Global Gene Regulation Research Articles

P-1087. Novel Antisense Wide-Range Peptide Nucleic Acids Coated on Polystyrene Plates to Prevent Bacterial Biofilm

Abstract Background Biofilms are three dimensional communities of microorganisms that are encased in self-produced extracellular polymeric substances. Biofilms play a key role in device-associated infections, including catheter-associated urinary tract infections, largely because they protect microorganisms from standard antimicrobial therapies. Antisense synthetic peptide nucleic acids (PNAs) are an emerging approach to inhibiting specific components of gene expression. Several regulatory and motility genes are relatively conserved across biofilm forming Gram-negative bacilli, such as rpoS, amrZ, rsmA, and motA. In the present study, wide range PNAs (wrPNAs) were developed by our group to exploit these conserved targets, offering a mechanism for precise, yet broad, biofilm inhibition. The impact of wrPNAs on Gram-negative biofilms and bacterial viability were investigated. Methods wrPNAs: Antisense-wrPNAs (patent pending) were designed using NCBI Blast to select a region inclusive of the start codon. The wrPNAs ranged from 12 to 14 basepairs, conjugated with a cell-penetrating peptide and O-linker, and were synthesized by PNA Bio (Figure 1). Quantify biofilm biomass: Bacterial isolates were grown in a 96-well polystyrene plate in minimal salt medium with wrPNA treatment (10 µM each) and incubated at 30˚C for 24hr in a humid chamber. Biofilms were stained with 0.1% crystal violet, solubilized in 33% glacial acetic acid, and measured at OD590 nm. Quantify bacterial viability: 10-fold serial dilutions and direct stamping onto agar plates. Statistical analysis: P values were determined using unpaired t-tests or one-way ANOVA with followup tests comparing the mean of each column to the control column (alpha = 0.05). Results wrPNA treatment significantly reduced biofilm biomass for their intended species (Figure 2). A cocktail of PNAs had a bactericidal effect on Pseudomonas aeruginosa, but wrPNA treatment had minimal effect on bacterial viability for the other Gram-negative bacilli tested (Figure 2). Conclusion wrPNAs against global regulator genes and a motility regular gene reduced biofilm biomass for their intended species. Future work will look at combination therapy to improve bactericidal effects as well as methods for coating treatments onto catheter and device surfaces. Disclosures All Authors: No reported disclosures

An environmental isolate of Pseudomonas, 20EI1, reduces Aspergillus flavus growth in an iron-dependent manner and alters secondary metabolism.

Aspergillus flavus is an opportunistic pathogenic fungus that infects oilseed crops worldwide. When colonizing plants, it produces mycotoxins, including carcinogenic compounds such as aflatoxins. Mycotoxin contamination results in an important economic and health impact. The design of new strategies to control A. flavus colonization and mycotoxin contamination is paramount. The biocontrol potential of a promising new isolate of Pseudomonas spp., 20EI1 against A. flavus was assessed using bioassays and microscopy. To further elucidate the nature of this bacterial-fungal interaction, we also performed chemical and transcriptomics analyses. In the present study, Pseudomonas spp., 20EI1 was able to reduce the growth of A. flavus. Furthermore, we determined that this growth inhibition is iron-dependent. In addition, Pseudomonas 20EI1 reduced or blocked the production of aflatoxin, as well as cyclopiazonic acid and kojic acid. Expression of iron-related genes was altered in the presence of the bacteria and genes involved in the production of aflatoxin were down-regulated. Iron supplementation partially reestablished their expression. Expression of other secondary metabolite (SM) genes was also reduced by the bacteria, including genes of clusters involved in cyclopiazonic acid, kojic acid and imizoquin biosynthesis, while genes of the cluster corresponding to aspergillicin, a siderophore, were upregulated. Interestingly, the global SM regulatory gene mtfA was significantly upregulated by 20EI1, which could have contributed to the observed alterations in SM. Our results suggest that Pseudomonas 20EI1 is a promising biocontrol against A. flavus, and provide further insight into this iron-dependent bacterial-fungal interaction affecting the expression of numerous genes, among them those involved in SM.

