Regulon Genes Research Articles

ProPr54 web server: predicting σ54 promoters and regulon with a hybrid convolutional and recurrent deep neural network.

σ54 serves as an unconventional sigma factor with a distinct mechanism of transcription initiation, which depends on the involvement of a transcription activator. This unique sigma factor σ54 is indispensable for orchestrating the transcription of genes crucial to nitrogen regulation, flagella biosynthesis, motility, chemotaxis and various other essential cellular processes. Currently, no comprehensive tools are available to determine σ54 promoters and regulon in bacterial genomes. Here, we report a σ54 promoter prediction method ProPr54, based on a convolutional neural network trained on a set of 446 validated σ54 binding sites derived from 33 bacterial species. Model performance was tested and compared with respect to bacterial intergenic regions, demonstrating robust applicability. ProPr54 exhibits high performance when tested on various bacterial species, highly surpassing other available σ54 regulon identification methods. Furthermore, analysis on bacterial genomes, which have no experimentally validated σ54 binding sites, demonstrates the generalization of the model. ProPr54 is the first reliable in silico method for predicting σ54 binding sites, making it a valuable tool to support experimental studies on σ54. In conclusion, ProPr54 offers a reliable, broadly applicable tool for predicting σ54 promoters and regulon genes in bacterial genome sequences. A web server is freely accessible at http://propr54.molgenrug.nl.

C-di-AMP accumulation disrupts glutathione metabolism in Listeria monocytogenes.

C-di-AMP homeostasis is critical for bacterial stress response, cell wall integrity, and virulence. Except for osmotic stress response, the molecular mechanisms underlying other processes are not well defined. A Listeria monocytogenes mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, is significantly attenuated in the mouse model of systemic infection. We utilized the ΔPDE mutant to define the molecular functions of c-di-AMP. RNAseq revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes regulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Subsequent quantitative gene expression analyses revealed that the ΔPDE strain is defective for PrfA-regulated gene expression both at the basal level and upon activation by GSH. We further found the ΔPDE strain to be significantly depleted for cytoplasmic GSH and impaired for GSH uptake. The ΔPDE strain was also deficient in GSH under conditions that activate GSH synthesis by the synthase GshF and upon constitutive expression of gshF, suggesting that c-di-AMP accumulation inhibits GSH synthesis activity or promotes GSH catabolism. A constitutively active PrfA* variant restored virulence gene expression in ΔPDE in broth cultures supplemented with GSH but did not rescue virulence defect in vivo. Therefore, virulence attenuation at high c-di-AMP is likely associated with defects outside of the PrfA regulon. For instance, the ΔPDE strain was sensitive to oxidative stress, a phenotype exacerbated in the absence of GshF. Our data reveal GSH metabolism as another pathway that is regulated by c-di-AMP.IMPORTANCEC-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the molecular functions of c-di-AMP are still not fully defined, and the underlying mechanisms for attenuated virulence at high c-di-AMP levels are unclear. Our analyses in Listeria monocytogenes indicate that virulence-related defects are likely outside of the virulence gene regulon. We found c-di-AMP accumulation to impair L. monocytogenes virulence gene expression and disrupt GSH metabolism. Further studies are necessary to establish the relative contributions of these regulations to virulence and host adaptation.

Nuclear receptor-SINE B1 network modulates expanded pluripotency in blastoids and blastocysts

Embryonic stem cells possess the remarkable ability to self-organize into blastocyst-like structures upon induction. These stem cell-based embryo models serve as invaluable platforms for studying embryogenesis and therapeutic developments. Nevertheless, the specific intrinsic regulators that govern this potential for blastoid formation remain unknown. Here we demonstrate an intrinsic program that plays a crucial role in both blastoids and blastocysts across multiple species. We first establish metrics for grading the resemblance of blastoids to mouse blastocysts, and identify the differential activation of gene regulons involved in lineage specification among various blastoid grades. Notably, abrogation of nuclear receptor subfamily 1, group H, member 2 (Nr1h2) drastically reduces blastoid formation. Nr1h2 activation alone is sufficient to rewire conventional ESC into a distinct pluripotency state, enabling them to form blastoids with enhanced implantation capacity in the uterus and contribute to both embryonic and extraembryonic lineages in vivo. Through integrative multi-omics analyses, we uncover the broad regulatory role of Nr1h2 in the transcriptome, chromatin accessibility and epigenome, targeting genes associated with embryonic lineage and the transposable element SINE-B1. The Nr1h2-centred intrinsic program governs and drives the development of both blastoids and early embryos.

