient strain) or ribN. Notably, many genes responding to riboflavin have been previously reported to belong to the iron regulon. Real time PCR analysis confirmed this effect and further documented that reciprocally, iron regulates RBP and ribN genes in a riboflavin-dependent way. A subset of genes were responding to both ribD and ribN deletions. Nonetheless, a group of genes was specifically affected in the ∆ribD strain, on which the functional terms protein folding and oxidation reduction process were enriched. Also, a subset of genes was affected specifically in the ∆ribN strain, on which the cytochrome complex assembly functional term was enriched. Results indicate that iron and riboflavin interrelate to regulate its respective provision genes and suggest that both common and specific effects of biosynthesized and internalized riboflavin exist. Overall design: Strains and growth conditions. V. cholerae N16961 strain and its ∆ribD and ∆ribN derivatives were grown overnight in LB plates at 37 °C. 5 ml of LB broth were inoculated with a colony of the plate cultures and incubated at 37 °C in an orbital shaker at 150 rpm until they reached an OD600 nm of 1.0. Next, cultures were centrifuged and pellet washed twice with T minimal medium (Wyckoff and Payne, 2011) and resuspended in 1 ml of fresh T medium. 10 ml of plain T medium or T + 2 µM riboflavin were inoculated with 10 µl of the resuspensions and incubated at 37 °C and 180 rpm until an OD600 nm of 0.8. 1 ml of each culture was centrifuged and subjected to RNA extraction. When indicated, iron was omitted in T media, in which case 3 ml of cultures at OD600 nm = 0.3 were harvested for RNA extraction. This growth protocol was similar for RNA subjected to transcriptomics and RT-PCR. RNA extractions, retrotranscription, RNAseq and RT-PCR. RNA extraction was performed with the Thermo Scientific Genejet RNA purification kit according to manufacturer´s instructions. RNA extracts were digested with Turbo DNA-free DNAase at 37 °C for 1 hour. For RNAseq, rRNA was removed using the Ribo-Zero removal kit and cDNA libraries were constructed using the TruSeq mRNA stranded kit, according to manufacturer´s instructions. Next, RNA was sequenced using the Illumina HiSeq 2500 platform to produce 100bp paired-end reads, with ~40 million reads (per sample). rRNA removal, cDNA libraries generation and RNAseq were performed at Genoma Mayor (Santiago, Chile). For RT-PCR analysis, the AffinityScript QPCR cDNA Synthesis kit (Agilent Technologies) was used for cDNA synthesis according to manufacturer´s instructions. As a control, a reaction with no reverse transcriptase was included for each sample in each run. RT-PCR was performed using the Brilliant II SYBR Green QPCR Master Mix kit in a One-Step Applen Biosystems (Life Technologies) thermocycler. Relative expression in the indicated conditions was determined through the ∆∆Ct method as developed before (Livak and Schmittgen, 2001). The 16s RNA subunit gene was used for normalization. For the assessment of the relative expression by RT-PCR of ribB, ribN, ribD and gyrB, the sets of primers used were ribB Fw/ribB Rv, ribN Fw/ribN Rv, ribD Fw/ribD Rv and gyrB Fw/gyrB Rv (Cisternas et al., 2017), respectively. Other RT-PCR primers are as follow: for tonB1, tonB1 Fw (5´- ggtgtttgccatgcctgctgg-3´) / tonB1 Rv (5´-GCGGCTTCACCTTCGGCTTAG-3´); for sodA, sodA Fw (5´-GCCAAGCGATATTCATCCAAGG-3´) / sodA Rv (5´-GCTCAGTGGCCTATCTTCATGC-3´). RNAseq data analysis. Quality control visualization and analysis (adapter and quality trimming) was performed using FastQC version 0.11.2 (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) and Trim_galore version 0.4.1 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), respectively. Reads were mapped to the genome of Vibrio cholerae 01 biovar El Tor str. N16961 (RefSeq, NCBI) using Bowtie2 version 2.1.0 (Langmead and Salzberg, 2012). In all the samples the alignment percentage of reads were above 98%. Differential expression analysis between samples was performed with the Bioconductor package edgeR version 3.18.1 (Robinson et al., 2010) using negative binomial model and exact test based on quantile-adjusted conditional maximum likelihood method (qCML). Genes with a statistically significant change in expression (P<0.05) were selected for further analysis. Analyses of enrichment of Gene Ontology (GO) terms of biological processes in the indicated subsets of genes were performed on the online platform of the Gene Ontology Consortium (www.geneontology.org), and statistically significant (P<0.05) functional terms were retrieved. This series contains only the RNA-seq data from wildtype, ∆ribD and ∆ribN strains grown in the presence or absence of riboflavin.