In-depth comparative transcriptome analysis of Purpureocillium sp. CB1 under cadmium stress.

Fungal bioremediation is a very attractive tool to cope with environmental pollution. We aimed to decipher the cadmium (Cd) response of Purpureocillium sp. CB1, isolated from polluted soil, at transcriptome level by RNA-sequencing (RNA-seq). We used 500 and 2500 mg/L of Cd2+ concentrations at two time points (t6;36). RNA-seq determined 620 genes that were co-expressed in all samples. The highest number of differentially expressed genes (DEGs) was obtained within the first six h of exposure to 2500 mg/L of Cd2+. Several genes encoding transcriptional regulators, transporters, heat shock proteins, and oxidative stress-related genes were differentially expressed under Cd2+ stress. Remarkably, the genes that encode salicylate hydroxylase, which is involved in naphthalene biodegradation pathway, were significantly overexpressed. Utilization of diesel as the sole carbon source by CB1 even in the presence of Cd2+ supported concomitant upregulation of hydrocarbon degradation pathway genes. Furthermore, leucinostatin-related gene expression levels increased under Cd2+ stress. In addition, leucinostatin extracts from Cd2+-treated CB1 cultures showed higher antifungal activity than the control. Notably, Cd2+ in CB1 was mainly found as bound to the cell wall, thus confirming its adsorption potential. Cd2+ stress slightly reduced growth and led to mycelial malformation due to Cd2+ adsorption, especially at a concentration of 2500 mg/L at t36. A strong correlation was recorded between RNA-seq and reverse-transcriptase-quantitative polymerase chain reaction (RT-qPCR) data. In conclusion, the study represents the first transcriptome analysis of Purpureocillium sp. under Cd2+ stress, providing insights into the primary targets for rational engineering to construct strains with remarkable bioremediation potency. KEY POINTS: • Upregulation of genes encoding salicylate hydroxylases under Cd2+ stress • Maximum Cd2+ adsorption at 500 mg/L at t36 as tightly bound to the cell wall • Concordant bioremediation potential of CB1 on Cd2+ and diesel.

The regulatory role of Fur-encoding SCLAV_3199 in iron homeostasis in Streptomyces clavuligerus.

Iron homeostasis is strictly regulated by complex cascades connected with secondary metabolism in bacteria. Ferric uptake regulators ('Fur's), siderophores, efflux systems, and two-component signal transduction systems are the leading players in response stimuli. However, these regulatory mechanisms remain to be elucidated in Streptomyces clavuligerus. Our study focused on unraveling a possible role of SCLAV_3199 which encodes a Fur family transcriptional regulator, particularly in iron regulation and at the global level in this species. We deleted the SCLAV_3199 gene in S. clavuligerus and compared gene expression differences with the wild-type strain based on iron availability by RNA-seq. We found a potential regulatory effect of SCLAV_3199 on many transcriptional regulators and transporters. Besides, the genes encoding iron sulfur binding proteins were overexpressed in the mutant in the presence of iron. Notably, catechol (SCLAV_5397), and hydroxamate-type (SCLAV_1952, SCLAV_4680) siderophore-related genes were upregulated in the mutant strain in iron scarcity. Concomitantly, S. clavuligerus Δ3199 produced 1.65 and 1.9 times more catechol and hydroxamate-type siderophores, respectively, than that of the wild type strain under iron depletion. Iron containing chemically defined medium did not favor antibiotic production in S. clavuligerus Δ3199 while fermentation in starch-asparagine medium led to improved cephamycin C (2.23-fold) and clavulanic acid (2.56-fold) production in the mutant compared to the control. However, better tunicamycin yield (2.64-fold) was obtained in trypticase soy broth-grown cultures of S. clavuligerus Δ3199. Our findings demonstrate that the SCLAV_3199 gene plays a significant role in regulating both iron homeostasis and secondary metabolite biosynthesis in S. clavuligerus.

Whole genome analysis of Flavobacterium aziz-sancarii sp. nov., isolated from Ardley Island (Antarctica), revealed a rich resistome and bioremediation potential.

