ResearchPad - mutant-strains https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Substantial improvement of tetraene macrolide production in <i>Streptomyces diastatochromogenes</i> by cumulative drug resistance mutations]]> https://www.researchpad.co/article/elastic_article_7861 Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.

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<![CDATA[Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system]]> https://www.researchpad.co/article/5c50c486d5eed0c4845e8885

Many bacterial pathogens and symbionts use type III secretion machines to interact with their hosts by injecting bacterial effector proteins into host target cells. A central component of this complex machine is the cytoplasmic sorting platform, which orchestrates the engagement and preparation of type III secreted proteins for their delivery to the needle complex, the substructure of the type III secretion system that mediates their passage through the bacterial envelope. The sorting platform is thought to be a dynamic structure whose components alternate between assembled and disassembled states. However, how this dynamic behavior is controlled is not understood. In S. Typhimurium a core component of the sorting platform is SpaO, which is synthesized in two tandemly translated products, a full length (SpaOL) and a short form (SpaOS) composed of the C-terminal 101 amino acids. Here we show that in the absence of SpaOS the assembly of the needle substructure of the needle complex, which requires a functional sorting platform, can still occur although with reduced efficiency. Consistent with this observation, in the absence of SpaOS secretion of effectors proteins, which requires a fully assembled injectisome, is only slightly compromised. In the absence of SpaOS we detect a significant number of fully assembled needle complexes that are not associated with fully assembled sorting platforms. We also find that although binding of SpaOL to SpaOS can be detected in the absence of other components of the sorting platform, this interaction is not detected in the context of a fully assembled sorting platform suggesting that SpaOS may not be a core structural component of the sorting platform. Consistent with this observation we find that SpaOS and OrgB, a component of the sorting platform, share the same binding surface on SpaOL. We conclude that SpaOS regulates the assembly of the sorting platform during type III secretion.

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<![CDATA[PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c5df336d5eed0c484580f0d

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.

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<![CDATA[CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon]]> https://www.researchpad.co/article/5c3d00f6d5eed0c484037189

Intercellular communication and self-recognition are critical for coordinating cooperative and competitive behaviors during sociomicrobiological community development. Contact-dependent growth inhibition (CDI) proteins are polymorphic toxin delivery systems that inhibit the growth of non-self neighboring bacteria that lack the appropriate immunity protein. In Burkholderia thailandensis, CDI system proteins (encoded by bcpAIOB genes) also induce cooperative behaviors among sibling (self) cells, a phenomenon called contact-dependent signaling (CDS). Here we describe a mobile genetic element (MGE) that carries the bcpAIOB genes in B. thailandensis E264. It is a ~210 kb composite transposon with insertion sequence (IS) elements at each end. Although the ISs are most similar to IS2 of Escherichia coli, the transposase-dependent intermediate molecule displays characteristics more similar to those of the IS26 translocatable unit (TU). A reaction requiring only the “left” IS-encoded transposase results in formation of an extrachromosomal circular dsDNA intermediate (“the megacircle”) composed of the left IS and the sequences intervening between the ISs. Insertion of the megacircle into the chromosome occurs next to a pre-existing copy of an IS2-like element, recreating a functional composite transposon. We found that BcpA activity is required for megacircle formation, and in turn, megacircle formation is required for CDS phenotypes. Our data support a model in which the bcpAIOB genes function as both helping and harming greenbeard genes, simultaneously enhancing the fitness of self bacteria that possess the same allele plus tightly linked genes that mediate cooperative behaviors, and killing non-self bacteria that do not possess the same bcpAIOB allele. Mobility of the megacircle between cells could allow bacteria invading a community to be converted to self, and would facilitate propagation of the bcpAIOB genes in the event that the invading strain is capable of overtaking the resident community.

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<![CDATA[Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027]]> https://www.researchpad.co/article/5c5b52e7d5eed0c4842bd237

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.

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<![CDATA[Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator]]> https://www.researchpad.co/article/5c521834d5eed0c484797785

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

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<![CDATA[Sugar and iron: Toward understanding the antibacterial effect of ciclopirox in Escherichia coli]]> https://www.researchpad.co/article/5c42438cd5eed0c4845e0573

New antibiotics are needed against antibiotic-resistant gram-negative bacteria. The repurposed antifungal drug, ciclopirox, equally blocks antibiotic-susceptible or multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates, indicating that it is not affected by existing resistance mechanisms. Toward understanding how ciclopirox blocks growth, we screened E. coli mutant strains and found that disruption of genes encoding products involved in galactose salvage, enterobacterial common antigen synthesis, and transport of the iron binding siderophore, enterobactin, lowered the minimum inhibitory concentration of ciclopirox needed to block growth of the mutant compared to the isogenic parent strain. We found that ciclopirox induced enterobactin production and that this effect is strongly affected by the deletion of the galactose salvage genes encoding UDP-galactose 4-epimerase, galE, or galactose-1-phosphate uridylyltransferase, galT. As disruption of ECA synthesis activates the regulation of capsular synthesis (Rcs) phosphorelay, which inhibits bacterial swarming and promotes biofilm development, we test whether ciclopirox prevents activation of the Rcs pathway. Sub-inhibitory concentrations of ciclopirox increased swarming of the E. coli laboratory K12 strain BW25113 but had widely varying effects on swarming or surface motility of clinical isolate E. coli, A. baumannii, and K. pneumoniae. There was no effect of ciclopirox on biofilm production, suggesting it does not target Rcs. Altogether, our data suggest ciclopirox-mediated alteration of lipopolysaccharides stimulates enterobactin production and affects bacterial swarming.

