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Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, and GAL10) for galactose catabolism through the Leloir pathway, and a "GALLAC" cluster containing the transcriptional activator gene LAC9, second copies of GAL1 and GAL10, and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences. IMPORTANCE This study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia. Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products. [ABSTRACT FROM AUTHOR]

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing different ial transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted. IMPORTANCE Chromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology. [ABSTRACT FROM AUTHOR]

Serratia sp. ATCC 39006 is an important model strain for the study of prodigiosin production, whose prodigiosin biosynthesis genes (pigA-O) are arranged in an operon. Several transcription factors have been shown to control the transcription of the pig operon. However, since the regulation of prodigiosin biosynthesis is complex, the regulatory mechanism for this process has not been well established. In most γ-proteobacteria, the ROK family regulator NagC acts as a global transcription factor in response to N-acetylglucosamine (GlcNAc). In Serratia sp. ATCC 39006, NagC represses the transcription of two divergent operons, nagE and nagBAC, which encode proteins involved in the transport and metabolism of GlcNAc. Moreover, NagC directly binds to a 21-nt region that partially overlaps the -10 and -35 regions of the pig promoter and promotes the transcription of prodigiosin biosynthesis genes, thereby increasing prodigiosin production. Although NagC still acts as both repressor and activator in Serratia sp. ATCC 39006, its transcriptional regulatory activity is independent of GlcNAc. NagC was first found to regulate antibiotic biosynthesis in Gram-negative bacteria, and NagC-mediated regulation is not responsive to GlcNAc, which contributes to future studies on the regulation of secondary metabolism by NagC in other bacteria. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between X. nematophila secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new N-acyl amides, GKA-Arg (1) and GKA-Pro (2) which harbor a γ-keto acyl appendage. A lrhA null mutant produced elevated levels of compound 1 and reduced levels of compound 2 relative to wild type. N-acyl amides 1 and 2 were shown to be selective agonists for the human Gprotein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist 2 deleteriously affected the hatch rate and length of Steinernema nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. [ABSTRACT FROM AUTHOR]

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance. [ABSTRACT FROM AUTHOR]

ABSTRACT Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa, this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2, but not lae-1, leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2, suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/VE-2 complex is a key element in transcriptional regulation that occurs during sexual development.IMPORTANCESexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa. VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes.

ABSTRACT Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1, established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1Δ/Δ mutation is most prominent under static or “biofilm-like” growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1: hypha-associated gene expression levels are substantially reduced in a brg1Δ/Δ mutant and partially restored in a brg1Δ/Δ rme1Δ/Δ double mutant. An rme1Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1, whose activity is most prominent under biofilm-like growth conditions.

ABSTRACT The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described β-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.IMPORTANCEAlgae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans. This bacterium uses the recently characterized β-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions.

ABSTRACT Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.IMPORTANCEAspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.

ABSTRACT 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.

ABSTRACT Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3′-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3′-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3′-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3′-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Chronic hepatitis B virus (HBV) infection (CHB) is a risk factor for the development of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Covalently closed circular DNA serves as the sole transcription template for all viral RNAs and viral transcription is driven and enhanced by viral promoter and enhancer elements, respectively. Interactions between transcription factors and these cis-elements regulate their activities and change the production levels of viral RNAs. Here, we report the identification of homeobox protein MSX-1 (MSX1) as a novel host restriction factor of HBV in liver. In both HBV-transfected and HBV-infected cells, MSX1 suppresses viral gene expression and genome replication. Mechanistically, MSX1 downregulates enhancer II/core promoter (EnII/Cp) activity via direct binding to an MSX1 responsive element within EnII/Cp, and such binding competes with hepatocyte nuclear factor 4α binding to EnII/Cp due to partial overlap between their respective binding sites. Furthermore, CHB patients in immune active phase express higher levels of intrahepatic MSX1 but relatively lower levels of serum and intrahepatic HBV markers compared to those in immune tolerant phase. Finally, MSX1 was demonstrated to induce viral clearance in two mouse models of HBV persistence, suggesting possible therapeutic potential for CHB. IMPORTANCE Covalently closed circular DNA plays a key role for the persistence of hepatitis B virus (HBV) since it serves as the template for viral transcription. Identification of transcription factors that regulate HBV transcription not only provides insights into molecular mechanisms of viral life cycle regulation but may also provide potential antiviral targets. In this work, we identified host MSX1 as a novel restriction factor of HBV transcription. Meanwhile, we observed higher intrahepatic MSX1 expression in chronic hepatitis B virus (CHB) patients in immune active phase compared to those in immune tolerant phase, suggesting possible involvement of MSX1 in the regulation of HBV activity by the host. Lastly, intrahepatic overexpression of MSX1 delivered by recombinant adenoviruses into two mouse models of HBV persistence demonstrated MSX1-mediated repression of HBV in vivo, and MSX1-induced clearance of intrahepatic HBV DNA in treated mice suggested its potential as a therapeutic target for the treatment of CHB. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman’s diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development. IMPORTANCE The reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases. [ABSTRACT FROM AUTHOR]

