Search

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.

This review constitutes a summary of current knowledge on GlnR, a global regulator, that assumes a critical function in the regulation of nitrogen metabolism of Actinomycetes. In cross-regulation with other regulators, GlnR was also shown to play a role in the regulation of carbon and phosphate metabolisms as well as of secondary metabolism. A description of the structure of the GlnR protein and of its binding sites in various genes promoters regions is also provided. This review thus provides a global understanding of the critical function played by GlnR in the regulation of primary and secondary metabolism in Actinomycetes.

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.

ABSTRACT DNA methylation is an important factor in the regulation of gene expression. In analyzing genomic data of Stereum hirsutum FP-91666, we found a hypothetical bifunctional transcription regulator/O6Meguanine-DNA methyltransferase (named SaraC), which is widely present in both bacteria and fungi, and confirmed that its function in bacteria is mainly for DNA reparation. In this paper, we confirmed that SaraC has the function of DNA binding and demethylation through surface plasma resonance and reaction experiments in vitro. Then, we achieved the overexpression of SaraC (OES) in S. hirsutum, sequenced the methylation and transcription levels of the whole-genome, and further conducted untargeted metabolomics analyses of the OES transformants and the wild type (WT). The results confirmed that the overall-methylation levels of the transformants were significantly downregulated, and various genes related to secondary metabolism were upregulated. Through comparative untargeted metabolomic analyses, it showed that OES SA6 transformant produced a greater number of hybrid polyketides, and we identified 2 novel hybrid polyketides from the fermentation products of SA6. Our results show that overexpression SaraC can effectively stimulate the expression of secondary-metabolism-related genes, which could be a broad-spectrum tool for discovery of metabolites due to its cross-species conservation. IMPORTANCE Fungi are one of the important sources of active compounds. However, in fungi, most of the secondary metabolic biosynthetic gene clusters are weakly expressed or silenced under conventional culture conditions. How to efficiently excavate potential new compounds contained in fungi is becoming a research hot spot in the world. In this study, we found a DNA demethylation protein (SaraC) and confirmed that it is a global secondary metabolism regulator in Stereum hirsutum FP-91666. In the past, SaraC-like proteins were mainly regarded as DNA repair proteins, but our findings proved that it will be a powerful tool for mining secondary metabolites for overexpression of SaraC, which can effectively stimulate the expression of genes related to secondary metabolism.

Six angucyclines including three unreported compounds (1–3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 μM.

Butenyl-spinosyn, a highly effective biological insecticide, is produced by Saccharopolyspora pogona. However, its application has been severely hampered by its low yield. Recent studies have shown that PhoU plays a pivotal role in regulating cell growth, secondary metabolite biosynthesis and intracellular phosphate levels. Nevertheless, the function of PhoU remains ambiguous in S. pogona. In this study, we investigated the effects of PhoU on the growth and the butenyl-spinosyn biosynthesis of S. pogona by constructing the mutants. Overexpression of phoU increased the production of butenyl-spinosyn to 2.2-fold that of the wild-type strain. However, the phoU deletion resulted in a severe imbalance of intracellular phosphate levels, and suppression of the growth and butenyl-spinosyn biosynthesis. Quantitative Real-time PCR (qRT-PCR) analysis, distinctive protein detection and mass spectrometry revealed that PhoU widely regulated primary metabolism, energy metabolism and DNA repair, which implied that PhoU influences the growth and butenyl-spinosyn biosynthesis of S. pogona as a global regulator.

ABSTRACT Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational ihfA-mVenus fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of ihf produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the lac operon, and show that a knockout of the lacI repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions. IMPORTANCE Persisters are phenotypic variants that survive exposure to antibiotics through temporary dormancy. Mutants with increased levels of persisters have been identified in clinical isolates, and evidence suggests these cells contribute to chronic infections and antibiotic treatment failure. Understanding the underlying mechanism of persister formation and tolerance is important for developing therapeutic approaches to treat chronic infections. In this study, we examine a global regulator, IHF, that plays a role in persister formation. We find that noise in expression of IHF contributes to persister formation, likely by regulating the switch between the TCA cycle that efficiently produces energy and the glyoxylate bypass. We extend this study to a simple model lac operon and show that when grown on lactose as the sole carbon source, noise in its expression influences ATP levels and determines persister formation. This noise is quenched by overexpression of the lac operon, providing a simple approach to test the involvement of a gene in persister formation.

Abstract Background Bacillus subtilis 916 has been identified as an effective biocontrol agent against Rhizoctonia solani, the causal pathogen of rice sheath blight, under greenhouse and field conditions. HPLC analysis showed that surfactin, a member of the lipopeptide family produced by B. subtilis, was the major antimicrobial substance. Results Previously, we obtained a mutant strain of B. subtilis 916, Bs-H74, which produced significantly more surfactin than the wild type and presented 10% stronger inhibitory activity against R. solani. To explore the molecular mechanism underlying the higher surfactin productivity in the mutant, high-throughput proteomic analysis was carried out to analyze the differential protein expression. Our results showed that several differentially expressed proteins are involved in OppA, DegU and Carbon Catabolite Repression (CCR) regulatory pathways, which could be positively or negatively associated with surfactin biosynthesis. At both transcriptional and translational levels, we suggested that OppA may play a key role in surfactin synthesis regulation. Based on the above findings, we proposed the hypothesis that a point mutation in the oppA gene may lead to changes in oligopeptides acquisition in B. subtilis, and then the changed oligopeptides may activate or suppress the global regulatory protein, CcpA in the CCR pathway, and ComA and DegU may indirectly regulate surfactin synthesis in Bs-H74. To further explore the regulatory mechanisms in Bs-H74, metabolomics analysis was performed in this study. Interestingly, only 16 metabolites showed changes in abundance in Bs-H74 compared to Bs-916. Neohesperidin, a type of natural flavanone glycosides from citrus with a range of biological activities, increased by 18 times over the wild type Bs-916. This result implied exciting findings in regulatory mechanisms by OppA protein. Conclusions In summary, this study has revealed the mechanisms underlying the improved antagonistic property with increased surfactin production in Bs-H74 at the gene, protein and metabolic levels, which may help to comprehend the map of the regulatory networks in B. subtilis. Findings from our work have provided a solid physical and theoretical basis for practically applying metabolic and genetic engineering to achieve improved and high-yielding biocontrol strains.