Discovery of 15-deoxynaphthomycins activating the antioxidant NRF2-ARE pathway from Streptomyces sp. N50 via genome mining, global regulator introduction, and molecular networking

Genome mining is a promising avenue for expanding the repertoire of microbial natural products, which are important for drug development. This approach involves predicting genetically encoded small molecules by examining bacterial genomes via accumulated knowledge of microbial biosynthesis. However, it is also important that the microbes produce the predicted molecule in practice. Here, we introduce an endophytic Streptomyces sp. N50, which was isolated from the medicinal plant Selaginella tamariscina. Upon sequencing its entire genome, 33 biosynthetic gene clusters (BGCs) were identified in a chromosome and a megaplasmid. Subsequent genome mining revealed that the new 15-deoxynaphthomycin could be produced due to the presence of an enoyl reductase domain, which is absent in the known BGC of naphthomycin, a type of ansamycin antibiotics. In addition, the engineered strain with the introduction of the global regulatory gene afsR2 into N50 successfully produced 15-deoxynaphthomycins. Furthermore, molecular network analysis via MS/MS selectively confirmed the presence of additional sulfur-containing 15-deoxynaphthomycin congeners. Eventually, six new 15-deoxynaphthomycins were isolated and elucidated from the engineered strain N50. This family of compounds is known to exhibit various biological activities. Also, the presence of quinone moieties in these compounds, which are known to activate NRF2, they were tested for their ability to activate NRF2. Among the new compounds, three (1, 5, and 6) activated the antioxidant NRF2-ARE signaling pathway. Treatment with these compounds significantly elevated NRF2 levels in HepG2 cells and further induced the expression of NRF2 target genes associated with the antioxidant response. This study suggests that the combination of genome mining, gene engineering and molecular networking is helpful for generating new small molecules as pharmaceutical candidates from microorganisms.

Global chromatin reorganization and regulation of genes with specific evolutionary ages during differentiation and cancer.

Cancer cells are highly plastic, allowing them to adapt to changing conditions. Genes related to basic cellular processes evolved in ancient species, while more specialized genes appeared later with multicellularity (metazoan genes) or even after mammals evolved. Transcriptomic analyses have shown that ancient genes are up-regulated in cancer, while metazoan-origin genes are inactivated. Despite the importance of these observations, the underlying mechanisms remain unexplored. Here, we study local and global epigenomic mechanisms that may regulate genes from specific evolutionary periods. Using evolutionary gene age data, we characterize the epigenomic landscape, gene expression regulation, and chromatin organization in three cell types: human embryonic stem cells, normal B-cells, and primary cells from Chronic Lymphocytic Leukemia, a B-cell malignancy. We identify topological changes in chromatin organization during differentiation observing patterns in Polycomb repression and RNA Polymerase II pausing, which are reversed during oncogenesis. Beyond the non-random organization of genes and chromatin features in the 3D epigenome, we suggest that these patterns lead to preferential interactions among ancient, intermediate, and recent genes, mediated by RNA Polymerase II, Polycomb, and the lamina, respectively. Our findings shed light on gene regulation according to evolutionary age and suggest this organization changes across differentiation and oncogenesis.

Metatranscriptomics provide insights into the role of the symbiont Midichloria mitochondrii in Ixodes ticks.

Ticks are important vectors of bacterial, viral, and protozoan pathogens of humans and animals worldwide. Candidatus Midichloria mitochondrii is a highly abundant bacterial endosymbiont found in many tick species, including two medically important ticks respectively found in Europe and Australia, Ixodes ricinus and Ixodes holocyclus. The present study aimed to determine the symbiont's biological role by identifying lateral gene transfer (LGT) events, characterizing the transcriptome, and performing differential expression analyses. Metatranscriptomic data revealed that M. mitochondrii species in I. ricinus and I. holocyclus were equipped with the metabolic potential and were actively transcribing the genes for several important roles including heme, biotin and folate synthesis, oxidative stress response, osmotic regulation, and ATP production in microaerobic conditions. Differential expression analyses additionally showed an upregulation in stringent response and DNA repair genes in M. mitochondrii of I. holocyclus nymphs compared to adults. Low rates of differential expression suggest the symbiont may lack global gene regulation, as observed in other endosymbionts. Moreover, the identification of an LGT event and the proposed specialization of the M. mitochondrii strains, mIxholo1 and mIxholo2, for different I. holocyclus life stages highlight the complex interactions between M. mitochondrii and their tick hosts.