The Kaposi’s sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body. IMPORTANCE Enhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes’ spatiotemporal and quantitative expression. Here, we show that Kaposi’s sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression. [ABSTRACT FROM AUTHOR]

ABSTRACTStaphylococcus aureus is a Gram-positive pathogen responsible for the majority of skin and soft tissue infections (SSTIs). S. aureus colonizes the anterior nares of approximately 20%–30% of the population and transiently colonizes the skin, thereby increasing the risk of developing SSTIs and more serious infections. Current laboratory models that mimic the skin surface environment are expensive, require substantial infrastructure, and limit the scope of bacterial physiology studies under human skin conditions. To overcome these limitations, we developed a cost-effective, open-source, chemically defined media recipe termed skin-like medium (SLM) that incorporates key aspects of the human skin surface environment and supports growth of several staphylococcal species. We utilized SLM to investigate the transcriptional response of methicillin-resistant Staphylococcus aureus (MRSA) following growth in SLM compared to a commonly used laboratory media. Through RNA-seq analysis, we observed the upregulation of several virulence factors, including genes encoding functions involved in adhesion, proteolysis, and cytotoxicity. To further explore these findings, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments to determine the influence of media composition, pH, and temperature on the transcriptional response of key factors involved in adhesion and virulence. We also demonstrated that MRSA primed in SLM adhered better to human corneocytes and demonstrated adhesin-specific phenotypes that previously required genetic manipulation. This improved adherence to corneocytes was dependent on both acidic pH and growth in SLM. These results support the potential utility of SLM as an in vitro model for assessing staphylococcal physiology and metabolism on human skin.IMPORTANCEStaphylococcus aureus is the major cause of skin diseases, and its increased prevalence in skin colonization and infections present a need to understand its physiology in this environment. The work presented here outlines S. aureus upregulation of colonization and virulence factors using a newly developed medium that strives to replicate the human skin surface environment and demonstrates roles for adhesins clumping factor A (ClfA), serine-rich repeat glycoprotein adhesin (SraP), and the fibronectin binding proteins (Fnbps) in human corneocyte adherence.

ABSTRACTAcinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumannii’s transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

ABSTRACTPseudomonas aeruginosa is a common, biofilm-forming pathogen that exhibits complex pathways of redox metabolism. It produces four different types of terminal oxidases for aerobic respiration, and for one of these, the cbb3-type terminal oxidases, it has the capacity to produce at least 16 isoforms encoded by partially redundant operons. It also produces small-molecule virulence factors that interact with the respiratory chain, including the poison cyanide. Previous studies had indicated a role for cyanide in activating expression of an “orphan” terminal oxidase subunit gene called ccoN4 and that the product contributes to P. aeruginosa cyanide resistance, fitness in biofilms, and virulence, but the mechanisms underlying this process had not been elucidated. Here, we show that the regulatory protein MpaR, which is predicted to be a pyridoxal phosphate-binding transcription factor and is encoded just upstream of ccoN4, controls ccoN4 expression in response to endogenous cyanide. Paradoxically, we find that cyanide production is required to support CcoN4’s contribution to respiration in biofilms. We identify a palindromic motif required for cyanide- and MpaR-dependent expression of ccoN4 and co-expressed adjacent loci. We also characterize the regulatory logic of this region of the chromosome. Finally, we identify residues in the putative cofactor-binding pocket of MpaR, which are required for ccoN4 expression. Together, our findings illustrate a novel scenario in which the respiratory toxin cyanide acts as a signal to control gene expression in a bacterium that produces the compound endogenously.IMPORTANCECyanide is an inhibitor of heme-copper oxidases, which are required for aerobic respiration in all eukaryotes and many prokaryotes. This fast-acting poison can arise from diverse sources, but mechanisms by which bacteria sense it are poorly understood. We investigated the regulatory response to cyanide in the pathogenic bacterium Pseudomonas aeruginosa, which produces cyanide as a virulence factor. Although P. aeruginosa has the capacity to produce a cyanide-resistant oxidase, it relies primarily on heme-copper oxidases and even makes additional heme-copper oxidase proteins specifically under cyanide-producing conditions. We found that the protein MpaR controls expression of cyanide-inducible genes in P. aeruginosa and elucidated the molecular details of this regulation. MpaR contains a DNA-binding domain and a domain predicted to bind pyridoxal phosphate (vitamin B6), a compound that is known to react spontaneously with cyanide. These observations provide insight into the understudied phenomenon of cyanide-dependent regulation of gene expression in bacteria.

ABSTRACTChlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression to create Chlamydia trachomatis with conditional GrgA deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fails to maintain its virulence plasmid. Results of RNA-Seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator, AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity.IMPORTANCEHallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

ABSTRACTFluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in C. glabrata.

Alphavirus infections cause multiple alterations in the intracellular environment that can have both positive and negative effects on viral replication. The Old World alphaviruses, such as Sindbis (SINV), chikungunya (CHIKV), and Semliki Forest viruses, hinder the ability of vertebrate cells to form stress granules (SGs). Previously, this inhibitory function was attributed to the hypervariable domain (HVD) of nsP3, which sequesters the key SG components, G3BP1 and G3BP2, and to the nsP3 macro domain. The macro domain possesses ADP-ribosylhydrolase activity, which can diminish the ADP-ribosylation of G3BP1 during viral replication. However, our recent findings do not support the prevailing notions. We demonstrate that the interactions between SINV- or CHIKV-specific nsP3s and G3BPs, and the ADP-ribosylhydrolase activity are not major contributors to the inhibitory process, at least when nsP3 is expressed at biologically relevant levels. Instead, the primary factors responsible for suppressing SG formation are virus-induced transcriptional and translational shutoffs that rapidly develop within the first few hours post infection. Poorly replicating SINV variants carrying mutated nsP3 HVD still inhibit SG development even in the presence of sodium arsenite (NaAs). Conversely, SINV mutants lacking transcription and/or translation inhibitory functions lose their ability to inhibit SG formation, despite expressing high levels of wt nsP3. Moreover, we found that stable cell lines expressing GFP-nsP3 fusions retain the capacity to form SGs when exposed to NaAs. However, our results do not rule out the possibility that additional alphavirus-induced changes in cell biology may contribute to the suppression of SG formation. [ABSTRACT FROM AUTHOR]

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria, and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ54) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ54 binds to a promoter positioned -24 (GG) and -12 (TGC) upstream between mmoG and mmoX1. The binding affinity and selectivity are lower (Kd = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity (Kd = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate (Kd = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ54 that regulate transcription in M. sporium 5 depend on the copper ion concentration. This study provides biochemical evidence of transcriptional regulation of soluble methane monooxygenase (sMMO) in methanotrophs that control methane levels in ecological systems. Previous studies have proposed transcriptional regulation of MMOs, including sMMO and pMMO, while we provide further evidence to elucidate its mechanism using a purified enhancer-binding protein (MmoR) and transcription factor (σ54). The characterization studies of σ54 and MmoR identified the promoter binding sites and enhancer-binding sequences essential for sMMO expression. Our findings also demonstrate that MmoR functions as a trigger for sMMO expression due to the high specificity and selectivity for enhancer-binding sequences. The UV-visible spectrum of purified MmoR suggested an iron coordination like other GAF domain, and that ATP is essential for the initiation of enhancer elements. Binding assays indicated that these interactions are blocked by the copper ion. These results provide novel insights into gene regulation of methanotrophs. [ABSTRACT FROM AUTHOR]

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effec ts of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids. [ABSTRACT FROM AUTHOR]

ABSTRACT Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess nosZ genes and the ability to respire N2O. However, the selective advantage that may favor these nosZ-harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N2O on Nrf-mediated DNRA was examined in a soil isolate, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C2H2, a NosZ inhibitor, with or without N2O, and the results were compared with C2H2-free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N2O. The NO2 −-to-NH4 + reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C2H2-amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possibility of C2H2 involvement was dismissed since the cultures continuously flushed with C2H2/N2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N2O-containing gas. The findings suggest a possibility that the oft-observed nosZ presence in DNRA-catalyzing microorganisms secures an early transcription of their DNRA genes by scavenging N2O, thus enhancing their capacity to compete with denitrifiers at oxic-anoxic interfaces. IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3 − and/or NO2 − to NH4 +. Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.