Antimicrobial resistance and genotypic attributes of virulence among Vibrio spp. isolated from Japanese retail seafood

Bacterial diseases caused by Vibrio spp. are a significant impediment to the aquaculture industry owing to difficult-to-treat infections and mortality in farmed animals, affecting socioeconomic development. This study aimed to evaluate the role of Japanese retail seafood in the production of antimicrobial-resistant Vibrio spp. and pathogenic factors. One hundred seafood samples purchased from various retail supermarkets in Hiroshima, Japan, were examined using microbiological, phenotypic, and molecular techniques. One hundred and twenty-eight Vibrio isolates belonging to more than 11 species were identified and characterized. Among the 100 samples, 26 (26 %) tested positive for Vibrio spp. with antimicrobial resistance, while 44 (44 %) were positive for those carrying virulence determinants. Out of 128 isolates, V. alginolyticus was predominant (38 %), followed by V. parahaemolyticus (29 %), V. neocaledonicus (7.0 %), V. cholerae (6.3 %), among others. For virulence gene assessment, only six of the 10 virulence attributes, tlh (34.4 %), VPI (29.7 %), ompW (7.0 %), toxR (5.5 %), hlyA (3.9 %), and ompU (3.1 %), were detected in the isolates. tcpA, ctxA, ctxB, and tdh are absent. Eight V. cholerae isolates of international origin were VPI-positive, seven of which harbored the toxR regulon and other virulence genes. The blaTEM resistance gene was identified in V. alginolyticus (22 isolates). All 46 antimicrobial resistance gene-harboring isolates were phenotypically susceptible to cephalosporins, aztreonam, meropenem, tetracycline, chloramphenicol, and ciprofloxacin. Ampicillin resistance was the highest (91.3 %), followed by colistin (30.4 %) and fosfomycin (15.2 %). The multiple-antibiotic resistance index ranged from 0.2 to 0.47, suggesting that some isolates originate from high-risk contamination sources. Our results shed light on the incidence of potentially pathogenic Vibrio spp. and highlighted the importance of continuous monitoring to improve food safety in the seafood supply chain. These findings will be useful for developing microbiological risk assessments for retail seafood management.

Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells

BackgroundCells and tissues have a remarkable ability to adapt to genetic perturbations via a variety of molecular mechanisms. Nonsense-induced transcriptional compensation, a form of transcriptional adaptation, has recently emerged as one such mechanism, in which nonsense mutations in a gene trigger upregulation of related genes, possibly conferring robustness at cellular and organismal levels. However, beyond a handful of developmental contexts and curated sets of genes, no comprehensive genome-wide investigation of this behavior has been undertaken for mammalian cell types and conditions. How the regulatory-level effects of inherently stochastic compensatory gene networks contribute to phenotypic penetrance in single cells remains unclear.ResultsWe analyze existing bulk and single-cell transcriptomic datasets to uncover the prevalence of transcriptional adaptation in mammalian systems across diverse contexts and cell types. We perform regulon gene expression analyses of transcription factor target sets in both bulk and pooled single-cell genetic perturbation datasets. Our results reveal greater robustness in expression of regulons of transcription factors exhibiting transcriptional adaptation compared to those of transcription factors that do not. Stochastic mathematical modeling of minimal compensatory gene networks qualitatively recapitulates several aspects of transcriptional adaptation, including paralog upregulation and robustness to mutation. Combined with machine learning analysis of network features of interest, our framework offers potential explanations for which regulatory steps are most important for transcriptional adaptation.ConclusionsOur integrative approach identifies several putative hits—genes demonstrating possible transcriptional adaptation—to follow-up on experimentally and provides a formal quantitative framework to test and refine models of transcriptional adaptation.

