Genome-wide Transcriptional Response during the Shift to N2-fixing Conditions in Heliobacterium modesticaldum

Abstract

Heliobacteria are the only known phototrophic Firmicute; all known members of this group appear to be capable of N2 fixation but incapable of CO2 fixation. They are anoxygenic and possess the simplest photosynthetic apparatus known. The sequence of the 3.1-Mb genome of Heliobacterium modesticaldum, a moderate thermophile within the family Heliobacteriaceae, is publicly available. The focus of this study is to understand how this organism operates at a fundamental level by examining changes in its transcriptome during a shift from ammonium-containing medium to N2-fixing conditions. RNA was purified from cells grown with pyruvate as the carbon source and ammonia or N2 as the nitrogen source. After rRNA depletion, the RNA pool was sequenced using the Ion Torrent PGM platform. We found that the nitrogenase gene cluster was only expressed under N2-fixing conditions, concomitant with increased expression of the high-affinity ammonium transporter. Most genes were down-regulated in N2-fixing conditions by a factor of at least three. A drastic down-regulation of the highly expressed genes encoding proteins involved in the cyclic electron transport chain also occurred. The photosynthetic pshA transcript also decreased more than 100-fold but subsequent photochemical analysis demonstrated no large drop in the concentration of the reaction center protein complex. This indicates that there is a role for substantial translational regulation in some genes. The transcriptomic analyses revealed a network of differentially expressed genes in H. modesticaldum. This study represents the first step in the creation of a quantitative genome-scale metabolic model establishing H. modesticaldum as a model organism for the Heliobacteriaceae family. temperatures under which it grows [9]. Nitrogen fixation is catalyzed by the nitrogenase enzyme complex, and results in the reduction of atmospheric dinitrogen (N2) to ammonium (NH4 +) and the production of molecular hydrogen [12]. This process requires large amounts of chemical energy (16 ATP) and reducing power (8 Fdred) to convert one N2 to two molecules of NH4 + [13], which is then assimilated into many biomolecules. Sequence similarity predicts the use of a Mo-Fe group I nitrogenase consisting of a homodimer of NifD/K polypeptides [14]. The primary pathway for NH4 + assimilation in heliobacteria is the glutamine synthetase/glutamate synthase pathway [15]. This pathway is essential for growth, because glutamine is the primary intracellular nitrogen donor for purine and pyrimidine synthesis. Both ATP and reducing power are required in carbon metabolism, nitrogen assimilation, and hydrogen production, inextricably linking these pathways. In H. modesticaldum, the high-energy demand required for nitrogen fixation during diazotrophic growth has resulted in strict regulation of the nif genes encoding for nitrogenase. Thus, the addition of NH4 + to cultures Citation: Sheehy D, Lu YK, Osman F, Alattar Z, Flores C, et al. (2018) Genome-wide Transcriptional Response during the Shift to N2-fixing Conditions in Heliobacterium modesticaldum. J Proteomics Bioinform 11: 143-160. doi: 10.4172/jpb.1000481 Volume 11(8) 143-160 (2018) 144 J Proteomics Bioinform, an open access journal ISSN: 0974-276X results in repression of nitrogenase activity [15,16]. However, the effects of N2-fixing versus non-fixing conditions on genome-wide expression and metabolism in Heliobacterium remained to be determined. The sequencing of the genome of H. modesticaldum, along with recent proteomic studies, have provided insights into the energy metabolism of this phototroph. However, metabolic studies have been correlated to only a few genes related to energy and carbon metabolism at the transcriptomic level [17]. To enhance our understanding of the energy metabolism of H. modesticaldum, it is necessary to explore the entire mRNA link between genome and proteome. We report here a singlenucleotide resolution map of the H. modesticaldum Ice1 transcriptome under N2-fixing and non-fixing conditions. In general, we observed low-level repression of transcription genome-wide upon a shift to N2-fixing conditions. On the contrary, a few genes, such as the genes involved in N2-fixation and ammonium scavenging, were upregulated. There were also several cases of