ABSTRACT Escherichia coli strains of phylogenetic group B2 are often associated with urinary tract infections (UTIs) and several other diseases. Recent genomic and transcriptomic analyses have not suggested or identified specific genes required for virulence, but have instead suggested multiple virulence strategies and complex host-pathogen interactions. Previous analyses have not compared core gene expression between phylogenetic groups or between pathogens and nonpathogens within phylogenetic groups. We compared the core gene expression of 35 strains from three phylogenetic groups that included both pathogens and nonpathogens after growth in a medium that allowed comparable growth of both types of strains. K-means clustering suggested a B2 cluster with 17 group B2 strains and two group A strains; an AD cluster with six group A strains, five group D strains and one B2 strain; and four outliers which included the highly studied model uropathogenic E. coli strains UTI89 and CFT073. Half of the core genes were differentially expressed between B2 and AD cluster strains, including transcripts of genes for all aspects of macromolecular synthesis—replication, transcription, translation, and peptidoglycan synthesis—energy metabolism, and environmental-sensing transcriptional regulators. Notably, core gene expression between nonpathogenic and uropathogenic transcriptomes within phylogenetic groups did not differ. If differences between pathogens and nonpathogens exist, then the differences do not require transcriptional reprogramming. In summary, B2 cluster strains have a distinct transcription pattern that involves hundreds of genes. We propose that this transcription pattern is one factor that contributes to virulence. IMPORTANCE Escherichia coli is a diverse species and an opportunistic pathogen that is associated with various diseases, such as urinary tract infections. When examined, phylogenetic group B2 strains are more often associated with these diseases, but the specific properties that contribute to their virulence are not known. From a comparative transcriptomic analysis, we found that group B2 strains grown in a nutrient-rich medium had a distinct transcription pattern, which is the first evidence that core gene expression differs between phylogenetic groups. Understanding the consequences of group B2 transcription pattern will provide important information on basic E. coli biology, the basis for E. coli virulence, and possibly for developing therapies for a majority of urinary tract infections and other group B2-associated diseases.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl- CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. [ABSTRACT FROM AUTHOR]

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. [ABSTRACT FROM AUTHOR]

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion. [ABSTRACT FROM AUTHOR]

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (,10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria. [ABSTRACT FROM AUTHOR]

ABSTRACT The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification are unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein, TgBDP1, which is conserved among Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using cleavage under targets and tagmentation, we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodeling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite. IMPORTANCE Histone acetylation is critical for proper regulation of gene expression in the single-celled eukaryotic pathogen Toxoplasma gondii. Bromodomain proteins are “readers” of histone acetylation and may link the modified chromatin to transcription factors. Here, we show that the bromodomain protein TgBDP1 is essential for parasite survival and that loss of TgBDP1 results in global dysregulation of gene expression. TgBDP1 is recruited to the promoter region of a large proportion of parasite genes, forms a core complex with two other bromodomain proteins, and interacts with different transcriptional regulatory complexes. We conclude that TgBDP1 is a key factor for sensing specific histone modifications to influence multiple facets of transcriptional regulation in Toxoplasma gondii.

ABSTRACT The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii-infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing. IMPORTANCE The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite’s ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii.

ABSTRACT Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination. IMPORTANCE Aspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