Whole genome regulatory effect of MoISW2 and consequences for the evolution of the rice plant pathogenic fungus Magnaporthe oryzae.

Isw2 proteins, ubiquitous across eukaryotes, exhibit a propensity for DNA binding and exert dynamic influences on local chromosome condensation in an ATP-dependent fashion, thereby modulating the accessibility of neighboring genes to transcriptional machinery. Here, we report the deletion of a putative MoISW2 gene, yielding substantial ramifications on plant pathogenicity. Subsequent gene complementation and chromatin immunoprecipitation sequencing (ChIP-seq) analyses were conducted to delineate binding sites. RNA sequencing (RNA-seq) assays revealed discernible impacts on global gene regulation along chromosomes in both mutant and wild-type strains, with comparative analyses against 55 external RNA-seq data sets corroborating these findings. Notably, MoIsw2-mediated binding and activities delineate genomic loci characterized by pronounced gene expression variability proximal to MoIsw2 binding sites, juxtaposed with comparatively stable expression in surrounding regions. The contingent genes influenced by MoIsw2 activity predominantly encompass niche-determinant genes, including those encoding secreted proteins, secondary metabolites, and stress-responsive elements, alongside avirulence genes. Furthermore, our investigations unveil a spatial correlation between MoIsw2 binding motifs and known transposable elements (TEs), suggesting a potential interplay wherein TE transposition at these loci could modulate the transcriptional landscape of Magnaporthe oryzae in a strain-specific manner. Collectively, these findings position MoIsw2 as a plausible master regulator orchestrating the delicate equilibrium between genes vital for biomass proliferation, akin to housekeeping genes, and niche-specific determinants crucial for ecological adaptability. Stress-induced TE transposition, in conjunction with MoIsw2 activity, emerges as a putative mechanism fostering enhanced mutagenesis and accelerated evolution of niche-determinant genes relative to housekeeping counterparts.IMPORTANCEIsw2 proteins are conserved in plants, fungi, animals, and other eukaryotes. We show that a fungal Isw2 protein in the rice pathogen Magnaporthe oryzae binds to retrotransposon (RT) DNA motifs and affects the epigenetic gene expression landscape of the fungal genome. Mainly ecological niche determinant genes close to the binding motifs are affected. RT elements occur frequently in DNA between genes in most organisms. They move place and multiply in the genome, especially under physiological stress. We further discuss the Isw2 and RT combined activities as a possible sought-after mechanism that can cause biased mutation rates and faster evolution of genes necessary for reacting to abiotic and biotic challenges. The most important biotic challenges for plant pathogens are the ones from the host plants' innate immunity. The overall result of these combined activities will be an adaptation-directed evolution of niche-determinant genes.

Host and antibiotic jointly select for greater virulence in Staphylococcus aureus.

