Streptomyces monashensis MSK03-mediated synthesis of gold nanoparticles: characterization and antibacterial activity.

Nanotechnology is a cutting-edge field with diverse applications, particularly in the utilization of gold nanoparticles (AuNPs) due to their stability and biocompatibility. AuNPs serve as pivotal components in medical applications, with a specific emphasis on their significant antibacterial efficacy. This study focuses on synthesizing AuNPs using the cell-free supernatant of Streptomyces monashensis MSK03, isolated from terrestrial soil in Thailand. The biosynthesis process involved utilizing the cell-free supernatant of S. monashensis MSK03 and hydrogen tetrachloroauric acid (HAuCl4) under controlled conditions of 37 °C and 200 rpm agitation. Characterization studies revealed spherical AuNPs with sizes ranging from 7.1 to 40.0 nm (average size: 23.2 ± 10.7 nm), as confirmed by TEM. UV-Vis spectroscopy indicated a localized surface plasmon resonance (LSPR) band at 545 nm, while XRD analysis confirmed a crystalline structure with characteristics of cubic lattice surfaces. The capping molecules on the surface of AuNPs carry a negative charge, indicated by a Zeta potential of -26.35 mV, and FTIR analysis identified functional groups involved in reduction and stabilization. XANES spectra further confirmed the successful reduction of Au3+ to Au0. Moreover, the synthesized AuNPs demonstrated antibacterial activity against drug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Interestingly, the AuNPs showed non-toxicity to Vero cell lines. These significant antibacterial properties of the produced nanoparticles mean they hold great promise as new antimicrobial treatments for tackling the increasing issue of antibiotic resistance.

Draft genome sequence data of the endophytic actinobacterium Streptomyces justiciae WPN32, a potential bioactive compounds producer.

Streptomyces justiciae WPN32 is an endophytic actinobacterium isolated from the rhizosphere of turmeric field soil at the Botanical Garden of Suranaree University of Technology (Nakhon Ratchasima Province, Thailand). Here we present the draft genome sequence of S. justiciae WPN32. It was sequenced on the Illumina NextSeq 550 sequencer. The draft genome consisted of 123 contigs with a total size of 9,832,147 base pairs, an N50 of 237,572 base pairs and a GC content of 70.87%. The dDDH between WPN32 and Streptomyces justiciae 3R004T was 80.1%, identifying the strain as Streptomyces justiciae. The data presented here may aid microbial taxonomy, comparative genomics and identification of gene clusters associated with the synthesis of bioactive compounds. The draft genome sequence data has been deposited at NCBI under Bioproject accession number PRJNA680432.

Isolation and Identification of Bioactive Compounds from Streptomyces actinomycinicus PJ85 and Their In Vitro Antimicrobial Activities against Methicillin-Resistant Staphylococcus aureus.

Antibiotic-resistant strains are a global health-threatening problem. Drug-resistant microbes have compromised the control of infectious diseases. Therefore, the search for a novel class of antibiotic drugs is necessary. Streptomycetes have been described as the richest source of bioactive compounds, including antibiotics. This study was aimed to characterize the antibacterial compounds of Streptomyces sp. PJ85 isolated from dry dipterocarp forest soil in Northeast Thailand. The 16S rRNA gene sequence and phylogenetic analysis showed that PJ85 possessed a high similarity to Streptomyces actinomycinicus RCU-197T of 98.90%. The PJ85 strain was shown to produce antibacterial compounds that were active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The active compounds of PJ85 were extracted and purified using silica gel column chromatography. Two active antibacterial compounds, compound 1 and compound PJ85_F39, were purified and characterized with spectroscopy, including liquid chromatography and mass spectrometry (LC-MS). Compound 1 was identified as actinomycin D, and compound PJ85_F39 was identified as dihomo-γ-linolenic acid (DGLA). To the best of our knowledge, this is the first report of the purification and characterization of the antibacterial compounds of S. actinomycinicus.

Streptomyces chiangmaiensis SSUT88A mediated green synthesis of silver nanoparticles: characterization and evaluation of antibacterial action against clinical drug-resistant strains.

This study involved the characterization of AgNPs synthesized from soil isolate Streptomyces sp. SSUT88A and their antimicrobial activities. The strain SSUT88A revealed 98.8% similarity of the 16s rRNA gene to Streptomyces chiangmaiensis TA4-1T. The AgNPs were synthesized by mixing either intracellular or extracellular cell-free supernatant of strain SSUT88A with AgNO3. The synthesized AgNPs from intracellular cell-free supernatant and extracellular cell-free supernatant were designated as IS-AgNPs and ES-AgNPs, respectively. The IS-AgNPs showed maximum absorbance of UV-vis spectra at 418 nm, while ES-AgNPs revealed maximum absorbance at 422 nm. The TEM observation of synthesized AgNPs revealed a spherical shape with an average diameter of 13.57 nm for IS-AgNPs and 30.47 nm for ES-AgNPs. The XRD and XANES spectrum profile of both synthesized AgNPs exhibited similar spectrum energy, which corresponded to AgNPs. The IS-AgNPs revealed antimicrobial activity against clinical isolate drug-resistant bacteria (Acinetobacter baumannii, Escherichia coli 8465, Klebsiella pneumoniae 1617, and Pseudomonas aeruginosa N90PS), while ES-AgNPs had no antimicrobial activity. When compared to commercial AgNPs, IS-AgNPs exhibited antibacterial efficacy against all clinical isolate bacteria including A. baumannii, one of the most threatening multi-drug resistant strains, while commercial AgNPs did not. Thus, IS-AgNPs has potential to be further developed as an antimicrobial agent against drug-resistant bacteria.

Potassium ion leakage impairs thermotolerance in Corynebacterium glutamicum.

Corynebacterium glutamicum, a gram-positive bacterium, can produce amino acids such as glutamic acid and lysine. The heat generated during cell growth and/or glutamate fermentation disturbs both the cell growth and fermentation. To overcome such a negative effect of the fermentation heat, we have tried to establish a high temperature fermentation. One of the approach is to create a thermotolerant strains, while the other is to create an optimum culture conditions able for the strain to grow at higher temperatures. In this study, we focused on the latter approach, where we examined the effect of potassium ion on cell growth at high growth temperatures of C. glutamicum. The supplementation of high concentrations of potassium chloride (300 mM) (or sorbitol, an osmolyte) mitigated the repressed cell growth induced by high temperature at 39 °C or 40 °C. The intracellular potassium concentration declines from 300 mM to ∼150 mM by increasing the growth temperature but not by supplementing potassium chloride or sorbitol. Furthermore, in vitro experiments revealed that the potassium ion leakage occurs at high temperatures, which was mitigated in the presence of high concentrations of extracellular potassium chloride. This suggested that the presence of high osmolyte in the culture medium could inhibit the potassium ion leakage induced by high temperature and subsequently support cell growth at high temperatures.

Corynebacterium suranareeae sp. nov., a glutamate producing bacterium isolated from soil and its complete genome-based analysis.

Strain N24T was isolated from soil contaminated with starling's feces collected from Roi-Et province, Thailand. Cells of N24T were Gram-stain-positive rods, aerobic and non-spore-forming. N24T was positive for catalase, urease, citrate utilization, nitrate reduction and Methyl Red (MR) test but negative for oxidase, casein, gelatin liquefaction, tyrosine, Voges-Proskauer (VP) reaction and starch hydrolysis. Meso-diaminopimelic acid, rhamnose, ribose, arabinose and galactose were detected in its whole-cell hydrolysates. The results of the 16S rRNA gene sequence analysis indicated that N24T represented a member of the genus Corynebacterium. N24T was closely related to Corynebacterium glutamicum ATCC 13032T, with 99.0 % 16S rRNA gene sequence similarity. According to results obtained using in silico DNA-DNA hybridization approaches, N24T showed highest DNA-DNA relatedness (27.6 %) and average nucleotide identity (84.1 %) to Corynebacterium glutamicum ATCC 13032T. The DNA G+C content of N24T was 51.8 mol% (genome based). The major cellular fatty acids of N24T were C16 : 0, and C18 : 1ω9c. N24T had the nine isoprenes unit, MK-9(H2) as the predominant menaquinone. The predominant polar lipids were phosphatidylglycerol, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were also present. According to the complete genome sequence data, strain N24T and C. glutamicum ATCC 13032T are close phylogenetic neighbours, but have different genome characteristics. On the basis of the results of the genotypic and genomic studies and phenotypic characteristics including chemotaxonomy, strain N24T should be classified as representing a novel species of the genus Corynebacterium, for which the name Corynebacterium suranareeae sp. nov. is proposed. The type strain is N24T (TBRC 5845T=NBRC 113465T).

The effect of reactive oxygen species (ROS) and ROS-scavenging enzymes, superoxide dismutase and catalase, on the thermotolerant ability of Corynebacterium glutamicum.

The function of two reactive oxygen species (ROS) scavenging enzymes, superoxide dismutase (SOD) and catalase, on the thermotolerant ability of Corynebacterium glutamicum was investigated. In this study, the elevation of the growth temperature was shown to lead an increased intracellular ROS for two strains of Corynebacterium glutamicum, the wild-type (KY9002) and the temperature-sensitive mutant (KY9714). In order to examine the effects of ROS-scavenging enzymes on cell growth, either the SOD or the catalase gene was disrupted or overexpressed in KY9002 and KY9714. In the case of the KY9714 strain, it was shown that the disruption of SOD and catalase disturbs cell growth, while the over-productions of both the enzymes enhances cell growth with a growth temperature of 30 °C and 33 °C. Whereas, in the relatively thermotolerant KY9002 strain, the disruption of both enzymes exhibited growth defects more intensively at higher growth temperatures (37 °C or 39 °C), while the overexpression of at least SOD enhanced the cell growth at higher temperatures. Based on the correlation between the cell growth and ROS level, it was suggested that impairment of cell growth in SOD or catalase-disrupted strains could be a result of an increased ROS level. In contrast, the improvement in cell growth for strains with overexpressed SOD or catalase resulted from a decrease in the ROS level, especially at higher growth temperatures. Thus, SOD and catalase might play a crucial role in the thermotolerant ability of C. glutamicum by reducing ROS-induced temperature stress from higher growth temperatures.

Fabrication of silver chloride nanoparticles using a plant serine protease in combination with photoactivation and investigation of their biological activities.

Recently, the development of "green" methods for fabrication of silver nanoparticles (Ag-NPs) has been emphasized, in view of their environmental safety, feasibility, and low cost. In this study, a serine protease, EuP-82 from Euphorbia cf. lactea latex, was used to fabricate silver chloride nanoparticles (AgCl-NPs) in phosphate-buffered saline (pH 7.2), under the influence of visible light. The fabricated nanoparticles had a maximal surface plasmon resonance absorption peak at 435 nm. The size of the AgCl-NPs, estimated by scanning electron microscopy, was 57 ± 14.7 nm. Energy dispersive X-ray spectroscopy, X-ray absorption spectroscopy, and X-ray diffraction analysis confirmed that the fabricated Ag-NPs were of the AgCl type. The fabricated nanoparticles had antioxidant activity, scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals with IC50 of 204 ± 1.8 µg/mL. The fabricated AgCl-NPs had broad-spectrum in vitro antimicrobial activities, acting against the Gram-positive bacteria Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Bacillus cereus, and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. AgCl-NPs also showed antifungal activity against Candida albicans and C. tropicalis. In addition, AgCl-NPs showed antiprotozoal activity against Giardia lamblia, with IC50 202 ± 2.1 µg/mL. Based on the biological activities of the fabricated AgCl-NPs, they have the potential for widespread application in medicine and industry.

Complete genome sequencing of newly isolated thermotolerant Corynebacterium glutamicum N24 provides a new insights into its thermotolerant phenotype.

To understand the genetic background of thermotolerance, we determined the complete genome sequence of a thermotolerant Corynebacterium glutamicum N24 strain isolated from soil. The whole genome based phylogenetic analysis between N24 and other related species revealed that N24 diverged from other C. glutamicum strains at earlier stages. Comparisons of thermotolerance between N24 and its related species showed that N24 and Corynebacterium efficiens YS-314 have a higher thermotolerance than Corynebacterium callunae DSM 20147 and C. glutamicum KY9002. In order to understand the link between a particular genetic background and thermotolerance, we compared the genomes of these four strains by mapping their genomes onto the N24 chromosome. We then determined the genes that were conserved in the thermotolerant species. Our results indicated the specific presence of glutathione-dependent aldehyde dehydrogenase, two sortase homologs, and lipid synthesis-related genes in the genomes of thermotolerant strains, YS-314 and N24. Therefore, these genes may be responsible for the differences in their thermotolerant phenotypes.

Biodiversity of Antimicrobial-Producing Actinomycetes Strains Isolated from Dry Dipterocarp Forest Soil in Northeast Thailand

The increasing use of antibiotics has led to the development of drug-resistant microorganisms. The emergence of resistant strains has compromised the treatment and control of infectious diseases. As a result, the search and development of a novel class of antibiotic drugs are required. Actinomycetes have been recognized as a richest source of secondary metabolites including antibiotics. The aim of this study was to investigate the diversity of antibiotic-producing soil Actinomycetes from dry dipterocarp forest in Northeast of Thailand. The soil from this area has been known for its poor in nutrients and highly acidic with pH values around 4.5. In such a harsh condition, soil-inhabiting Actinomycetes elevate their protective mechanisms by inducing the production of antimicrobials and other defense compounds. Therefore, dry dipterocarp forest could be served as a potential source for the screening of the novel antimicrobial drugs. Twelve antibiotic-producing strains isolated from soil in Suranaree University of Technology produce antimicrobial agents which are active against wide range of test pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Based on 16S rRNA gene analysis, these strains are close affiliated with the genus Streptomyces (11 isolates) and Nonomuraea (1 isolate). Most of soil isolates show narrow antimicrobial spectrum activity against Gram-positive bacteria. Two isolates, PJ36 and PJ95, exhibit broad antimicrobial spectrum against Gram-positive bacteria, Gram-negative bacteria and yeasts. Phylogenetic tree analysis of 16S rDNA reveals that isolates PJ33, PJ36, PJ43, PJ51, PJ75, PJ76, PJ77, PJ85, PJ88 and PJ95 strains are not cluster with others strain of Streptomyces. They represent a distinct phyletic line which might be suggested the novel strains. This study was the first attempted to isolate antibiotic-producing Actinomycetes from dry dipterocarp forest soil in Northeast Thailand. Most of soil isolates (8 strains) obtained from this study were active against methicillin-resistant Staphylococcus aureus (MRSA). These isolates could be used for the development of new drugs to combat antibiotic resistances.

Antibacterial activity of Aquilaria crassna leaf extract against Staphylococcus epidermidis by disruption of cell wall.

BACKGROUND: Aquilaria crassna Pierre ex Lecomte has been traditionally used in Thailand for treatment of infectious diseases such as diarrhoea and skin diseases for a long time. The main objectives of this study were to examine antibacterial activity of the Aquilaria crassna leaf extract against Staphylococcus epidermidis and its underlying mechanism. The antioxidant activity and acute toxicity were studied as well. METHODS: Antioxidant activities were examined by FRAP, ABTS and DPPH scavenging methods. Antibacterial activity was conducted using disc diffusion assay and the minimum inhibitory concentration (MIC) was determined by dilution method. The minimum bactericidal concentration (MBC) was reported as the lowest concentration producing no growth of microbes in the subcultures. Morphological changes of the microbe were observed by scanning electron microscopy, while an inhibitory effect on biofilm formation was evaluated by phase contrast microscopic analysis. Bacterial cell wall integrity was assessed by transmission electron microscopy. Acute toxicity was conducted in accordance with the OECD for Testing of Chemicals (2001) guidelines. RESULTS: The extract exhibited considerable antioxidant activity. Staphylococcus epidermidis was susceptible to the extract with the MIC and MBC of 6 and 12 mg/ml, respectively. The extract caused swelling and distortion of bacterial cells and inhibited bacterial biofilm formation. Rupture of bacterial cell wall occurred after treated with the extract for 24 h. Acute toxicity test in mice showed no sign of toxicity or death at the doses of 2,000 and 15,000 mg/kg body weight. CONCLUSION: The aqueous extract of Aquilaria crassna leaves possesses an in vitro antibacterial activity against Staphylococcus epidermidis, with no sign of acute oral toxicity in mice, probably by interfering with bacterial cell wall synthesis and inhibiting biofilm formation.

Electron transfer ability from NADH to menaquinone and from NADPH to oxygen of type II NADH dehydrogenase of Corynebacterium glutamicum.

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.

Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in Corynebacterium glutamicum KY9714.

The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H(+)/O ratio. However, in the strain that lacked NDH-2, membrane L-lactate oxidase activity increased, while NDH-2 over-expression led to decreased L-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, L-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane L-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum.

Stable integration and expression of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus into the chromosome of Enterobacter amnigenus: a potential breakthrough in mosquito biocontrol.

Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Omega). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Omega, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Omega region yielding two integrants designated An11S4::cry4B and An11S4::Omega::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Omega::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.

Development of a method for heterologous gene expression in Enterobacter amnigenus, a potential host for the biological control of mosquito larvi.

An integrative plasmid containing a 1.3 kb fragment of chromosomal DNA from Enterobacter amnigenus was constructed. The Omega fragment encoding spectinomycin/streptomycin resistance was cloned into the unique BglII site of the resulting plasmid, and the interrupted fragment was transferred via plasmid pMAK705 by electroporation into E. amnigenus with a selection for spectinomycin resistance. Cointegrants were resolved to generate an E. amnigenus strain that expressed spectinomycin resistance, but grew as rapidly as the parental strain. The cloned fragment encodes a putative homologue of the proW gene of Escherichia coli that is not essential for E. amnigenus growth. The integrative plasmid is now available to introduce any heterologous DNA into the E. amnigenus chromosome, for the construction of promoter-probe vectors for the studies of gene regulation, or to construct plasmids suitable for the isolation of secretion signals. Immediate applications of this system will include the expression and secretion of crystal toxins from bacilli for the biological control of mosquito larvae infected with the bacterial host.