Inference and prioritization of tissue-specific regulons in Arabidopsis and Oryza

A regulon refers to a group of genes regulated by a transcription factor binding to regulatory motifs to achieve specific biological functions. To infer tissue-specific gene regulons in Arabidopsis, we developed a novel pipeline named InferReg. InferReg utilizes a gene expression matrix that includes 3400 Arabidopsis transcriptomes to make initial predictions about the regulatory relationships between transcription factors (TFs) and target genes (TGs) using co-expression patterns. It further improves these anticipated interactions by integrating TF binding site enrichment analysis to eliminate false positives that are only supported by expression data. InferReg further trained a graph convolutional network with 133 transcription factors, supported by ChIP-seq, as positive samples, to learn the regulatory logic between TFs and TGs to improve the accuracy of the regulatory network. To evaluate the functionality of InferReg, we utilized it to discover tissue-specific regulons in 5 Arabidopsis tissues: flower, leaf, root, seed, and seedling. We ranked the activities of regulons for each tissue based on reliability using Borda ranking and compared them with existing databases. The results demonstrated that InferReg not only identified known tissue-specific regulons but also discovered new ones. By applying InferReg to rice expression data, we were able to identify rice tissue-specific regulons, showing that our approach can be applied more broadly. We used InferReg to successfully identify important regulons in various tissues of Arabidopsis and Oryza, which has improved our understanding of tissue-specific regulations and the roles of regulons in tissue differentiation and development.

The hallmarks of a tradeoff in transcriptomes that balances stress and growth functions.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

PhoB-regulated phosphate assimilation of Ralstonia solanacearum is cross-activated by VsrB in Pi-abundant rich medium

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate–Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.

The CpxRA two-component system of adherent and invasive Escherichia coli contributes to epithelial cell invasion and early-stage intestinal fitness in a dysbiotic mouse model mediated by type 1 fimbriae expression.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.

A comprehensive analysis of pneumococcal two-component system regulatory networks.

Two-component systems are key signal-transduction systems that enable bacteria to respond to a wide variety of environmental stimuli. The human pathogen, Streptococcus pneumoniae (pneumococcus) encodes 13 two-component systems and a single orphan response regulator, most of which are significant for pneumococcal pathogenicity. Mapping the regulatory networks governed by these systems is key to understand pneumococcal host adaptation. Here we employ a novel bioinformatic approach to predict the regulons of each two-component system based on publicly available whole-genome sequencing data. By employing pangenome-wide association studies (panGWAS) to predict genotype-genotype associations for each two-component system, we predicted regulon genes of 11 of the pneumococcal two-component systems. Through validation via next-generation RNA-sequencing on response regulator overexpression mutants, several top candidate genes predicted by the panGWAS analysis were confirmed as regulon genes. The present study presents novel details on multiple pneumococcal two-component systems, including an expansion of regulons, identification of candidate response regulator binding motifs, and identification of candidate response regulator-regulated small non-coding RNAs. We also demonstrate a use for panGWAS as a complementary tool in target gene identification via identification of genotype-to-genotype links. Expanding our knowledge on two-component systems in pathogens is crucial to understanding how these bacteria sense and respond to their host environment, which could prove useful in future drug development.

Transcription Factor Regulation of Gene Expression Network by ZNF385D and HAND2 in Carotid Atherosclerosis.