Widespread antibiotic usage has resulted in the rapid evolution of drug-resistant bacterial pathogens and poses significant threats to public health. Resolving how pathogens respond to antibiotics under different contexts is critical for understanding disease emergence and evolution going forward. The impact of antibiotics has been demonstrated most directly through in vitro pathogen passaging experiments. Independent from antibiotic selection, interactions with hosts have also altered the evolutionary trajectories and fitness landscapes of pathogens, shaping infectious disease outcomes. However, it is unclear how interactions between hosts and antibiotics impact the evolution of pathogen virulence. Here, we evolved and re-sequenced Staphylococcus aureus , a major bacterial pathogen, varying exposure to host and antibiotics to tease apart the contributions of these selective pressures on pathogen adaptation. After 12 passages, S. aureus evolving in Caenorhabditis elegans nematodes exposed to a sub-minimum inhibitory concentration of antibiotic (oxacillin) became highly virulent, regardless of whether the ancestral pathogen was methicillin-resistant (MRSA) or methicillin-sensitive (MSSA). Host and antibiotic exposure selected for reduced drug susceptibility in MSSA lineages while increasing MRSA total growth outside hosts. We identified mutations in genes involved in complex regulatory networks linking virulence and metabolism, including codY , agr , and gdpP , suggesting that rapid adaptation to infect hosts may have pleiotropic effects. In particular, MSSA populations under selection from host and antibiotic accumulated mutations in the global regulator gene codY , which controls biofilm formation in S. aureus. These populations had indeed evolved more robust biofilms-a trait linked to both virulence and antibiotic resistance-suggesting evolution of one trait can confer multiple adaptive benefits. Despite evolving in similar environments, MRSA and MSSA populations proceeded on divergent evolutionary paths, with MSSA populations exhibiting more similarities across replicate populations. Our results underscore the importance of considering multiple and concurrent selective pressures as drivers of pervasive pathogen traits.

Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Ketoprofen promotes the conjugative transfer of antibiotic resistance among antibiotic resistant bacteria in natural aqueous environments

The emergence and spread of antibiotic resistance in the environment pose a serious threat to global public health. It is acknowledged that non-antibiotic stresses, including disinfectants, pharmaceuticals and organic pollutants, play a crucial role in horizontal transmission of antibiotic resistance genes (ARGs). Despite the widespread presence of non-steroidal anti-inflammatory drugs (NSAIDs), notably in surface water, their contributions to the transfer of ARGs have not been systematically explored. Furthermore, previous studies have primarily concentrated on model strains to investigate whether contaminants promote the conjugative transfer of ARGs, leaving the mechanisms of ARG transmission among antibiotic resistant bacteria in natural aqueous environments under the selective pressures of non-antibiotic contaminants remains unclear. In this study, the Escherichia coli (E. coli) K12 carrying RP4 plasmid was used as the donor strain, indigenous strain Aeromonas veronii containing rifampicin resistance genes in Taihu Lake, and E. coli HB101 were used as receptor strains to establish inter-genus and intra-genus conjugative transfer systems, examining the conjugative transfer frequency under the stress of ketoprofen. The results indicated that ketoprofen accelerated the environmental spread of ARGs through several mechanisms. Ketoprofen promoted cell-to-cell contact by increasing cell surface hydrophobicity and reducing cell surface charge, thereby mitigating cell-to-cell repulsion. Furthermore, ketoprofen induced increased levels of reactive oxygen species (ROS) production, activated the DNA damage-induced response (SOS), and enhanced cell membrane permeability, facilitating ARG transmission in intra-genus and inter-genus systems. The upregulation of outer membrane proteins, oxidative stress, SOS response, mating pair formation (Mpf) system, and DNA transfer and replication (Dtr) system related genes, as well as the inhibition of global regulatory genes, all contributed to higher transfer efficiency under ketoprofen treatment. These findings served as an early warning for a comprehensive assessment of the roles of NSAIDs in the spread of antibiotic resistance in natural aqueous environments.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Single-cell level LasR-mediated quorum sensing response of Pseudomonas aeruginosa to pulses of signal molecules

Quorum sensing (QS) is a communication form between bacteria via small signal molecules that enables global gene regulation as a function of cell density. We applied a microfluidic mother machine to study the kinetics of the QS response of Pseudomonas aeruginosa bacteria to additions and withdrawals of signal molecules. We traced the fast buildup and the subsequent considerably slower decay of a population-level and single-cell-level QS response. We applied a mathematical model to explain the results quantitatively. We found significant heterogeneity in QS on the single-cell level, which may result from variations in quorum-controlled gene expression and protein degradation. Heterogeneity correlates with cell lineage history, too. We used single-cell data to define and quantitatively characterize the population-level quorum state. We found that the population-level QS response is well-defined. The buildup of the quorum is fast upon signal molecule addition. At the same time, its decay is much slower following signal withdrawal, and the quorum may be maintained for several hours in the absence of the signal. Furthermore, the quorum sensing response of the population was largely repeatable in subsequent pulses of signal molecules.

