An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter.

IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

A comprehensive spectral assay library to quantify the Halobacterium salinarum NRC-1 proteome by DIA/SWATH-MS.

Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requires a priori generated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish, E. coli and few other organisms. However, a spectral assay library for the extreme halophilic archaeon Halobacterium salinarum NRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotated H. salinarum NRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. The H. salinarum NRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

A Genome-Scale Atlas Reveals Complex Interplay of Transcription and Translation in an Archaeon.

The scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation in environmental acclimation are underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating the transcriptome-wide locations of transcript processing sites (TPSs) and SmAP1 binding, the genome-wide locations of antisense RNAs (asRNAs), and the consequences of RNase_2099C knockout on the differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely being regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (transcriptome sequencing [RNA-Seq]) and protein levels (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry [SWATH-MS]) for 2,579 genes over four phases of batch culture growth in complex medium generated additional evidence for the conditional post-transcriptional regulation of 7% of all protein-coding genes. We demonstrate that post-transcriptional regulation may act to fine-tune specialized and rapid acclimation to stressful environments, e.g., as a switch to turn on gas vesicle biogenesis to promote vertical relocation under anoxic conditions and modulate the frequency of transposition by insertion sequence (IS) elements of the IS200/IS605, IS4, and ISH3 families. Findings from this study are provided as an atlas in a public Web resource (https://halodata.systemsbiology.net). IMPORTANCE While the transcriptional regulation landscape of archaea has been extensively investigated, we currently have limited knowledge about post-transcriptional regulation and its driving mechanisms in this domain of life. In this study, we collected and integrated omics data from multiple sources and technologies to infer post-transcriptionally regulated genes and the putative mechanisms modulating their expression at the protein level in Halobacterium salinarum NRC-1. The results suggest that post-transcriptional regulation may drive environmental acclimation by regulating hallmark biological processes. To foster discoveries by other research groups interested in the topic, we extended our integrated data to the public in the form of an interactive atlas (https://halodata.systemsbiology.net).

Halobacterium salinarum and Haloferax volcanii Comparative Transcriptomics Reveals Conserved Transcriptional Processing Sites.

Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.

Cold Regulation of Genes Encoding Ion Transport Systems in the Oligotrophic Bacterium Caulobacter crescentus.

In this study, we characterize the response of the free-living oligotrophic alphaproteobacterium Caulobacter crescentus to low temperatures by global transcriptomic analysis. Our results showed that 656 genes were upregulated and 619 were downregulated at least 2-fold after a temperature downshift. The identified differentially expressed genes (DEG) belong to several functional categories, notably inorganic ion transport and metabolism, and a subset of these genes had their expression confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Several genes belonging to the ferric uptake regulator (Fur) regulon were downregulated, indicating that iron homeostasis is relevant for adaptation to cold. Several upregulated genes encode proteins that interact with nucleic acids, particularly RNA: cspA, cspB, and the DEAD box RNA helicases rhlE, dbpA, and rhlB. Moreover, 31 small regulatory RNAs (sRNAs), including the cell cycle-regulated noncoding RNA (ncRNA) CcnA, were upregulated, indicating that posttranscriptional regulation is important for the cold stress response. Interestingly, several genes related to transport were upregulated under cold stress, including three AcrB-like cation/multidrug efflux pumps, the nitrate/nitrite transport system, and the potassium transport genes kdpFABC. Further characterization showed that kdpA is upregulated in a potassium-limited medium and at a low temperature in a SigT-independent way. kdpA mRNA is less stable in rho and rhlE mutant strains, but while the expression is positively regulated by RhlE, it is negatively regulated by Rho. A kdpA-deleted strain was generated, and its viability in response to osmotic, acidic, or cold stresses was determined. The implications of such variation in the gene expression for cold adaptation are discussed. IMPORTANCE Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow. Here, we show that in Caulobacter crescentus, the expression of the genes involved in cation transport and homeostasis is altered in response to cold, which could lead to a decrease in iron uptake and an increase in nitrogen and high-affinity potassium transport by the Kdp system. This previously uncharacterized regulation of the Kdp transporter has revealed a new mechanism for adaptation to low temperatures that may be relevant for oligotrophic bacteria.