ABSTRACT Ceragenins, including CSA-13, are cationic antimicrobials that target the bacterial cell envelope differently than colistin. However, the molecular basis of their action is not fully understood. Here, we examined the genomic and transcriptome responses by Enterobacter hormaechei after prolonged exposure to either CSA-13 or colistin. Resistance of the E. hormaechei 4236 strain (sequence type 89 [ST89]) to colistin and CSA-13 was induced in vitro during serial passages with sublethal doses of tested agents. The genomic and metabolic profiles of the tested isolates were characterized using a combination of whole-genome sequencing (WGS) and transcriptome sequencing (RNA-seq), followed by metabolic mapping of differentially expressed genes using Pathway Tools software. The exposure of E. hormaechei to colistin resulted in the deletion of the mgrB gene, whereas CSA-13 disrupted the genes encoding an outer membrane protein C and transcriptional regulator SmvR. Both compounds upregulated several colistin-resistant genes, such as the arnABCDEF operon and pagE, including genes coding for DedA proteins. The latter proteins, along with beta-barrel protein YfaZ and VirK/YbjX family proteins, were the top overexpressed cell envelope proteins. Furthermore, the l-arginine biosynthesis pathway and putrescine-ornithine antiporter PotE were downregulated in both transcriptomes. In contrast, the expression of two pyruvate transporters (YhjX and YjiY) and genes involved in pyruvate metabolism, as well as genes involved in generating proton motive force (PMF), was antimicrobial specific. Despite the similarity of the cell envelope transcriptomes, distinctly remodeled carbon metabolism (i.e., toward fermentation of pyruvate to acetoin [colistin] and to the glyoxylate pathway [CSA-13]) distinguished both antimicrobials, which possibly reflects the intensity of the stress exerted by both agents. IMPORTANCE Colistin and ceragenins, like CSA-13, are cationic antimicrobials that disrupt the bacterial cell envelope through different mechanisms. Here, we examined the genomic and transcriptome changes in Enterobacter hormaechei ST89, an emerging hospital pathogen, after prolonged exposure to these agents to identify potential resistance mechanisms. Interestingly, we observed downregulation of genes associated with acid stress response as well as distinct dysregulation of genes involved in carbon metabolism, resulting in a switch from pyruvate fermentation to acetoin (colistin) and the glyoxylate pathway (CSA-13). Therefore, we hypothesize that repression of the acid stress response, which alkalinizes cytoplasmic pH and, in turn, suppresses resistance to cationic antimicrobials, could be interpreted as an adaptation that prevents alkalinization of cytoplasmic pH in emergencies induced by colistin and CSA-13. Consequently, this alteration critical for cell physiology must be compensated via remodeling carbon and/or amino acid metabolism to limit acidic by-product production.

ABSTRACT The two-component system (TCS) PhoPQ has been demonstrated to be crucial for the formation of resistance to quinolones and cephalosporins in Salmonella Enteritidis (S. Enteritidis). However, the mechanism underlying PhoPQ-mediated antibiotic resistance formation remains poorly understood. Here, it was shown that PhoP transcriptionally regulated an assortment of genes associated with envelope homeostasis, the osmotic stress response, and the redox balance to confer resistance to quinolones and cephalosporins in S. Enteritidis. Specifically, cells lacking the PhoP regulator, under nalidixic acid and ceftazidime stress, bore a severely compromised membrane on the aspects of integrity, fluidity, and permeability, with deficiency to withstand osmolarity stress, an increased accumulation of intracellular reactive oxygen species, and dysregulated redox homeostasis, which are unfavorable for bacterial survival. The phosphorylated PhoP elicited transcriptional alterations of resistance-associated genes, including the outer membrane porin ompF and the aconitate hydratase acnA, by directly binding to their promoters, leading to a limited influx of antibiotics and a well-maintained intracellular metabolism. Importantly, it was demonstrated that the cavity of the PhoQ sensor domain bound to and sensed quinolones/cephalosporins via the crucial surrounding residues, as their mutations abrogated the binding and PhoQ autophosphorylation. This recognition mode promoted signal transduction that activated PhoP, thereby modulating the transcription of downstream genes to accommodate cells to antibiotic stress. These findings have revealed how bacteria employ a specific TCS to sense antibiotics and combat them, suggesting PhoPQ as a potential drug target with which to surmount S. Enteritidis. IMPORTANCE The prevalence of quinolone and cephalosporin-resistant S. Enteritidis is of increasing clinical concern. Thus, it is imperative to identify novel therapeutic targets with which to treat S. Enteritidis-associated infections. The PhoPQ two-component system is conserved across a variety of Gram-negative pathogens, by which bacteria adapt to a range of environmental stimuli. Our earlier work has demonstrated the importance of PhoPQ in the resistance formation in S. Enteritidis to quinolones and cephalosporins. In the current work, we identified a global profile of genes that are regulated by PhoP under antibiotic stresses, with a focus on how PhoP regulated downstream genes, either positively or negatively. Additionally, we established that PhoQ sensed quinolones and cephalosporins in a manner of directly binding to them. These identified genes and pathways that are mediated by PhoPQ represent promising targets for the development of a drug potentiator with which to neutralize antibiotic resistance in S. Enteritidis.