Unveiling the Role of SlRNC1 in Chloroplast Development and Global Gene Regulation in Tomato Plants.

RNC1, a plant-specific gene, is known for its involvement in splicing group II introns within maize chloroplast. However, its role in chloroplast development and global gene expression remains poorly understood. This study aimed to investigate the role of RNC1 in chloroplast development and identify the genes that mediate its function in the development of entire tomato plants. Consistent with findings in maize, RNC1 silencing induced dwarfism and leaf whitening in tomato plants. Subcellular localization analysis revealed that the RNC1 protein is localized to both the nucleus and cytoplasm, including the stress granule and chloroplasts. Electron microscopic examination of tomato leaf transverse sections exposed significant disruptions in the spatial arrangement of the thylakoid network upon RNC1 silencing, crucial for efficient light energy capture and conversion into chemical energy. Transcriptome analysis suggested that RNC1 silencing potentially impacts tomato plant development through genes associated with all three categories (biological processes, cellular components, and molecular functions). Overall, our findings contribute to a better understanding of the critical role of RNC1 in chloroplast development and its significance in plant physiology.

PfHDAC1 is an essential regulator of P. falciparum asexual proliferation and host cell invasion genes with a dynamic genomic occupancy responsive to artemisinin stress.

Malaria is a major public health problem, with the parasite Plasmodium falciparum causing most of the malaria-associated mortality. It is spread by the bite of infected mosquitoes and results in symptoms such as cyclic fever, chills, and headache. However, if left untreated, it can quickly progress to a more severe and life-threatening form. The World Health Organization currently recommends the use of artemisinin combination therapy, and it has worked as a gold standard for many years. Unfortunately, certain countries in southeast Asia and Africa, burdened with a high prevalence of malaria, have reported cases of drug-resistant infections. One of the major problems in controlling malaria is the emergence of artemisinin resistance. Population genomic studies have identified mutations in the Kelch13 gene as a molecular marker for artemisinin resistance. However, several reports thereafter indicated that Kelch13 is not the main mediator but rather hinted at transcriptional deregulation as a major determinant of drug resistance. Earlier, we identified PfGCN5 as a global regulator of stress-responsive genes, which are known to play a central role in artemisinin resistance generation. In this study, we have identified PfHDAC1, a histone deacetylase as a cell cycle regulator, playing an important role in artemisinin resistance generation. Taken together, our study identified key transcriptional regulators that play an important role in artemisinin resistance generation.

Ubiquitous nanocolloids suppress the conjugative transfer of plasmid-mediated antibiotic resistance in aqueous environment

Nanocolloids (Nc) are widespread in natural water environment, whereas the potential effects of Nc on dissemination of antibiotic resistance remain largely unknown. In this study, Nc collected from the Yellow River in Henan province was tested for its ability to influence the conjugative transfer of resistant plasmid in aqueous environment. The results revealed that the conjugative transfer of RP4 plasmid between Escherichia coli was down-regulated by 52%–91% upon exposure to 1–10 mg/L Nc and the reduction became constant when the dose became higher (20−200 mg/L). Despite the exposure of Nc activated the anti-oxidation and SOS response in bacteria through up-regulating genes involved in glutathione biosynthesis and DNA recombination, the inhibition on the synthesis and secretion of extracellular polysaccharide induced the prevention of cell-cell contact, leading to the reduction of plasmid transfer. This was evidenced by the decreased bacterial adhesion and lowered levels of genes and metabolites relevant to transmembrane transport and D-glucose phosphorylation, as clarified in phenotypic, transcriptomics and metabolomics analysis of E. coli. The significant down-regulation of glycolysis/gluconeogenesis and TCA cycle was associated with the shortage of ATP induced by Nc. The up-regulation of global regulatory genes (korA and trbA) and the reduction of plasmid genes (trfAp, trbBp, and traG) expression also contributed to the suppressed conjugation of RP4 plasmid. The obtained findings remind that the role of ubiquitous colloidal particles is nonnegligible when practically and comprehensively assessing the risk of antibiotic resistance in the environment.