Selective Translation of Low Abundance and Upregulated Transcripts in Halobacterium salinarum.

When organisms encounter an unfavorable environment, they transition to a physiologically distinct, quiescent state wherein abundant transcripts from the previous active growth state continue to persist, albeit their active transcription is downregulated. In order to generate proteins for the new quiescent physiological state, we hypothesized that the translation machinery must selectively translate upregulated transcripts in an intracellular milieu crowded with considerably higher abundance transcripts from the previous active growth state. Here, we have analyzed genome-wide changes in the transcriptome (RNA sequencing [RNA-seq]), changes in translational regulation and efficiency by ribosome profiling across all transcripts (ribosome profiling [Ribo-seq]), and protein level changes in assembled ribosomal proteins (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) to investigate the interplay of transcriptional and translational regulation in Halobacterium salinarum as it transitions from active growth to quiescence. We have discovered that interplay of regulatory processes at different levels of information processing generates condition-specific ribosomal complexes to translate preferentially pools of low abundance and upregulated transcripts. Through analysis of the gene regulatory network architecture of H. salinarum, Escherichia coli, and Saccharomyces cerevisiae, we demonstrate that this conditional, modular organization of regulatory programs governing translational systems is a generalized feature across all domains of life.IMPORTANCE Our findings demonstrate conclusively that low abundance and upregulated transcripts are preferentially translated, potentially by environment-specific translation systems with distinct ribosomal protein composition. We show that a complex interplay of transcriptional and posttranscriptional regulation underlies the conditional and modular regulatory programs that generate ribosomes of distinct protein composition. The modular regulation of ribosomal proteins with other transcription, translation, and metabolic genes is generalizable to bacterial and eukaryotic microbes. These findings are relevant to how microorganisms adapt to unfavorable environments when they transition from active growth to quiescence by generating proteins from upregulated transcripts that are in considerably lower abundance relative to transcripts associated with the previous physiological state. Selective translation of transcripts by distinct ribosomes could form the basis for adaptive evolution to new environments through a modular regulation of the translational systems.

The Primary Antisense Transcriptome of Halobacterium salinarum NRC-1.

Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3' ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin⁻antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.

OxyR and the hydrogen peroxide stress response in Caulobacter crescentus.

Oxidative stress generated by hydrogen peroxide is faced by bacteria when encountering hostile environments. In order to define the physiological and regulatory networks controlling the oxidative stress response in the free-living bacterium Caulobacter crescentus, a whole transcriptome analysis of wild type and ΔoxyR strains in the presence of hydrogen peroxide for two different exposure times was carried out. The C. crescentus response to H2O2 includes a decrease of the assimilative sulfate reduction and a shift in the amino acid synthesis pathways into favoring the synthesis of histidine. Moreover, the expression of genes encoding enzymes for the depolymerization of polyhydroxybutyrate was increased, and the RpoH-dependent genes were severely repressed. Based on the expression pattern and sequence analysis, we postulate that OxyR is probably directly required for the induction of three genes (katG, ahpCF). The putative binding of OxyR to the ahpC regulatory region could be responsible for the use of one of two alternative promoters in response to oxidative stress. Nevertheless, OxyR is required for the expression of 103 genes in response to H2O2. Fur and part of its regulon were differentially expressed in response to hydrogen peroxide independently of OxyR. The non-coding RNA OsrA was upregulated in both strains, and an in silico analysis indicated that it may have a regulatory role. This work characterizes the physiological response to H2O2 in C. crescentus, the regulatory networks and differentially regulated genes in oxidative stress and the participation of OxyR in this process. It is proposed that besides OxyR, a second layer of regulation may be achieved by a small regulatory RNA and other transcriptional regulators.

Iron Deficiency Generates Oxidative Stress and Activation of the SOS Response in Caulobacter crescentus.

In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.