Streptomyces can produce a wealth of pharmaceutically valuable antibiotics and other bioactive compounds. Production of most antibiotics is generally low due to the rigorously controlled regulatory networks, in which global/pleiotropic and cluster-situated regulatory proteins coordinate with various intra- and extracellular signals. Thus, mining new antibiotic regulatory proteins, particularly the ones that are widespread, is essential for understanding the regulation of antibiotic biosynthesis. Here, in the biopesticide milbemycin producing strain Streptomyces bingchenggensis, a novel global/pleiotropic regulatory protein, SspH, a single domain protein containing only the HATPase domain, was identified as being involved in controlling antibiotic biosynthesis. The sspH overexpression inhibited milbemycin production by repressing the expression of milbemycin biosynthetic genes. The sspH overexpression also differentially influenced the expression of various antibiotic biosynthetic core genes. Site-directed mutagenesis revealed that the HATPase domain was essential for SspH’s function, and mutation of the conserved amino acid residues N54A and D84A led to the loss of SspH function. Moreover, cross-overexpression experiments showed that SspH and its orthologs, SCO1241 from Streptomyces coelicolor and SAVERM_07097 from Streptomyces avermitilis, shared identical functionality, and all exerted a positive effect on actinorhodin production but a negative effect on avermectin production, indicating that SspH-mediated differential control of antibiotic biosynthesis may be widespread in Streptomyces. This study extended our understanding of the regulatory network of antibiotic biosynthesis and provided effective targets for future antibiotic discovery and overproduction.

Transcription regulation caused by global regulators exerts important effects on fungal secondary metabolism. By overexpression of the global regulator Talae1 in a Ficus elastica-associated fungus Trichoderma afroharzianum, two structurally new polyketides (1 and 2) that were newly produced in the transformant were isolated and identified. Their structures, including the absolute configurations, were elucidated through a combination of high resolution mass spectrometer (HRMS), NMR, and electronic circular dichroism (ECD) calculations. The growth inhibitory activities of compounds 1 and 2 were evaluated against four bacteria and six plant-pathogenic fungi. Compound 1 showed the highest antifungal activity against Botrytis cinerea and Fusarium oxysporum f. sp. nicotianae with MIC of 8 μg/ml. To the best of our knowledge, this is the first study to report on the application of the global regulator in T. afroharzianum to activate the biosynthesis of bioactive secondary metabolites.

Integrons are considered hot spots for bacterial evolution, since these platforms allow one-step genomic innovation by capturing and expressing genes that provide advantageous novelties, such as antibiotic resistance. The acquisition and shuffling of gene cassettes featured by integrons enable the population to rapidly respond to changing selective pressures. However, in order to avoid deleterious effects and fitness burden, the integron activity must be tightly controlled, which happens in an elegant and elaborate fashion, as discussed in detail in the present review. Here, we aimed to provide an up-to-date overview of the complex regulatory networks that permeate the expression and functionality of integrons at both transcriptional and translational levels. It was possible to compile strong shreds of evidence clearly proving that these versatile platforms include functions other than acquiring and expressing gene cassettes. The well-balanced mechanism of integron expression is intricately related with environmental signals, host cell physiology, fitness, and survival, ultimately leading to adaptation on the demand.

The extensive use of acids in a variety of manufacturing industries results in the increase of discharged acidic waste stream into the environment. Such co-pollution of acids and other organic pollutants limits the biodegradation capability of neutrophilic degraders. With high-throughput genetic techniques, we aim to improve the acid tolerance of a pollutant-degrading bacterium, Pseudomonas putida S16 by genetically engineering it with the glutamate decarboxylase (GAD)-dependent system and the global regulator (IrrE) of extreme radiation resistance. The engineered strains holding either GAD system or irrE regulator could grow under pH 4.5, compared to the wild type. They could also degrade over 90% of a selected pollutant (benzoate or nicotine) under pH 5.0 in 48 h, while no biodegradation was detected with the wild type under the same conditions. We conclude that acid stress tolerance by the possession of the GAD system or IrrE regulator in pollutant-degrading bacteria would be a promising approach to enhance their viability and biodegrading activities in bioremediation of acidic wastes.

Overexpression of the global regulator LaeA in a marine-derived fungal strain of Penicillium dipodomyis YJ-11 induced obvious morphological changes and metabolic variations. Further chemical investigation of the mutant strain afforded a series of sorbicillinoids including two new ones named 10,11-dihydrobislongiquinolide (1) and 10,11,16,17-tetrahydrobislongiquinolide (2), as well as four known analogues, bislongiquinolide (3), 16,17-dihydrobislongiquinolide (4), sohirnone A (5), and 2′,3′-dihydrosorbicillin (6). The results support that the global regulator LaeA is a useful tool in activating silent gene clusters in Penicillium strains to obtain previously undiscovered compounds.