Despite being one of the most isolated regions in the world, Antarctica is at risk of increased contamination with potentially toxic elements and other toxic chemicals through anthropogenic interventions. In this study, a psychrotolerant bacterium was isolated using the lake water collected from Ardley Island (Antarctica), which can grow at temperatures between 4 and 30 °C and pH values between 6.0 and 9.0. The isolate, named AC, had protease, amylase, and lipase activities with no NaCl tolerance and could degrade 1-5% diesel fuel. Multilocus sequence analysis (MLSA) using 16S rRNA, gyrB, tuf, and rpoD genes resulted in 92.91-98.6% sequence similarities between the isolate AC and other Flavobacterium spp. Whole genome analysis indicated that the genome length of Flavobacterium sp. AC is 5.8 Mbp with a GC content of 34.04% and 1274 genes predicted. The strain AC branched independently from other Flavobacterium spp. in the phylogenetic and phylogenomic trees and ranked a new species named Flavobacterium aziz-sancarii. Genome mining identified several cold-inducible genes, including stress-associated genes such as cold-shock proteins, chaperones, carotenoid biosynthetic genes, or oxidative-stress response genes. In addition, virulence, gliding motility, and biofilm-related genes were determined. Its genome contains 35 and 88 open-reading frames related to potentially toxic element and antibiotic resistance, respectively. F. aziz-sancarii showed a remarkable tolerance of Cr and Ni, with minimal inhibitory concentration values of 2.88 and 2.81 mM, respectively. Pb, Cu, and Zn exposure resulted in moderate toxicity (2.14-2.41 mM), while Cd showed the highest inhibitory effect in bacterial growth (0.74 mM). Antibiotic susceptibility testing indicated multidrug-resistant phenotype in correlation to in silico prediction of antibiotic resistance genes. Overall, our results contribute to biodiversity of Antarctica and provide new insights into resistome profile of Antarctic microorganisms. Additionally, the diesel degradation feature of F. aziz-sancarii offers potential use for the bioremediation of hydrocarbon-contaminated polar ecosystems.

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris).

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks < 1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume.

A Novel 4H-Chromen-4-One Derivative from Marine Streptomyces ovatisporus S4702T as Potential Antibacterial and Anti-Cancer Agent.

BACKGROUND: Marine actinomycetes are among indispensable sources of natural bioactive compounds with unique antimicrobial and anti-cancer activities. OBJECTIVE: Herein, it was aimed to elucidate the bioactive potential of a marine-derived Streptomyces ovatisporus S4702T, isolated previously. METHODS: Streptomyces ovatisporus S4702T was cultured in N-Z Amine broth, and extraction was carried out using different organic solvents. Bioassay-guided purification was followed by chemical characterization using NMR and LC-MS/MS. The compound was then evaluated for its antibacterial, antioxidant and cytotoxic activities. RESULTS: Etyl acetate extracts gave the highest antibacterial activity, and chemical characterization of this extract indicated the formula as C15H29O5N3 and the corresponding possible molecular structure as 4H-chromen-4-one derivative. It was found highly potent against Bacillus subtilis ATCC 6633 (MIC: 0.25 µg ml-1) and Micrococcus luteus ATCC 9341 (MBC: 0.5 µg ml-1). It has no remarkable antioxidant activity, but a higher EC50 value and less cytotoxicity against normal cells. The EC50 values of this chromen derivative were found as 9.68 µg ml-1 for human colon carcinoma, 9.93 µg ml-1 for human prostate adenocarcinoma and 25.5 µg ml-1 for human embryonic kidney cells. CONCLUSION: Overall, the presented 4H-chromen-4-one derivative is a remarkable bioactive compound with potent antibacterial and cytotoxic activity. With its high bioactive potential, it is proposed as a good candidate in medicine.

Identification of 3-Bromo-1-Ethyl-1H-Indole as a Potent Anticancer Agent with Promising Inhibitory Effects on GST Isozymes.

