Positive regulation of MarR-type regulator slnO and improving salinomycin production of Streptomyces albus by multiple transcriptional regulations

The purpose of this study is to explore the function of MarR-family regulator slnO. In addition, the high-yield strain of salinomycin was constructed by using combined regulation strategies. Firstly the slnO gene over-expression strain (GO) was constructed in Streptomyces albus. Compared to wild type (WT) strain，salinomycin production in GO strain was increased about 28%. Electrophoretic mobility gel shift assays (EMSAs) confirmed that SlnO protein can bind specifically to the intergenic region of slnN-slnO, slnQ-slnA1 and slnF-slnT. qRT-PCR experiments also showed that slnA1, slnF, and slnT1 were significantly up-regulated, while the expression level of the slnN gene was down-regulated in GO strain. Secondly, slnN gene deletion strain (slnNDM) was used as the starting strain, and the pathway specific gene slnR in salinomycin gene cluster was over expressed in slnNDM. The new strain was named ZJUS01. The yield of salinomycin in ZJUS01 strain was 25% and 56% higher than that in slnNDM strain and WT strain. Above results indicate that the slnO gene has a positive regulation effect on the biosynthesis of salinomycin. Meanwhile, the yield of salinomycin could be greatly increased by manipulating multiple transcriptional regulations.

Characterization of the Biosynthetic Gene Cluster of Enterocin F4-9, a Glycosylated Bacteriocin

Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection. Article Summary Hsp90 is a conserved molecular chaperone that modulates virulence traits and drug resistance in fungal pathogens. Despite the potential of Hsp90 as a target for antifungal development, genetic characterization remains lacking in Cryptococcus neoformans. Here, we report generation of a C. neoformans HSP90 conditional expression strain. Utilizing this genetic tool, we found depletion of Hsp90 impacted tolerance to environmental stresses, growth at physiological temperature, and virulence in vivo. Thus, we suggest targeting Hsp90 is a viable strategy for treating cryptococcosis.

A yceI Gene Involves in the Adaptation of Ralstonia solanacearum to Methyl Gallate and Other Stresses

Ralstonia solanacearum is a plant-pathogenic bacterium causing plant bacterial wilt, and can be strongly inhibited by methyl gallate (MG). Our previous transcriptome sequencing of MG-treated R. solanacearum showed that the yceI gene AVT05_RS03545 of Rs-T02 was up-regulated significantly under MG stress. In this study, a deletion mutant (named DM3545) and an over-expression strain (named OE3545) for yceI were constructed to confirm this hypothesis. No significant difference was observed among the growth of wild-type strain, DM3545, and OE3545 strains without MG treatment. Mutant DM3545 showed a lower growth ability than that of the wild type and OE3545 strains under MG treatment, non-optimal temperature, or 1% NaCl. The ability of DM3545 for rhizosphere colonization was lower than that of the wild-type and OE3545 strains. The DM3545 strain showed substantially reduced virulence toward tomato plants than its wild-type and OE3545 counterpart. Moreover, DM3545 was more sensitive to MG in plants than the wild-type and OE3545 strains. These results suggest that YceI is involved in the adaptability of R. solanacearum to the presence of MG and the effect of other tested abiotic stresses. This protein is also possibly engaged in the virulence potential of R. solanacearum.

The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve the Resistance of Zebrafish to Aeromonas Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of A. veronii were tested in vitro, and the results showed that this two had different effects on A. veronii: wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 was evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve The Resistance of Zebrafish To A. Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. The bacteriostatic effect of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 in vitro showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Therefore simple combination of B. subtilis wt55 strain and AiiO-AIO6 was used to evaluate potential synergistic effect. The results showed this combination could significantly reduce the number of invasive A. veronii in gut after challenge, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Manufacturing of probiotics and functional foods using lactic acid bacteria that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analyzed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for lactic acid bacteria, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~6-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavine, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve the Resistance of Zebrafish to A. Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. The bacteriostatic effect of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 in vitro showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Therefore simple combination of B. subtilis wt55 strain and AiiO-AIO6 was used to evaluate potential synergistic effect. The results showed this combination could significantly reduce the number of invasive A. veronii in gut after challenge, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

Recent Progress in the Study of Peroxiredoxin in the Harmful Algal Bloom Species Chattonella marina

Peroxiredoxin (Prx) is a relatively recently discovered antioxidant enzyme family that scavenges peroxides and is known to be present in organisms from biological taxa ranging from bacteria to multicellular eukaryotes, including photosynthetic organisms. Although there have been many studies of the Prx family in higher plants, green algae, and cyanobacteria, few studies have concerned raphidophytes and dinoflagellates, which are among the eukaryotic algae that cause harmful algal blooms (HABs). In our proteomic study using 2-D electrophoresis, we found a highly expressed 2-Cys peroxiredoxin (2-CysPrx) in the raphidophyte Chattonella marina var. antiqua, a species that induces mass mortality of aquacultured fish. The abundance of the C. marina 2-CysPrx enzyme was highest in the exponential growth phase, during which photosynthetic activity was high, and it then decreased by about a factor of two during the late stationary growth phase. This pattern suggested that 2-CysPrx is a key enzyme involved in the maintenance of high photosynthesis activity. In addition, the fact that the depression of photosynthesis by excessively high irradiance was more severe in the 2-CysPrx low-expression strain (wild type) than in the normal-expression strain (wild type) of C. marina suggested that 2-CysPrx played a critical role in protecting the cell from oxidative stress caused by exposure to excessively high irradiance. In the field of HAB research, estimates of growth potential have been desired to predict the population dynamics of HABs for mitigating damage to fisheries. Therefore, omics approaches have recently begun to be applied to elucidate the physiology of the growth of HAB species. In this review, we describe the progress we have made using a molecular physiological approach to identify the roles of 2-CysPrx and other antioxidant enzymes in mitigating environmental stress associated with strong light and high temperatures and resultant oxidative stress. We also describe results of a survey of expressed Prx genes and their growth-phase-dependent behavior in C. marina using RNA-seq analysis. Finally, we speculate about the function of these genes and the ecological significance of 2-CysPrx, such as its involvement in circadian rhythms and the toxicity of C. marina to fish.