ABSTRACT Autophagy is a conserved degradation and recycling pathway in eukaryotes and is important for their normal growth and development. An appropriate status of autophagy is crucial for organisms which is tightly regulated both temporally and continuously. Transcriptional regulation of autophagy-related genes (ATGs) is an important layer in autophagy regulation. However, the transcriptional regulators and their mechanisms are still unclear, especially in fungal pathogens. Here, we identified Sin3, a component of the histone deacetylase complex, as a transcriptional repressor of ATGs and negative regulator of autophagy induction in the rice fungal pathogen Magnaporthe oryzae. A loss of SIN3 resulted in upregulated expression of ATGs and promoted autophagy with an increased number of autophagosomes under normal growth conditions. Furthermore, we found that Sin3 negatively regulated the transcription of ATG1, ATG13, and ATG17 through direct occupancy and changed levels of histone acetylation. Under nutrient-deficient conditions, the transcription of SIN3 was downregulated, and the reduced occupancy of Sin3 from those ATGs resulted in histone hyperacetylation and activated their transcription and in turn promoted autophagy. Thus, our study uncovers a new mechanism of Sin3 in modulating autophagy through transcriptional regulation. IMPORTANCE Autophagy is an evolutionarily conserved metabolic process and is required for the growth and pathogenicity of phytopathogenic fungi. The transcriptional regulators and precise mechanisms of regulating autophagy, as well as whether the induction or repression of ATGs is associated with autophagy level, are still poorly understood in M. oryzae. In this study, we revealed that Sin3 acts as a transcriptional repressor of ATGs to negatively regulate autophagy level in M. oryzae. Under the nutrient-rich conditions, Sin3 inhibits autophagy with a basal level through directly repressing the transcription of ATG1-ATG13-ATG17. Upon nutrient-deficient treatment, the transcriptional level of SIN3 would decrease and dissociation of Sin3 from those ATGs associates with histone hyperacetylation and activates their transcriptional expression and in turn contributes to autophagy induction. Our findings are important as we uncover a new mechanism of Sin3 for the first time to negatively modulate autophagy at the transcriptional level in M. oryzae.

ABSTRACT Feast-famine response proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the Escherichia coli leucine-responsive regulatory protein (Lrp). Lrp senses the environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium toward the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrp’s regulatory activity and that, contrary to previous speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also show evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation. IMPORTANCE Leucine-responsive regulatory protein (Lrp) is one of the most impactful regulators in E. coli and other bacteria. Lrp senses nutrient conditions and responds by controlling strategies for virulence, cellular motility, and nutrient acquisition. Despite its importance and being evolutionarily highly conserved across bacteria and archaea, several mysteries remain regarding Lrp, including how it actually responds to leucine to change its regulation of targets. Previous studies have led to the hypothesis that Lrp switches between two states, an octamer (8 Lrp molecules together) and a hexadecamer (16 Lrp molecules together), upon exposure to leucine; these are referred to as different oligomerization states. Here, we show that contrary to previous expectations, it is Lrp’s propensity to bind DNA, rather than its oligomerization state, that is directly affected by leucine in the cell’s environment. Our new understanding of Lrp activity will aid in identifying and disrupting pathways used by bacteria to cause disease.

The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. IMPORTANCE The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals. [ABSTRACT FROM AUTHOR]

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/ 106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/ 106 cells; 5.6%) or intact (0.6 copies/106 cells; <1%) HIV RNA. Likewise, the frequency of CD4+ T cells with 39 defective HIV RNA was greater than the frequency with 59 defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 39 defective RNA over 59 defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. [ABSTRACT FROM AUTHOR]