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<![CDATA[Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c3e4f3cd5eed0c484d733f0

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

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<![CDATA[Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking]]> https://www.researchpad.co/article/5b600752463d7e39c5526205

Outcome of host-pathogen encounter is determined by the complex interplay between protective bacterial and host defense strategies. This complexity further amplifies with the existence of cell-to-cell phenotypic heterogeneity in pathogens which remains largely unexplored. In this study, we illustrated that heterogeneous expression of pneumolysin (Ply), a pore-forming toxin of the meningeal pathogen, S. pneumoniae (SPN) gives rise to stochastically different bacterial subpopulations with variable fate during passage across blood-brain barrier (BBB). We demonstrate that Ply mediated damage to pneumococcus containing vacuolar (PCV) membrane leads to recruitment of cytosolic “eat-me” signals, galectin-8 and ubiquitin, targeting SPN for autophagic clearance. However, a majority of high Ply producing subset extensively damages autophagosomes leading to pneumococcal escape into cytosol and efficient clearance by host ubiquitination machinery. Interestingly, a low Ply producing subset halts autophagosomal maturation and evades all intracellular defense mechanisms, promoting its prolonged survival and successful transcytosis across BBB, both in vitro and in vivo. Ply therefore acts as both, sword and shield implying that its smart regulation ensures optimal disease manifestation. Our elucidation of heterogeneity in Ply expression leading to disparate infection outcomes attempts to resolve the dubious role of Ply in pneumococcal pathogenesis.

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<![CDATA[Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level]]> https://www.researchpad.co/article/5b498f9c463d7e0897c6e017

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

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<![CDATA[Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori]]> https://www.researchpad.co/article/5aafc676463d7e7d7e2e8752

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

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<![CDATA[Effects of Pyrogallol on Growth and Cytotoxicity of Wild-Type and katG Mutant Strains of Vibrio vulnificus]]> https://www.researchpad.co/article/5989da0aab0ee8fa60b77589

Vibrio vulnificus is a causative agent of fatal septicemia and necrotic wound infection and the pathogen infection became an important public health problem in many counties. Vibrio vulnificus causes RtxA1 toxin-induced acute cell death. We tried to identify natural products that inhibit the acute cytotoxicity of V. vulnificus using a lactate hydrogenase assay. A polyphenol pyrogallol protected HeLa cells from V. vulnificus-induced cytotoxicity. Pyrogallol also decreased the growth of V. vulnificus; this inhibitory effect was more significant during log phase than stationary phase. To further elucidate the inhibitory mechanism, pyrogallol-induced toxicity was compared between a V. vulnificus catalase-peroxidase mutant (katG) and the isogenic wild-type MO6-24/O strains. No growth was observed for the katG mutant in the presence of pyrogallol (50 μg/mL) even after 24 h, whereas the wild-type strain demonstrated growth recovery following a prolonged lag phase. Pyrogallol-mediated growth inhibition of the katG mutant strain was partially rescued by exogenous catalase treatment. These results indicate that the mechanism by which pyrogallol inhibits the growth and cytotoxicity of V. vulnificus likely involves polyphenol-induced prooxidant damage. Taken together, these results suggest that pyrogallol has potential for development as a new paradigm drug to treat infectious diseases.

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<![CDATA[A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis]]> https://www.researchpad.co/article/5989da07ab0ee8fa60b76097

Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen’s virulence.

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<![CDATA[Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization]]> https://www.researchpad.co/article/5989dac6ab0ee8fa60bb2649

Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5’ end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis.

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<![CDATA[YvqE and CovRS of Group A Streptococcus Play a Pivotal Role in Viability and Phenotypic Adaptations to Multiple Environmental Stresses]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc871

Streptococcus pyogenes (group A Streptococcus, or GAS) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS utilizes TCSs to sense and respond to environmental changes and adapts its pathogenic traits accordingly; however, many GAS TCSs and their interactions remain uncharacterized. Here, we elucidated the roles of a poorly characterized TCS, YvqEC, and a well-studied TCS, CovRS, in 2 different GAS strain SSI-1 and JRS4, respectively. Deletion of yvqE and yvqC in JRS4 resulted in lower cell viability and abnormality of cell division when compared to the wild-type strain under standard culture conditions, demonstrating an important role for YvqEC. Furthermore, a double-deletion of yvqEC and covRS in SSI-1 and JRS4 resulted in a significantly impaired ability to survive under various stress conditions, as well as an increased sensitivity to cell wall-targeting antibiotics compared to that observed in either single mutant or wild-type strains suggesting synergistic interactions. Our findings provide new insights into the impact of poorly characterized TCS (YvqEC) and potential synergistic interactions between YvqEC and CovRS and reveal their potential role as novel therapeutic targets against GAS infection.

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<![CDATA[A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbee6

The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner.

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<![CDATA[Improving the Secretory Expression of an -Galactosidase from Aspergillus niger in Pichia pastoris]]> https://www.researchpad.co/article/5989daa6ab0ee8fa60ba7ab2

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.

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<![CDATA[Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum]]> https://www.researchpad.co/article/5989dad4ab0ee8fa60bb7502

Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress.

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<![CDATA[Loss-of-Function Mutants and Overexpression Lines of the Arabidopsis Cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS Exhibit Different Defects in Prophase-I Meiocytes but Produce the Same Meiotic Products]]> https://www.researchpad.co/article/5989db0eab0ee8fa60bcb570

In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products.

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<![CDATA[The Involvement of Mig1 from Xanthophyllomyces dendrorhous in Catabolic Repression: An Active Mechanism Contributing to the Regulation of Carotenoid Production]]> https://www.researchpad.co/article/5989da10ab0ee8fa60b79369

The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis.

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