Improving Surfactin Production in Bacillus subtilis 168 by Metabolic Engineering.

Surfactin is widely used in the petroleum extraction, cosmetics, biopharmaceuticals and agriculture industries. It possesses antibacterial and antiviral activities and can reduce interfacial tension. Bacillus are commonly used as production chassis, but wild-type Bacillus subtilis 168 cannot synthesise surfactin. In this study, the phosphopantetheinyl transferase (PPTase) gene sfp* (with a T base removed) was overexpressed and enzyme activity was restored, enabling B. subtilis 168 to synthesise surfactin with a yield of 747.5 ± 6.5 mg/L. Knocking out ppsD and yvkC did not enhance surfactin synthesis. Overexpression of predicted surfactin transporter gene yfiS increased its titre to 1060.7 ± 89.4 mg/L, while overexpression of yerP, ycxA and ycxA-efp had little or negative effects on surfactin synthesis, suggesting YfiS is involved in surfactin efflux. By replacing the native promoter of the srfA operon encoding surfactin synthase with three promoters, surfactin synthesis was significantly reduced. However, knockout of the global transcriptional regulator gene codY enhanced the surfactin titre to 1601.8 ± 91.9 mg/L. The highest surfactin titre reached 3.89 ± 0.07 g/L, with the yield of 0.63 ± 0.02 g/g DCW, after 36 h of fed-batch fermentation in 5 L fermenter. This study provides a reference for further understanding surfactin synthesis and constructing microbial cell factories.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Rok from B. subtilis: Bridging genome structure and transcription regulation.

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.

The contribution of BvgR, RisA, and RisS to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation in Bordetella bronchiseptica.

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation.

Genomic and Comparative Transcriptomic Analyses Reveal Key Genes Associated with the Biosynthesis Regulation of Okaramine B in Penicillium daleae NBP-49626.

Restricted production of fungal secondary metabolites hinders the ability to conduct comprehensive research and development of novel biopesticides. Okaramine B from Penicillium demonstrates remarkable insecticidal efficacy; however, its biosynthetic yield is low, and its regulatory mechanism remains unknown. The present study found that the yield difference was influenced by fermentation modes in okaramine-producing strains and performed genomic and comparative transcriptome analysis of P. daleae strain NBP-49626, which exhibits significant features. The NBP-49626 genome is 37.4 Mb, and it encodes 10,131 protein-encoding genes. Up to 5097 differentially expressed genes (DEGs) were identified during the submerged and semi-solid fermentation processes. The oka gene cluster, lacking regulatory and transport genes, displayed distinct transcriptional patterns in response to the fermentation modes and yield of Okaramine B. Although transcription trends of most known global regulatory genes are inconsistent with those of oka, this study identified five potential regulatory genes, including two novel Zn(II)2Cys6 transcription factors, Reg2 and Reg19. A significant correlation was also observed between tryptophan metabolism and Okaramine B yields. In addition, several transporter genes were identified as DEGs. These results were confirmed using real-time quantitative PCR. This study provides comprehensive information regarding the regulatory mechanism of Okaramine B biosynthesis in Penicillium and is critical to the further yield improvement for the development of insecticides.