Carotid intima-media thickness (CIMT) is a surrogate indicator for atherosclerosis and has been shown to predict cardiovascular risk in multiple large studies. Identification of molecular markers for carotid atheroma plaque formation can be critical for early intervention and prevention of atherosclerosis. This study performed transcription factor (TF) network analysis of global gene expression data focusing on two TF genes, ZNF385D and HAND2, whose polymorphisms have been recently reported to show association with CIMT. Genome-wide gene expression data were measured from pieces of carotid endarterectomy collected from 34 hypertensive patients (atheroma plaque of stages IV and above according to the Stary classification) each paired with one sample of distant macroscopically intact tissue (stages I and II). Transcriptional regulation networks or the regulons were reconstructed for ZNF385D (5644 target genes) and HAND2 (781 target genes) using network inference. Their association with the progression of carotid atheroma was examined using gene-set enrichment analysis with extremely high statistical significance for regulons of both ZNF385D and HAND2 (p < 6.95 × 10-7) suggesting the involvement of expression quantitative loci (eQTL). Functional annotation of the regulon genes found heavy involvement in the immune system's response to inflammation and infection in the development of atherosclerosis. Detailed examination of the regulation and correlation patterns suggests that activities of the two TF genes could have high clinical and interventional impacts on impairing carotid atheroma plaque formation and preventing carotid atherosclerosis.

The Staphylococcus aureus ArlS Kinase Inhibitor Tilmicosin Has Potent Anti-Biofilm Activity in Both Static and Flow Conditions.

Staphylococcus aureus can form biofilms on biotic surfaces or implanted materials, leading to biofilm-associated diseases in humans and animals that are refractory to conventional antibiotic treatment. Recent studies indicate that the unique ArlRS regulatory system in S. aureus is a promising target for screening inhibitors that may eradicate formed biofilms, retard virulence and break antimicrobial resistance. In this study, by screening in the library of FDA-approved drugs, tilmicosin was found to inhibit ArlS histidine kinase activity (IC50 = 1.09 μM). By constructing a promoter-fluorescence reporter system, we found that tilmicosin at a concentration of 0.75 μM or 1.5 μM displayed strong inhibition on the expression of the ArlRS regulon genes spx and mgrA in the S. aureus USA300 strain. Microplate assay and confocal laser scanning microscopy showed that tilmicosin at a sub-minimal inhibitory concentration (MIC) had a potent inhibitory effect on biofilms formed by multiple S. aureus strains and a strong biofilm-forming strain of S. epidermidis. In addition, tilmicosin at three-fold of MIC disrupted USA300 mature biofilms and had a strong bactericidal effect on embedded bacteria. Furthermore, in a BioFlux flow biofilm assay, tilmicosin showed potent anti-biofilm activity and synergized with oxacillin against USA300.

Dual functioning by the PhoR sensor is a key determinant to Mycobacterium tuberculosis virulence

PhoP-PhoR, one of the 12 two-component systems (TCSs) that empower M. tuberculosis to sense and adapt to diverse environmental conditions, remains essential for virulence, and therefore, represents a major target to develop novel anti-TB therapies. Although both PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. Here, we show that PhoR is a sensor of acidic pH/high salt conditions, which subsequently activate PhoP via phosphorylation. In keeping with this, transcriptomic data uncover that acidic pH- inducible expression of PhoP regulon is significantly inhibited in a PhoR-deleted M. tuberculosis. Strikingly, a set of PhoP regulon genes displayed a low pH-dependent activation even in the absence of PhoR, suggesting the presence of non-canonical mechanism(s) of PhoP activation. Using genome-wide interaction-based screening coupled with phosphorylation assays, we identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how level of P~PhoP is regulated, we discovered that in addition to its kinase activity PhoR functions as a phosphatase of P~PhoP. Our subsequent results identify the motif/residues responsible for kinase/phosphatase dual functioning of PhoR. Collectively, these results uncover that contrasting kinase and phosphatase functions of PhoR determine the homeostatic mechanism of regulation of intra-mycobacterial P~PhoP which controls the final output of the PhoP regulon. Together, these results connect PhoR to pH-dependent activation of PhoP with downstream functioning of the regulator. Thus, PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome, and a PhoR-deleted M. tuberculosis remains significantly attenuated in macrophages and animal models.

