Bioactive potentiality of secondary metabolites from endophytic bacteria against SARS-COV-2: An in-silico approach.

Five endophytic bacterial isolates were studied to identify morphologically and biochemically, according to established protocols and further confirmed by 16S rDNA Sanger sequencing, as Priestia megaterium, Staphylococcus caprae, Neobacillus drentensis, Micrococcus yunnanensis, and Sphingomonas paucimobiliz, which were then tested for phytohormone, ammonia, and hydrolytic enzyme production. Antioxidant compounds total phenolic content (TPC), and total flavonoid content (TFC) were assessed by using bacterial crude extracts obtained from 24-hour shake-flask culture. Phylogenetic tree analysis of those identified isolates shared sequence similarities with the members of Bacillus, Micrococcus, Staphylococcus, and Pseudomonas species, and after GenBank submission, accession numbers for the nucleotide sequences were found to be MW494406, MW494408, MW494401, MW494402, and MZ021340, respectively. In silico analysis was performed to identify their bioactive genes and compounds in the context of bioactive secondary metabolite production with medicinal value, where nine significant bioactive compounds according to six different types of bioactive secondary metabolites were identified, and their structures, gene associations, and protein-protein networks were analyzed by different computational tools and servers, which were reported earlier with their antimicrobial, anti-infective, antioxidant, and anti-cancer capabilities. These compounds were then docked to the 3-chymotrypsin-like protease (3CLpro) of the novel SARS-COV-2. Docking scores were then compared with 3CLpro reference inhibitor (lopinavir), and docked compounds were further subjected to ADMET and drug-likeness analyses. Ligand-protein interactions showed that two compounds (microansamycin and aureusimine) interacted favorably with coronavirus 3CLpro. Besides, in silico analysis, we also performed NMR for metabolite detection whereas three metabolites (microansamycin, aureusimine, and stenothricin) were confirmed from the 1H NMR profiles. As a consequence, the metabolites found from NMR data aligned with our in-silico analysis that carries a significant outcome of this research. Finally, Endophytic bacteria collected from medicinal plants can provide new leading bioactive compounds against target proteins of SARS-COV-2, which could be an effective approach to accelerate drug innovation and development.

SHANK3 genetic polymorphism and susceptibility to ASD: evidence from molecular, in silico, and meta-analysis approaches.

BACKGROUND: The SHANK3 gene encodes a master synaptic scaffolding protein at the excitatory synapse's postsynaptic density, which is predominantly responsible for constructing a synapse, maintaining synaptic structure, and functions. Recently, evidence from rare mutations and copy number variation provided an important clue about SHANK3 which acts as a strong candidate gene in the pathogenesis of Autism Spectrum Disorder (ASD). MATERIALS AND METHODS: To investigate potential allelic variants for the SHANK3 (rs9616915) gene as a genetic risk factor, we performed PCR-RFLP analysis and Sanger sequencing for 90 ASD and 90 healthy subjects. Moreover, to understand the functional and structural impacts of our selected non-synonymous SHANK3 SNP rs9616915, we have performed an in silico analysis. Subsequently, a meta-analysis of rs9616915 with a total of 6 eligible studies (including the present study) containing a total of 795 cases and 12,947 controls was obtained from a comprehensive online database search to evaluate the overall association with ASD. RESULTS: Our retrieved data, such as Pearson's chi-square test (p = 0.081) as well as logistic regression analysis of co-dominant (p = 0.1131), dominant (p = 0.3656) and recessive models (p = 0.0569) speculated no significant association between rs9616915 and our studied sample. Interestingly, by in silico analysis, we have observed two hydrogen bonds between amino acids instead of one hydrogen bond in the protein structure of rs9616915, which indicates this mutant structure could affect the proteins' stability. The findings of the meta-analysis revealed that four genetic association models were associated with ASD susceptibility. CONCLUSIONS: Our study suggested that targeted SHANK3 SNP of interest rs9616915 might not be associated with ASD in the southern part of the Bangladeshi population.

Optimization of some fermentation conditions for the production of extracellular amylases by using Chryseobacterium and Bacillus isolates from organic kitchen wastes.