Epigenetic Dissection of Human Blood Group Genes Reveals Unknown Regulatory Elements and Detailed Characteristics of KEL and Other Loci

Accurate blood group typing is essential for compatible blood transfusions and can often be achieved with the combination of serology and genotyping technologies. Currently, most genotyping platforms focus on variants in coding regions. In addition, variants in the non-coding region that can disrupt regulatory motifs in the promoter or enhancer regions are also important for blood group typing, as demonstrated in ABO, FY and XG systems for example. These blood group regulatory regions have yet to be fully characterized for all genes. Recently, we systematically explored the GATA1-dependent blood group regulome in silico and found 193 candidate regulators in 33 blood group genes. As proof of concept, we resolved in vitro the GATA1-dependent mechanism underlying the Helgeson phenotype of the Knops system, where there is very low expression of Complement Receptor 1 (CR1) on red blood cells. Aiming to improve the precision of our global blood group gene regulator prediction, we expanded the analysis to include preexisting epigenetic datasets such as ChIPseq with erythroid transcription factors (TFs; GATA1, KLF1, RUNX1, NFE2), histone modifications (H3K27ac, H3K4me1, H3K4me3), and ATAC-seq. In total, 96 candidate regions were identified where at least 2 TFs were bound. Four genes ( CR1, EMP3, ABCB6 and ABCC4) showed co-localization of ChIPseq peaks for all 4 TFs within their introns 37, 4, 1 and 1, respectively. Functional analysis with luciferase assay of these potential enhancers with their respective promoters showed different levels of enhancement. Among the more well-known and clinically important blood groups, the KEL proximal promoter was shown to co-localize GATA1 and KLF1. Previous studies suggested the presence of 3 GATA1-binding sites and an Sp1 site, but KLF1 was novel to our knowledge. Moreover, decreased KEL expression has been reported in a KLF1-null human and in mouse knockouts. Candidate GATA1 sites near the KLF1 motif were observed, with the GATA1 motif closest to the KLF1 motif having the highest motif score in JASPAR (939 vs. 913 for the 5‘ and 851 for the 3‘ GATA1 motifs using GATA1 logotype MA0035.3). In light of this, and the importance of KEL for transfusion and fetomaternal incompatibility, we carefully examined the KEL promoter region. A decrease in JASPAR score was used to select two natural variants that disrupt TF binding at the GATA1 and KLF1 motifs compared to the wild-type (wt): rs1204402835:A>G for GATA1 (939>815); rs1442994069:G>A for KLF1 (924>801, KLF1 logotype: MA0493.2). We showed binding of KLF1 and GATA1 to the −35 to +1 bp region (NM_000420.3) with electrophoretic mobility shift assay (EMSA) and mass spectrometry (MS) using synthetic probes and nuclear extract from HEL cells. Disruption of the KLF1 and/or GATA1-binding motifs with the aforementioned natural variants led to the loss of bands which, compared to the wt, represented nuclear extract binding to the probes in the EMSAs. The decreased abundance of bound TFs was confirmed by MS (−2.38 for KLF1 and −1.76 for GATA1, log 2-fold changes compared to wt). Luciferase assays of the KEL promoter exhibited the highest activity (set to 100%) with wt GATA1 and KLF1 motifs and omitting Sp1 and 3'-side GATA1 motifs (Figure 1). The whole region including Sp1 and 3' GATA1 sites gave 44% activity whilst the 3' GATA1 alone or with Sp1 site was <10% (similar to vector control). The promoter construct containing the 5' GATA1 site and the central GATA1 and KLF1 sites showed the highest activity, but reduced activity was observed with disrupted GATA1 motif at 44% and KLF1 motif at 48%. Activity decreased to 17% when both variants were present (Figure 1). In summary, we used a combined bioinformatics and experimental approach to unravel regions with potential to regulate blood group expression and explain weak phenotypes that may escape detection in clinical practice. Our studies are limited by the number of available datasets for relevant TFs but were able to detect regions where these TFs co-localize in CR1, EMP3, ABCB6 and ABCC4. Furthermore, we characterized the clinically important KEL blood group gene promoter in greater detail indicating that the central GATA1 motif is the most crucial. For the first time, we described that KLF1 and GATA1 jointly enhance KEL transcription. Finally, our updated pipeline provides multiple candidate regulatory regions to be explored to improve knowledge on the blood group regulome.