even more drastic down-regulation, including the core genes of cyclic photophosphorylation. Materials and Methods Growth of H. modesticaldum Isolated colonies of H. modesticaldum strain Ice1 were cultured in gel-rite media modified by Lin and Casida (1984) for thermophilic application [18] inside an anaerobic Coy glove box at 52°C under infrared lights at 780 nm. Cells were inoculated in Pyruvate-Yeast Extract (PYE) growth media [16], which contains 1 g/L of NH4SO4 as the source of nitrogen and “vitamin levels” of yeast extract (0.02%). The PYE-NH4 + medium was made by elimination of NH4SO4 and increasing the amount of Na2S2O3•5H2O from 0.2 g to 0.4 g. The pH was adjusted to 6.8 with H2SO4 prior to autoclaving. Growth was monitored spectroscopically at an Optical Density (OD) at 625-nm, as minimal photosynthetic pigments absorb at this wavelength in heliobacteria [3,17]. Cells were grown under anaerobic conditions at 52°C. From PYE or PYE-NH4 + conditions, 3 ml of cells in late exponential growth phase were inoculated into 300 ml of similar media. This ensured no traces of ammonia were carried over to the next generation of cells. Extraction was performed when cells were in mid-log phase of growth at an OD of 0.303 (PYE-NH4 +) and 0.410 (PYE). A biological replicate for each condition was also prepared. Isolation and purification of mRNA Total RNA was isolated using the Purelink total RNA isolation kit (Invitrogen, USA). Cells were anaerobically extracted and lysed using the needle homogenization method offered in the kit protocol. Once total RNA was extracted the solutions were treated with DNaseI via the Ambion RiboPure Bacteria procedure to remove all genomic DNA. Depletion of the 16S and 23S rRNAs was performed via the Ambion MICROBEExpress kit through subtractive hybridization with capture oligonucleotides. The resulting solution contained tRNAs, 5S rRNAs, and enriched mRNA. Library preparation and Ion Torrent sequencing Library preparation was performed using the Ion Total RNASeq Kit (Ambion, USA). Both sample and WT control RNAs (1 μg/ μL HeLa total RNA) were fragmented using RNase III at 37°C for 10 minutes. The fragmented RNA was purified via Ambion’s RiboMinus Concentration Module. The resulting mRNA yield was measured on the Bioanalyzer (Agilent, USA). These enriched mRNA samples were hybridized and ligated with Ion Torrent adaptors. Strand specificity was retained using the Ion Adaptor Mix containing oligonucleotides with a single-stranded degenerate sequence at the 3’ end and a defined sequence at the 5’ end. This effectively constrains the RNA orientation, with sequencing only performed from the 5’ end of the sense strand. Reverse transcription was performed and the cDNA was purified and size selected using Agencourt’s AMPure XP reagent for an optimal fragment size between 30-200 bp. The cDNA samples were amplified using the provided Ion 5’ and 3’ PCR Primers for 16 cycles. Fragment size was verified on the Bioanalyzer (Figure S1). The prepared cDNA libraries were loaded on the chip and sequenced using the Ion PGM sequencer per the manufacturer’s instructions (Life Technologies, USA). Bioinformatic analysis of RNA-seq data The sequenced reads captured on the Ion Torrent platform were analyzed by following procedure described here. Initially, the sequencing reads were subject to filtering out of those with the poor quality and more than 50% of Ns in reads using PRINSEQ [19]. Bowtie2 [20] with default parameters was used to align filtered RNA-seq reads against H. modesticaldum chromosome as the reference genome. The resulting sequence alignment files were imported into Partek Genomics Suite (Partek Inc., St. Louis, MO) to compute raw and fragments per kilobase of exon model per million mapped (RPKM) reads for the normalized expression values of each transcript. A stringent filtering criterion with RPKM value of 1.0 [21] was used to obtain expressed transcripts. The RPKM values of filtered transcripts were log-transformed using log2 (RPKM + offset) with an offset value of 1.0, and fold changes in transcript expression, differential expression, and p-values were generated from these using the Partek software with default settings. Metabolic pathways that were significantly enriched between +/-NH4 + were identified using Cytoscape [22] with the reference pathways of H. modesticaldum from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [23].