BACKGROUND: Indole-based heterocyclic compounds play important roles in pharmaceutical chemistry due to their unexpected biological and pharmacological properties. OBJECTIVE: Herein, we describe novel biological properties (antioxidant, antimicrobial and anti-cancer) of 3- bromo-1-ethyl-1H-indole (BEI) structure. METHODS: BEI was synthesized from 1-Methyl-2-phenylindole and N-bromosuccinimide and was characterized by using 1H and 13C NMR. Cytotoxicity was determined by MTT assay. Apoptosis analysis of BEI was determined by Arthur™ image-based Cytometer. Different methods were applied to assess the antioxidant activity of BEI. Molecular docking studies were conducted to determine the interactions of bonding between GST isozymes and BEI. RESULTS: According to the antioxidant and antimicrobial activity assays, BEI compound showed reduced total antioxidant activity compared to the Trolox standard, whereas it showed moderate antimicrobial activity against Aspergillus niger and Phytophora eryhtrospora. Notably, the BEI compound demonstrated substantial selective cytotoxicity for the first time towards cancer cell lines, and there existed a significant decrease in the percentage of live cells treated with BEI, in comparison to the control ones. Interestingly, BEI exhibited a promising glutathione S-transferase isozymes inhibition. CONCLUSION: The results of this study suggest that BEI seems to be a promising molecule to be used in the design of new anti-cancer agents that provide superiority to present commercial anti-cancer drugs.

Homologous expression of lysA encoding diaminopimelic acid (DAP) decarboxylase reveals increased antibiotic production in Streptomyces clavuligerus.

lysA gene encoding meso-diaminopimelic acid (DAP) decarboxylase enzyme that catalyzes L-lysine biosynthesis in the aspartate pathway in Streptomyces clavuligerus was overexpressed, and its effects on cephamycin C (CephC), clavulanic acid (CA), and tunicamycin productions were investigated. Multicopy expression of lysA gene under the control of glpF promoter (glpFp) in S. clavuligerus pCOlysA led to higher expression levels ranging from 2- to 6-fold increase at both lysA gene and CephC biosynthetic gene cluster at T36 and T48 of TSBG fermentation. These results accorded well with CephC production. Thus, 1.86- and 3.14-fold higher volumetric as well as 1.26- and 1.71-fold increased specific CephC yields were recorded in S. clavuligerus pCOlysA in comparison with the wild-type and its control strain, respectively, at 48th h. Increasing the expression of lysA provided 4.3 times more tunicamycin yields in the recombinant strain. These findings suggested that lysA overexpression in S. clavuligerus made the strain more productive for CephC and tunicamycin. The results also supported the presence of complex interactions among antibiotic biosynthesis pathways in S. clavuligerus.

Molecular and biochemical characterization of a recombinant endoglucanase rCKT3eng, from an extreme halophilic Haloarcula sp. strain CKT3.

Halophilic cellulases are indispensable enzymes of heavy industrial processes as resistant biocatalysts due to high level activity at extreme conditions. In this study, crude cellulase from an extreme halophilic Haloarcula sp. CKT3 was characterized. Then, recombinant expression of putative endo-1,4-ß-glucanase gene, of CKT3 strain, in E. coli BL21(DE3) was performed with the aim of obtaining highly pure, active and robust industrial enzyme for such industrial aplications. The crude cellulase had optimal activity (16.9 U/mg) at 70 °C, pH 7.0 and 4 M NaCl exhibiting good thermostability, high pH and halotolerance. Indeed, it is very stable in water-insoluble organic solvents with log Po/w ≥ 2.13 and highly resistant to SDS (10%). Recombinant CKT3eng has a molecular weight of 36.9 kDa and 99% aminoacid identity to endo-l,4-ß-D-glucanase from Haloarcula argentinensis. Its 3D structure was predicted using Phyre2 and I-TASSER. rCKT3eng enzyme provided 31.6 U/mg activity at optimal 50 °C, pH 7.0 and 3 M NaCl. In addition to its quite similar stability values and resistance to organic solvents and SDS, rCKT3eng has superiority over crude enzyme with 1.87-fold higher specific activity. Therefore, rCKT3eng offers a promising enzyme for industrial use with its valuable activity and stability in extreme conditions.