ABSTRACT Malaria transmission to humans begins with sporozoite infection of the liver. The elucidation of gene regulation during the sporozoite stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to the development of strategies for preventing malaria transmission. AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midguts of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes, comprising those involved in distinct processes parasites experience during this stage, from sporogony to development into the liver stage and representing the majority of genes highly expressed in the sporozoite stage. These results suggest that AP2-Sp determines basal patterns of gene expression by targeting a broad range of genes directly. The ChIP-seq analyses also showed that AP2-Sp maintains its own expression by a transcriptional autoactivation mechanism (positive-feedback loop) and induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP. The results showed that AP2-Sp exists at the top of the transcriptional cascade of this stage and triggers the formation of this stage as a master regulator. IMPORTANCE The sporozoite stage plays a central role in malaria transmission from a mosquito to vertebrate host and is an important target for antimalarial strategies. AP2-Sp is a candidate master transcription factor for the sporozoite stage. However, study of its role in gene regulation has been hampered because of difficulties in performing genome-wide studies of gene regulation in this stage. Here, we conquered this problem and revealed that AP2-Sp has the following prominent features as a master transcription factor. First, it determines the repertory of gene expression during this stage. Second, it maintains its own expression through a transcriptional positive-feedback loop and induces all other transcription factors specifically expressed in this stage. This study represents a major breakthrough in fully understanding gene regulation in this important malarial stage.

ABSTRACT Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5′-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5′ end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

ABSTRACT Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.

ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro, iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods.

Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly restricted, but during the productive lytic phase, more than 80 genes are expressed in a temporally coordinated cascade. In this study, we used Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to characterize early viral transcriptional events using HSV-1 immediate early (IE) gene mutants, corresponding genetically repaired viruses, and wild-type virus. Unexpectedly, in the absence of the IE genes ICP4, ICP22, and ICP0 at 1.5 hours postinfection (hpi), we observed high levels of aberrant transcriptional activity across the mutant viral genomes but substantially less on either wild-type or the congenic repaired virus genomes. This feature was particularly prominent in the absence of ICP4 expression. Cycloheximide treatment during infection with both the ICP4 and ICP22 mutants and their respective genetic repairs did not alter the relative distribution of Pol activity, but it increased overall activity across both viral genomes, indicating that both virion components and at least some de novo protein synthesis were required for full repression. Overall, these data reveal that prior to their role in transcriptional activation, IE gene products and virion components first repress transcription and that the HSV-1 lytic transcriptional cascade is mediated through subsequent derepression steps. [ABSTRACT FROM AUTHOR]

Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain (BET) family of proteins. Brd4 regulates human papillomavirus (HPV) transcription, genome replication, and segregation by binding to the E2 protein. The SETD6 methyltransferase binds to and methylates Brd4 at lysine 99. We investigated the interactions of SETD6 and Brd4 with E2 and their role in HPV transcription. SETD6 coimmunoprecipitated with the E2 transactivation domain, and its depletion in CIN612 episomal cells reduced human papillomavirus type 31 (HPV-31) transcription, whereas depletion of SETD6 in integrated HPV cell lines had no effect on viral gene expression. The mutant Brd4 K99R (bearing a change of K to R at position 99), which cannot be methylated by SETD6, displayed decreased binding to HPV-31 E2, suggesting that SETD6 methylation of Brd4 also influences E2 association with the Brd4 protein. Using chromatin immunoprecipitation, SETD6 was detected at the enhancer region of the HPV long control region. We propose that methylation of Brd4 at K99 by SETD6 is an important mechanism for E2-Brd4 association and HPV transcriptional activation. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is a major health burden worldwide, and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral trement. HBV core protein (HBc) has emerged as a promising antiviral target, as it plays important roles in critical steps of the viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains under debate. In this study, different approaches were used to address this question. In synthesized HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen (HBsAg) production in a hepatoma cell line and primary human hepatocytes. Reconstitution of HBc did not alter the expression of cccDNA-derived HBV markers. Similar results were obtained from an in vivo mouse model harboring cccDNA. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays revealed transcription regulation of HBc-deficient cccDNA chromatin similar to that of wild-type cccDNA. Furthermore, treatment with capsid assembly modulators (CAMs) dramatically reduced extracellular HBV DNA but could not alter viral RNA and HBsAg. Our results demonstrate that HBc neither affects histone modifications and transcription factor binding of cccDNA nor directly influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, they do not suppress cccDNA transcriptional activity. Thus, therapeutics targeting capsid or HBc should not be expected to sufficiently reduce cccDNA transcription. [ABSTRACT FROM AUTHOR]