Integrated genomics provides insights into the evolution of the polyphosphate accumulation trait of Ca. Accumulibacter

Candidatus Accumulibacter, a prominent polyphosphate-accumulating organism (PAO) in wastewater treatment, plays a crucial role in enhanced biological phosphorus removal (EBPR). The genetic underpinnings of its polyphosphate accumulation capabilities, however, remain largely unknown. Here, we conducted a comprehensive genomic analysis of Ca. Accumulibacter-PAOs and their relatives within the Rhodocyclaceae family, identifying 124 core genes acquired via horizontal gene transfer (HGT) at its least common ancestor. Metatranscriptomic analysis of an enrichment culture of Ca. Accumulibacter revealed active transcription of 44 of these genes during an EBPR cycle, notably including the polyphosphate kinase 2 (PPK2) gene instead of the commonly recognized polyphosphate kinase 1 (PPK1) gene. Intriguingly, the phosphate regulon (Pho) genes showed minimal transcriptions, pointing to a distinctive fact of Pho dysregulation, where PhoU, the phosphate signaling complex protein, was not regulating the high-affinity phosphate transport (Pst) system, resulting in continuous phosphate uptake. To prevent phosphate toxicity, Ca. Accumulibacter utilized the laterally acquired PPK2 to condense phosphate into polyphosphate, resulting in the polyphosphate-accumulating feature. This study provides novel insights into the evolutionary emergence of the polyphosphate-accumulating trait in Ca. Accumulibacter, offering potential advancements in understanding the PAO phenotype in the EBPR process.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that other competence induction stimulus, like norfloxacin, is not conveyed by the HtrA-dependent pathway. This finding strongly suggests that competence can be induced by at least two independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

Expression of the &lt;i&gt;soxRS&lt;/i&gt; regulon in bacterial cells exposed to various stress factors

Background. Some stress responses contribute to the formation of bacterial antibiotic resistance, including the soxRS oxidative defense regulon. Elevation of reactive oxygen species production and oxidative stress was detected in bacterial cells exposed to various environmental stresses. It can be supposed that a stress-mediated increase in the level of reactive oxygen species will activate the expression of the soxRS regulon genes, which may provide pre-adaptation to antibiotics.The aim. To study changes in the expression of soxRS regulon genes in Escherichia coli cells exposed to NaCl, acetic acid, and heating.Materials and methods. Gene expression was measured in cells bearing reporter gene fusions (soxS::lacZ, nfo::lacZ). An overnight broth culture was diluted in fresh LB broth to OD600 = 0.1 and cultivated at 37 °C without stirring until OD600 = 0.3, then the stressors were applied.Results. Exposure to NaCl and acetic acid activated the expression of soxRS regulon genes, while heating caused a decrease in gene expression. An increase in the expression level was observed in cells subjected to stresses of low intensity (which did not cause a decrease in the number of colony-forming units (CFU) by the 4th hour of exposure compared to the beginning of the stress exposure) and medium intensity (which caused a 10-fold decrease in the number of CFU), whereas high-intensity stresses (which caused a decrease in the number of CFU by more than 10 times), regardless of their nature, were accompanied by a decrease in the expression of the soxRS regulon genes.Conclusion. Under the conditions studied, only the osmotic stress caused by the addition of NaCl was accompanied by a significant activation of the soxRS regulon genes. Sublethal exposure to NaCl, causing an increase in the expression of soxRS regulon genes by 2–2.5 times, may provide pre-adaptation of bacteria to the factors that this regulon is aimed at counteracting, including antibacterial drugs.

Single-Cell RNA Sequencing of Sox17-Expressing Lineages Reveals Distinct Gene Regulatory Networks and Dynamic Developmental Trajectories.

During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia.

Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab's inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS Mab , a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRS Mtb , is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosR Mab regulon was predicted using bioinformatics based on DosR Mtb motifs however, the role and regulon of DosRS Mab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRS Mab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS Mab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.