Amylolytic bacterial isolates were obtained by starch-agar plate method from municipal solid wastes. Six amylolytic bacteria were isolated and the best two isolates, named as DY and W1, were selected based on clear zone ratio. The 16S rDNA sequence analysis identified DY and W1 isolates as Chryseobacterium sp. and Bacillus sp., respectively. Amylase production was optimized using basal media. The maximum level of amylase production was achieved from Chryseobacterium and Bacillus isolates after 60 h and 48 h of cultivation, respectively. The optimal temperature, initial pH of the media, agitation and inoculum size were determined for the both isolates. Increased amylase production was observed when basal media were substituted with organic carbon and nitrogen sources. The optimum pH and temperature for amylase activity of the crude amylase of Chryseobacterium sp. were 5.0 and 50 °C, respectively and those of amylase from Bacillus sp. were pH 7.0 and 50 °C, correspondingly. The crude amylase from the Chryseobacterium sp. was stable at pH 5.0-6.0 and up to 40 °C but that from Bacillus sp. was stable at pH 7.0 and up to 30 °C. Amylases from both the isolates lost ∼50% activity when stored at room temperature for two days. Under the optimized fermentation conditions both Chryseobacterium and Bacillus isolates produced almost the similar amount of amylase with organic kitchen wastes compared to the basal media. Results reported herein support the notion that Chryseobacterium sp. and Bacillus sp. can be used to produce industrially important amylases by utilizing organic kitchen wastes.

Characterization of furnace oil bioremediation potential of hydrocarbonoclastic bacteria isolated from petroleum contaminated sites of the Sundarbans, Bangladesh.

Spillage of furnace oil is a more frequent event in recent times. In this study, environmental samples from furnace oil spillage sites of the Shela River, the Sundarbans, Bangladesh, were collected after three weeks of spillage. Serial dilution was applied and total seven bacterial isolates were separated as pure cultures. The oil-degrading potentiality of all seven isolates was further assessed, confirmed and compared with the growth pattern in furnace oil supplemented media, 2, 6-dichlorophenolindophenol test, and gravimetric analysis. After 7 days of incubation, isolates SS3, RW2, and SB degraded 56%, 43%, and 52% of supplemented furnace oil, respectively. The top three hydrocarbonoclastic bacterial isolates were selected as potential and identified as Pseudomonas aeruginosa (SS3), Bacillus sp. (RW2), and Serratia sp. (SB). All three isolates showed significant oil-degrading capacity compared to negative control, when incubated in sterile pond water supplemented with 2% furnace oil, suggesting them as potential bioremediation agents.

A single metabolite production by Escherichia coli BW25113 and its pflA.cra mutant cultivated under microaerobic conditions using glycerol or glucose as a carbon source.

Abundant, low prices and a highly reduced nature make glycerol to be an ideal feedstock for the production of reduced biochemicals and biofuels. Escherichia coli has been paid much attention as the platform of microbial cell factories due to its high growth rate (giving higher metabolite production rate) and the capability of utilizing a wide range of carbon sources. However, one of the drawbacks of using E. coli as a platform is its mixed metabolite formation under anaerobic conditions. In the present study, it was shown that ethanol could be exclusively produced from glycerol by the wild type E. coli, while d-lactic acid could be exclusively produced from glucose by pflA.cra mutant, where the glucose uptake rate could be increased by this mutant as compared to the wild type strain. It was also shown that the growth rate is significantly reduced in pflA.cra mutant for the case of using glycerol as a carbon source due to redox imbalance. The metabolic regulation mechanisms behind the fermentation characteristic were clarified to some extent.

Effect of acidic condition on the metabolic regulation of Escherichia coli and its phoB mutant.

Effect of acidic condition on the fermentation characteristics was investigated by the continuous culture of Escherichia coli. In accordance with down-regulation of crp gene transcript level as well as up-regulation of arcA, the expressions of the TCA cycle genes were down-regulated, which caused more acetate formation at acidic condition under aerobic condition. It was also shown that yfiD was up-regulated in accordance with up-regulation of fnr, and the respiratory pathway genes were up-regulated under acidic condition. The effect of phoB gene knockout on fermentation characteristics was also investigated. Under micro-aerobic condition, the fermentation pattern changed in such a way that formate and lactate were more produced at lower pH due to up-regulations of pflA, yfiD and ldhA genes, whereas ethanol was less produced as compared to the case at neutral pH. The overall regulation mechanism under acidic condition was clarified based on fermentation characteristics and gene transcript levels.

Metabolic regulation of an fnr gene knockout Escherichia coli under oxygen limitation.

In addition to our previous study on the effect of fnr gene knockout on the metabolism in Escherichia coli under aerobic conditions (Kumar and Shimizu, Microb Cell Fact 2011), here we further investigated the effect of fnr gene knockout on the metabolism under micro-aerobic condition based on gene expressions, enzyme activities and intracellular metabolic fluxes. The objective of the present research is to clarify the metabolic regulation mechanism on how the culture environment, such as oxygen level, affects the cell metabolism in relation to gene expressions, enzyme activities and fluxes via global regulators such as Fnr and ArcA/B systems. Under micro-aerobic condition, the flux through Pfl and Frd were reduced for the mutant, which are due to fnr gene knockout. The decreased flux through Pfl may have caused accumulation of PYR, which increased the flux through LDH. The fnr gene knockout caused arcA to be downregulated, and thus the TCA cycle was activated, and cyoA and cydB genes were upregulated. The downregulation of arcA caused lpdA and aceE, F to be upregulated where the flux through PDHc increased. The fnr gene knockout indirectly caused cra gene transcript level to be decreased, which in turn caused the glycolysis genes to be upregulated, which correspond to the increase in the specific glucose consumption rate. The fnr gene knockout also caused crp transcript level to be increased, where there might be some relationship between the two due to similar structure and gene sequence. It may be quite important to understand the metabolic regulation mechanism based on different levels of information for the efficient metabolic engineering and control of the culture environment for process optimization.

Metabolic regulation of Escherichia coli and its phoB and phoR genes knockout mutants under phosphate and nitrogen limitations as well as at acidic condition.

BACKGROUND: The phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. The phosphate is involved in many biochemical reactions by the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism. In particular, phosphorylation may play important roles in the metabolic regulation at acidic condition and nitrogen limiting condition, which typically appears at the late growth phase in the batch culture. Moreover, phosphate starvation is a relatively inexpensive means of gene induction in practice, and the phoA promoter has been used for overexpression of heterologous genes. A better understanding of phosphate regulation would allow for optimization of such processes. RESULTS: The effect of phosphate (P) concentration on the metabolism in Escherichia coli was investigated in terms of fermentation characteristics and gene transcript levels for the aerobic continuous culture at the dilution rate of 0.2 h-1. The result indicates that the specific glucose consumption rate and the specific acetate production rate significantly increased, while the cell concentration decreased at low P concentration (10% of the M9 medium). The increase in the specific glucose uptake rate may be due to ATP demand caused by limited ATP production under P-limitation. The lower cell concentration was also caused by less ATP production. The less ATP production by H+-ATPase may have caused less cytochrome reaction affecting in quinone pool, and caused up-regulation of ArcA/B, which repressed TCA cycle genes and caused more acetate production. In the case of phoB mutant (and also phoR mutant), the fermentation characteristics were less affected by P-limitation as compared to the wild type where the PhoB regulated genes were down-regulated, while phoR and phoU changed little. The phoR gene knockout caused phoB gene to be down-regulated as well as PhoB regulated genes, while phoU and phoM changed little. The effect of pH together with lower P concentration on the metabolic regulation was also investigated. In accordance with up-regulation of arcA gene expression, the expressions of the TCA cycle genes such as sdhC and mdh were down-regulated at acidic condition. The gene expression of rpoS was up-regulated, and the expression of gadA was up-regulated at pH 6.0. In accordance with this, PhoB regulated genes were up-regulated in the wild type under P-rich and P-limited conditions at pH 6.0 as compared to those at pH 7.0. Moreover, the effect of nitrogen limitation on the metabolic regulation was investigated, where the result indicates that phoB gene was up-regulated, and PhoB regulated genes were also up-regulated under N-limitation, as well as nitrogen-regulated genes. CONCLUSION: The present result shows the complicated nature of the metabolic regulation for the fermentation characteristics upon phosphate limitation, acidic condition, and nitrogen limitation based on the transcript levels of selected genes. The result implies that the regulations under phosphate limitation, acidic condition, and nitrogen limitation, which occur typically at the late growth phase of the batch culture, are interconnected through RpoS and RpoD together with Pho genes.