[This corrects the article DOI: 10.3389/fpsyg.2025.1669090.].

The onset of the chronic stage of infection by bacteria in the Burkholderia cepacia complex depends on the controlled transition to the biofilm-associated lifestyle. In this study, we systematically investigated the effect of all five hns genes of Burkholderia cenocepacia H111 on the motile-to-biofilm lifestyle switch and pathogenicity. The hns genes are distributed across the three replicons (hns1.1 and hns1.2 on chromosome 1, hns2 on chromosome 2, and hns3.1 and hns3.2 on the megaplasmid pC3). We showed that deletion of either hns1.1 or hns1.2 affects cell growth and promotes the cell transition to the biofilm, with the major impact observed in cells lacking hns1.2. According to a comparative transcriptome analysis, the motile-to-biofilm switch linked to the absence of hns1.2 is supported by downregulation of flagellar and chemotaxis genes and upregulation of several genes involved in biofilm formation. In contrast, biofilm-related genes were downregulated; biofilm formation was reduced; and motility was increased in cells lacking hns3.1. An opposite impact of hns1.2 and hns3.1 on the motile-to-biofilm transition was also supported by using cells expressing these hns genes at high levels. Furthermore, hns3.1 and hns1.1 were shown to play a role in bacterial virulence in the Galleria mellonella infection model. Therefore, this study uncovers hns genes as new players of the motile-to-biofilm transition in B. cenocepacia. IMPORTANCE:Several opportunistic pathogenic bacteria can thrive as motile, free-living cells in soil and water, as well as in biofilm in their hosts. This includes the closely related species comprising the Burkholderia cepacia complex, which chronically colonize the airway in individuals with cystic fibrosis. Over the last two decades, the number of genes involved in the control of the motile-to-biofilm lifestyle switch has increased in this bacterial group, particularly in Burkholderia cenocepacia. In the present study, we uncover three hns genes that participate in the motile-to-biofilm lifestyle switch in B. cenocepacia H111 under the conditions tested. While two chromosomally encoded hns genes work to maintain cells at the motile stage, a megaplasmid-encoded hns gene facilitates the cell transition to biofilm. By systematically investigating all five hns genes, we considerably expand our understanding of the control of the motile-to-biofilm lifestyle switch in the Burkholderia cepacia complex.

Circular RNAs (circRNAs) are RNA molecules with 5′ and 3′ ends covalently ligated. Their functions range from acting as genetic regulators to producing proteins, and they are often expressed in a tissue and condition-specific manner. Next-generation sequencing with prior RNA treatment with the RNase R exonuclease (circRNA-Seq) has been used to identify circRNAs in many organisms, especially in model eukaryotes. However, we know little about circRNAs in prokaryotes: they have not been consistently reported in bacteria and, to date, only a few circRNA-Seq studies have been done in archaea. We have developed a prokaryotic-specific computational pipeline, MonArch, that explores RNA-Seq reads for circRNA signatures. We annotated circRNAs in newly generated Halobacterium salinarum circRNA-Seq data and reanalyzed over 20 archaeal public RNA-Seq datasets with this tool. H. salinarum has 49 high-confidence circRNAs, with some validated by RT-PCR. We detected known circular ribosomal RNA and transfer RNA processing intermediates and novel circRNAs associated with ωRNAs (obligate mobile element–guided activity - OMEGA) and IS200/IS605 transposases. The ωRNAs circular isoforms have a growth-dependent expression pattern, distinct from the total ωRNAs levels. This is one of the few examples of prokaryotic circRNAs with a conditional expression pattern. In all the other public archaea circRNA-Seq data (Haloferax volcanii, Saccharolobus solfataricus, Sulfolobus acidocaldarius, and Pyrococcus abyssi), we found circRNAs associated with the same classes of transcripts as for H. salinarum, including circRNAs in IS200/IS605 transposases in the two Sulfolobales species. We broadened our search for circRNAs in representatives of major archaeal groups, and found that circRNAs associated with the rRNA and tRNA are widespread, indicating conserved processing of these transcripts. Circular ωRNAs are also present in other haloarchaeal species. Together, our results show that circRNAs appear to be conserved and abundant among archaea, maybe more than previously appreciated. The circular ωRNAs are present in different distant archaeal species, and are a new piece in the IS200/IS605 system.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

The role of the iron–sulfur [Fe-S] cluster transcriptional regulator IscR in maintaining [Fe-S] homeostasis in bacteria is still poorly characterized in many groups. Caulobacter crescentus and other Alphaproteobacteria have a single operon encoding [Fe-S] cluster biosynthesis enzymes. We showed that the expression of this operon increases in iron starvation, but not in oxidative stress, and is controlled mainly by IscR. Transcriptome analysis comparing an iscR null mutant strain with the wild-type (wt) strain identified 94 differentially expressed genes (DEGs), with 47 upregulated and 47 downregulated genes in the ΔiscR mutant. We determined the IscR binding sites in conditions of sufficient or scarce iron by Chromatin Immunoprecipitation followed by DNA sequencing (ChIP-seq), identifying two distinct putative DNA binding motifs. The estimated IscR regulon comprises 302 genes, and direct binding to several regulatory regions was shown by Electrophoresis Mobility Shift Assay (EMSA). The results showed that the IscR and Fur regulons partially overlap and that IscR represses the expression of the respiration regulator FixK, fine-tuning gene regulation in response to iron and redox balance.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum . Screening of a 10,000-transposon mutant library for siderophore halos on PSA-CAS plates identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum . Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS . Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutation of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum .Importance The iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum , an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS , which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum .

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.

ABSTRACT The RNA helicases from the DEAD-box family participate in several biochemical processes, with special relevance in the assembly of ribosome ribonucleoparticles and in RNA turnover as part of the multi-enzymatic RNA degradosome complex. Caulobacter crescentus has three DEAD-box RNA helicases, including RhlB, the main RNA helicase associated with the RNA degradosome. In this work, we evaluated the impact of RhlB on C. crescentus gene expression both at normal and low temperatures and its role in global RNA decay. The rhlB null mutant showed a freezing-sensitive phenotype after pre-incubation at low temperature, indicating a failure in assembling a proper cryotolerance response. A global transcriptomic profile of the wild-type and rhlB mutants was carried out at 30°C and 10°C and showed 73 and 477 differentially expressed genes, with the most affected gene categories being translation and posttranslational modifications. The rhlB mutant is more sensitive to streptonigrin, suggesting that the downregulation of iron-regulated genes may be due to a higher intracellular iron concentration. A high throughput screening for RhlB-binding RNAs identified 220 transcripts at 30°C, and global RNA decay analyses identified several mRNAs and sRNAs that displayed altered profiles of RNA decay rates. Analyses of the transcripts’ 5′-ends from the C. crescentus rhlB mutant and from Pseudomonas aeruginosa PAO1 rhlE1 and rhlE2 mutants confirm that in the absence of the DEAD-box RNA helicase associated with RNase E, there is an accumulation of RNA degradation intermediates. 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.

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E ) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC-family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC-type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator.

Halobacterium salinarum knockout strains (delta-Ura3: control; delta-Ura3/delta-SmAP1: SmAP1 knockout) were cultured in uracil-supplemented (50 μg/mL) complex medium (CM) until mid-exponential phase (OD600nm = 0.5). The cultures were grown at 37C, under light exposure, and with constant agitation (125 RPM). We collected 2 mL samples and submitted them to DNA extraction using the DNeasy Blood & Tissue kit (QIAGEN), according to the manufacturer's instructions for Gram-negative bacteria. We tested DNA samples for purity and quantity using spectrophotometric and fluorimetric methods, respectively. The samples were prepared for long-read sequencing following the 1D native barcoding genomic DNA protocol using SQK-LSK108 and EXP-NBD103 (Oxford Nanopore Technologies). The equimolar pool of barcoded samples was sequenced using a MinION Mk1B instrument (Oxford Nanopore Technologies) in an FLO-MIN106 flow cell for 24 hours. Three biological replicates were sequenced for each one of the strains (control and SmAP1 knockout). This repository stores the partitioned (eleven parts: aa-ak) compressed directory (tar.gz) containing all the raw reads (fast5 format) output by the MinKNOW software. Experimental design: Barcode Description Barcode 01 Control, biological replicate 1 Barcode 02 Control, biological replicate 2 Barcode 03 Control, biological replicate 3 Barcode 04 SmAP1 knockout, biological replicate 1 Barcode 05 SmAP1 knockout, biological replicate 2 Barcode 06 SmAP1 knockout, biological replicate 3 Instructions to merge files and extract: 1. Download all the files available in this Zenodo entry (smap1_ko_exp_fast5.tar.gz.part_a*; from aa to ak; eleven files) to your preferred directory; 2. Execute the following commands using a Linux or OSX terminal: # concatenate all the files into a single one cat smap1_ko_exp_fast5.tar.gz.part_a* > smap1_ko_exp_fast5.tar.gz # extract the merged file tar zxvf smap1_ko_exp_fast5.tar.gz

Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration. In spite of being an established model microorganism for the archaea domain, direct comparisons between its proteome and transcriptome during osmotic stress are still not available. Through RNA-seq-based transcriptomics, we compared a low salt (2.6 M NaCl) stress condition with 4.3 M of NaCl and found 283 differentially expressed loci. The more commonly found classes of genes were: ABC-type transporters and transcription factors. Similarities, and most importantly, differences between our findings and previously published datasets in similar experimental conditions are discussed. We validated three important biological processes differentially expressed: gas vesicles production (due to down-regulation of gvpA1b, gvpC1b, gvpN1b, and gvpO1b); archaellum formation (due to down-regulation of arlI, arlB1, arlB2, and arlB3); and glycerol metabolism (due to up-regulation of glpA1, glpB, and glpC). Direct comparison between transcriptomics and proteomics showed 58% agreement between mRNA and protein level changes, pointing to post-transcriptional regulation candidates. From those genes, we highlight rpl15e, encoding for the 50S ribosomal protein L15e, for which we hypothesize an ionic strength-dependent conformational change that guides post-transcriptional processing of its mRNA and, thus, possible salt-dependent regulation of the translation machinery.

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 highaffinity 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.

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.

BACKGROUND:Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown.RESULTS:Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment, as seem by growth curve assays. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins.CONCLUSIONS:Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense (ohrA/ohrR), anaerobic respiration (narK1K2 and narGHJI), and biofilm formation (putative RTX adhesins).

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.

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.

Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.