Chronic infection with hepatitis B virus (HBV) remains an important public health challenge. Viral covalently closed circular DNA (cccDNA) persists in infected hepatocytes and serves as the template for transcribing all viral RNA species. HBV regulatory protein X (HBx) interacts with other viral and cellular proteins to play diverse functions in viral life cycle, including modulation of cccDNA transcription activity. Here, we used proximity labeling coupled with proteomic analysis to screen for HBx-interacting host proteins. One of the identified candidates, deubiquitinating enzyme valosin-containing protein-interacting protein 1 (VCPIP1), directly binds HBx and stabilizes HBx by reducing its proteasomal degradation, which corroborated a recent report. VCPIP1-mediated upregulation of HBV transcription, antigen expression, and genome replication was demonstrated using a series of HBV replication and infection models. More importantly, VCPIP1 was found to upregulate HBV in the absence of HBx. Mechanistic studies revealed that VCPIP1 HBx-independently associates with HBV enhancer I/X promoter (EnI/Xp) and positively modulates both its promoter and enhancer activities, partially through promoting the binding of YY1 transcription factor to EnI/Xp. Results presented here expand the recently described role of VCPIP1 in HBV life cycle and establish it as a multi-functional positive regulator of this virus., Importance: Hepatitis B virus (HBV) encodes the regulatory protein HBx that plays crucial roles in viral life cycle and cellular processes through interacting with viral and cellular proteins. Identifying HBx-interacting proteins may reveal novel aspects of host-virus interactions. In this work, proximity labeling coupled with proteomic analysis identified multiple HBx-interacting host factors, and among these, valosin-containing protein-interacting protein 1 (VCPIP1) was confirmed to directly bind HBx and reduce its proteasomal degradation, corroborating a recent report. In addition to upregulating HBx-expressing HBV, we showed that VCPIP1 also positively regulates mutant HBV lacking HBx expression. This novel HBx-independent function of VCPIP1 was shown to involve its association with one viral promoter/enhancer element, which upregulated the latter's promoter and enhancer activities. These results establish VCPIP1 as a positive regulator of HBV that acts through multiple, diverse mechanisms and might also contribute toward revealing novel cellular functions of VCPIP1.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn 7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal att Tn 7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn 7 insertion vectors by the addition of inducible (P cym ) and constitutive (PcL and P rpsM ) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn 7 construct harboring these elements into the att Tn 7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and P rpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated P cym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the P cym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn 7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn 7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology., Competing Interests: The authors declare no conflict of interest.

Antimicrobial resistance is a growing health threat, but standard methods for determining antibiotic susceptibility are slow and can delay optimal treatment, which is especially consequential in severe infections such as bacteremia. Novel approaches for rapid susceptibility profiling have emerged that characterize either bacterial response to antibiotics (phenotype) or detect specific resistance genes (genotype). G en o typic and Ph enotypic AST through R NA detection (GoPhAST-R) is a novel assay, performed directly on positive blood cultures, that integrates rapid transcriptional response profiling with the detection of key resistance gene transcripts, thereby providing simultaneous data on both phenotype and genotype. Here, we performed the first clinical pilot of GoPhAST-R on 42 positive blood cultures: 26 growing Escherichia coli , 15 growing Klebsiella pneumoniae , and 1 with both. An aliquot of each positive blood culture was exposed to nine different antibiotics, lysed, and underwent rapid transcriptional profiling on the NanoString platform; results were analyzed using an in-house susceptibility classification algorithm. GoPhAST-R achieved 95% overall agreement with standard antimicrobial susceptibility testing methods, with the highest agreement for beta-lactams (98%) and the lowest for fluoroquinolones (88%). Epidemic resistance genes including the extended spectrum beta-lactamase bla CTX-M-15 and the carbapenemase bla KPC were also detected within the population. This study demonstrates the clinical feasibility of using transcriptional response profiling for rapid resistance determination, although further validation with larger and more diverse bacterial populations will be essential in future work. GoPhAST-R represents a promising new approach for rapid and comprehensive antibiotic susceptibility testing in clinical settings.IMPORTANCEExposure to antibiotics causes differential transcriptional signatures in susceptible vs resistant bacteria. These differences can be leveraged to rapidly predict resistance profiles of Escherichia coli and Klebsiella pneumoniae in clinically positive blood cultures.

Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa , this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2 , but not lae-1 , leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2 , suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/VE-2 complex is a key element in transcriptional regulation that occurs during sexual development., Importance: Sexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa . VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes., Competing Interests: The authors declare no conflict of interest.

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of C RP- m ediated d ual e nhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in p -coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of C RP- m ediated d ual e nhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies.