Strain DH5α Research Articles

Novel plasmid-mediated CMY variant (CMY-192) conferring ceftazidime-avibactam resistance in multidrug-resistant Escherichia coli.

The rapid rise of multidrug resistance (MDR) among Gram-negative bacteria has accelerated the development of novel therapies. Ceftazidime-avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor recently approved for the treatment of limited infectious diseases. Here, we describe a novel CMY variant, CMY-192, that confers high-level resistance to CZA. This gene was detected in a clinical MDR Escherichia coli strain (Ec73552) isolated from an outpatient with a community-acquired urinary tract infection who had not received prior CZA treatment. Ec73552 was typed as O101:H9-ST10, a high-risk clone associated with human and animal diseases. Ec73552 was able to colonize the bladder in a mouse model, suggesting that this strain was uropathogenic. CMY-192 shared the highest amino acid identity (98.95%) with CMY-172 and conferred at least a 32-fold increase in CZA MIC (from ≤0.125/4 to 8/4 mg/L) when cloned into a CZA-susceptible E. coli DH5α strain. Knockout of CMY-192 in Ec73552 resulted in a 256-fold reduction in CZA MIC (from 64/4 to 0.25/4 mg/L). CMY-192 was encoded on an IncB/O/K/Z-type plasmid (pCMY192). Conjugation assays confirmed that pCMY192 was self-transmissible, resulting in a 256-fold increase in the CZA MIC of the recipient. Notably, pCMY192 cured in Ec73552 did not confer a growth advantage, while the conjugant exhibited reduced biomass and growth rate, indicating that fitness costs imposed by pCMY192 may have been compensated in Ec73552. Our findings highlight the importance of continuous monitoring of CZA susceptibility to prevent the spread of resistance in clinical settings.

Bovine Lactoferrin Impairs in-vitro Growth of Neonatal Sepsis-causingEscherichia Coli Clinical Isolates

Abstract Background E. coli is a dominant cause of neonatal sepsis with no prevention currently available. Lactoferrin (LF) is an iron-sequestering antibacterial protein that has been shown to decrease all-cause neonatal late-onset sepsis. However, the impact of LF on impairing growth of neonatal E. coli septicemia isolates carrying various iron acquisition virulence genes has not been investigated. We aimed to determine the effects of bovine lactoferrin (bLF) on in vitro growth of neonatal E. coli septicemia strains and to determine the presence of iron-acquisition virulence genes in the isolates. Methods We tested six clinical E. coli strains obtained from blood of septic neonates, including the archetypal RS218 strain. Isolates represent the most frequent phylogroups (B2 and D) and multi-locus sequence types (ST95, ST69, and ST131) among neonatal E. coli septicemia strains. The nonpathogenic strain DH5α was used as control. Strains were grown in media containing 0, 0.1, 1.0, and 10 mg/mL bLF. Optical density (OD) readings were obtained every 30 min. for 20 h. Experiments were repeated 3 times. OD area under the curve (AUC) values were compared with ANOVA. Whole genome sequencing (WGS) was obtained to determine the presence of iron-acquisition virulence genes using VirulenceFinder v. 2.0.3 (J Clin Micro 2020. 58 (10):e01269-20). Results For all strains, the growth decrease with 10 mg/mL bLF compared to zero bLF was highly significant (p < 0.001) (Fig.1). Growth was also significantly reduced at 1 mg/mL for most strains (p ≤ 0.02). Growth decrease at 0.1 mg/mL was only significant for isolate SCB31. Growth suppression at 10 mg/mL was greatest in DH5α. WGS analyses showed that all pathogenic strains carried several iron acquisition virulence genes, with chuA (hemin receptor), fyuA (siderophore receptor) in irp2 (protein 2 peptide synthetase) and sitA (iron transport protein) found in all (Fig. 2). DH5α had no iron acquisition virulence genes. Conclusion Bovine LF is effective in impairing growth of neonatal sepsis-causing E. coli regardless of the presence of iron acquisition genes known to contribute to virulence. Future studies will examine additional isolates and the effect of greater bLF concentrations. These data have the potential to inform dosing regimens for additional in vivo studies to further assess bLF’s protective effects against neonatal sepsis. Figure 1. Comparison of growth curve data among E. coli strains. Optical density AUC values of six neonatal septicemia E. coli isolates growth with increasing concentrations of bovine lactoferrin. DH5α is a nonpathogenic control strain. Bars depict means and standard deviations for each condition tested. Asterisks indicate statistically significant differences compared to the 0 mg/mL bLF condition (*p < 0.001, **p < 0.05). Figure 2. Presence of iron acquisition virulence genes in E. coli isolates. WGS analyses detected several genes, which are indicated in black. White indicates absence of gene. ST, Sequence type; chuA, Outer membrane hemin receptor; fyuA, Siderophore receptor (yersiniabactin); iroN, Siderophore receptor; irp2, High-molecular-weight protein 2 nonribosomal peptide synthetase; iucC, Aerobactin siderophore synthetase; iutA, Ferric aerobactin siderophore receptor; sitA, Iron transport protein. *Multi-drug resistant isolate.

Electro-peroxone process for triclocarban and triclosan removal and reclaimed water disinfection

Water reclamation is a known solution to the water scarcity problem; however, concern over water contamination by emerging contaminants and pathogens is growing. This study investigates the simultaneous removal of emerging contaminants and pathogens by applying the continuous-flow electro-peroxone process. Triclocarban (TCC) and triclosan (TCS) were selected as model contaminants of emerging concern, while Escherichia coli strain DH5α was chosen as a representative pathogen. The effects of the initial concentration, water flow rate, and water matrices (direct reclaimed water (treated wastewater), indirect reclaimed water (groundwater), and natural surface water) on TCC, TCS, and E. coli removal were examined. The removal efficiency of TCC (2 mg/L) was up to 92.54 % in 120 min. TCS (non-ozone-resistant compound) was effectively removed in 15 min, while E. coli was instantly inactivated in 3 min. The increase in flow rate decreased the electrical energy per order (EEO) of TCC removal, with the lowest EEO of 19 kWh/m3 at 45 mL/min. The dissolved organic carbon content in water matrices (consuming O3 and H2O2) markedly inhibited the TCC removal, and the kinetic rate as well as reduced 3,4-dichloroaniline (intermediate product) formation. Overall, these findings show the use of the electro-peroxone process is effective in mitigating ozone- and non-ozone-resistant emerging contaminants (TCC and TCS) and pathogens with consideration of water matrices for reclaimed water application.

Innovative DIY drinking water disinfection for underserved communities

Waterborne pathogens threaten 2.2 billion people lacking access to safely managed drinking water services, causing over a million annual diarrheal deaths. Individuals without access to chlorine reagents or filtration devices often resort to do-it-yourself (DIY) methods, such as boiling or solar disinfection (SODIS). However, these methods are not simple to implement. In this study, we introduced an innovative and easily implemented disinfection approach. We discovered that immersing aluminum foil in various alkaline solutions produces alkali-treated aluminum foil (ATA foil) that effectively adsorbs Escherichia coli (E. coli), Salmonella, and Acinetobacter through the generated surface aluminum hydroxide. For example, a 25 cm2 ATA foil efficiently captures all 104E. coli DH5α strains in 100 mL water within 30 min. Using a saturated suspension of magnesium hydroxide, a type of fertilizer, as the alkaline solution, the properties of the saturated suspension eliminate the need for measuring reagents or changing solutions, making it easy for anyone to create ATA foil. ATA foils can be conveniently produced within mesh bags and placed in household water containers, reducing the risk of recontamination. Replacing the ATA foil with a foil improves the adsorption efficiency, and re-immersing the used foil in the production suspension restores its adsorption capacity. Consequently, ATA foil is an accessible and user-friendly alternative DIY method for underserved communities. Verification experiments covering variations in the water quality and climate are crucial for validating the efficacy of the foil. Fortunately, the ATA foil, with DIY characteristics similar to those of boiling and SODIS, is well-suited for testing under diverse global conditions, offering a promising solution for addressing waterborne pathogens worldwide.

Expression and purification of truncated recombinant human angiotensin converting enzyme 2 (trhACE2) capable of binding spike protein of SARS-CoV-2

SARS-CoV-2 infects the host through the interaction be-tween spike protein and the ACE2 (Angiotensin convert-ing enzyme 2) receptor on the host cell surface. There-fore, ACE2 is considered as a key target in drug devel-opment against COVID-19. In this study, we generated the truncated recombinant human ACE2 (trhACE2) expressed on Escherichia coli and evaluated its binding activity to spike protein of SARS-CoV-2. The sequence of ACE2 (18-119 aa) was cloned into pET28a(+) plasmid and transformed into E. coli DH5α strain and finally was transferred to E. coli BL21 (DE3) cells for protein expression. Protein trhACE2 was purified by affinity chromatography using a HisTrap HP column. Protein purity was above 95% and production yield was 75 mg/l. The purified protein was refolded by dialysis method. trhACE2 was evaluated for its ability to bind the spike protein by sandwich ELISA assay and surface plasmon resonance (SPR). The results showed that trhACE2 has the ability to bind spike protein of SARS-CoV-2 with the equilibrium dissociation constant Kd=15.7 nM. With a relatively simple and inexpensive expression and purifi-cation process on E. coli system, there is the potential to develop trhACE2 for preventive and supportive thera-pies against COVID-19.

Induction of proteome changes involved in the cloning of mcr-1 and mcr-2 genes in Escherichia coli DH5-α strain to evaluate colistin resistance

Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli-carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Plasmid extraction was performed using an alkaline lysis-based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2-mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms.

Characterization of sugarcane mutants developed through gamma irradiations for their lignin content and caffeic acid-O-methyl transferase (COMT) gene mutations

Purpose Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. Materials and methods The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. Results In comparison to the control (21.5%), the mutant plants’ lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. Conclusions Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The Km and Vm values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

Enhancing rural drinking water safety using an Mg–Al-type layered double hydroxide foil as a new point-of-use disinfection tool

Abstract Point-of-use (POU) drinking water treatment is crucial for residents in resource-constrained areas. This study introduces a layered double hydroxide (LDH) foil, an innovative POU device that eliminates waterborne pathogens via adsorption. It is an aluminum foil coated with magnesium–aluminum (Mg–Al)-type LDH, a recognized human-safe pathogen adsorbent. Its sheet-like morphology enables easy retrieval of inherently powdered LDH from post-disinfected water. Coating with Mg–Al LDH foils was accomplished by immersing aluminum foils in alkaline magnesium solutions, resulting in the on-surface formation of LDH. Using 12 distinct alkaline magnesium solutions, 12 types of LDH foils were prepared. Most LDH foils demonstrated >99% removal of Escherichia coli (DH5α strain) within 3–24 h when 100 mL of water was inoculated with DH5α at approximately 103 colony-forming units (CFU)/mL agitated with a 25-cm2 LDH foil piece. The LDH foil with the highest efficacy had a maximum adsorption capacity of approximately 1.3 × 106 CFU per 1-cm2 LDH foil. Furthermore, the LDH foil was regenerated by submersing it in the alkaline solution during its preparation, enabling multiple reuses. Owing to its straightforward production and application, the LDH foil holds substantial promise as a convenient-to-use adsorbent for batch water disinfection, such as jug water, in rural households.

Разработка и оценка эффективности ранозаживляющих соединений на основе катионных пептидов и фуллерена

Skin and soft tissue infections following surgical procedures are usually caused by a broad range of bacteria and are the major cause of septic complications and hospital mortality. Treatment of such wounds is a challenge often resulting from the transition from acute to chronic inflammation due to persistence of pathogenic microflora in the wound tissue. The study was aimed to assess the wound-healing activity of the ointment composition based on the dispersion of fullerene C60 (AFD) in the in vivo model of skin wound, to estimate the effects of AFD on the expression of cytokines as markers of regenerative processes, to determine antibacterial activity of the developed cationic peptides. AFD was obtained by tangential ultrafiltration and used to make an ointment composition. The BALB/c mice were used to model the skin injury. The cationic peptides (CPs) were synthesized by the solid-phase method using the Fmoc technology. Antibacterial effects of CPs and AFD were estimated by colony counting. It was found that the AFD-based ointment exerted wound-healing and anti-inflammatory activity. The minimum bactericidal concentrations (MBC) of the CPs most active against the E. coli Dh5α strain, AB-1, AB-2, AB-3, and ST-10, were 1.15, 0.11, 0.74, and 0.74 mM, respectively, while MBC of ampicillin was 0.7 mM. We assume that constructing the hybrid compounds/fullerene C60 conjugates with active CPs will be a promising area of the development of drugs for treatment of wounds complicated by bacterial infection.

Identification and characterization of a novel chromosomal aminoglycoside 3'-O-phosphotransferase, APH(3')-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm.

Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention. The bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3')-Id gene and its phylogenetic relationship with other AMEs. A novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'-O-phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3')-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M-1/s-1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M-1/s-1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M-1/s-1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M-1/s-1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3')-Id gene. In this work, a novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.

Pratylenchus zeae a causative agent of Corn Root Rot in Jiangsu Province of China.

Corn (Zea mays L.) plays an important role in China's cash crops, not only as food, but a vital raw material for animal husbandry and industry (Li et al. 2022). Pratylenchus zeae is one of the most damaging root-lesion nematodes (RLN) that can result in decreased yield and quality of crops (Liu et al. 2017). In September 2020, five root/soil samples were collected from the rhizosphere of corn (cv. Zhengdan 958), which had weak growth and root brown lesions in Chenzhou Village, Taolin Town, Donghai County, Lianyungang City, Jiangsu Province of China. Nematodes were extracted from the collected samples using the modified Baermann funnel method (Hooper et al. 2005). RLN were found in all samples, an average of 46 RLN per gram of root and 138 RLN per 100 cm3 of soil. The obtained RLN females were sterilized with 0.3% streptomycin sulfate and then inoculated on each carrot disks individually to obtain the purified population. RLN were examined by morphological and molecular characteristics to confirm the species indentification. The main morphological measurements of adult (n = 15) included body length = 524.7 μm (mean) ± 15.1 (standard deviation) (range = 490.7 to 543.6 μm), stylet = 15.2 μm ± 0.8 (14.2 to 16.8 μm), tail length = 30.3 μm ± 2.5 (26.3 to 35.3 μm), a = 25.6 ± 1.3 (24.4 to 29.3), b = 5.3 ± 0.3 (4.7 to 5.8), c = 17.4 ± 1.4 (14.9 to 19.3), two annules on the lip region. No males were found in the specimens. The morphological characters of this population are consistent with the description of P. zeae (Castillo and Vovlas, 2007). Furthermore, DNA was extracted from individual nematodes. The primers of TW81/AB28 and D2A/D3B (Subbotin et al. 2006) were used to amplified the rDNA-ITS region and rDNA 28S D2-D3 region, respectively. The purified PCR products were ligated into One step ZTOPO-Blunt/TA vector and transformed to Escherichia coli strain DH5α, and then sequenced by Sunya Biotechnology Co., Ltd (Henan, China). The obtained seqences were submitted to NCBI. The rDNA-ITS sequences (669 bp, GenBank Accession No: OP456372 and OP466367) exhibited 95.0% to 97.1% of identity with P. zeae sequences (KU198980 and KU198975). The obtained D2-D3 region of the 28S rDNA sequences (782 bp, OP441397 and OP448675) exhibited 99.7% to 100% identity with P. zeae sequences (EU130893 and KY424269). Consequently, both morphological and molecular data confirmed the identity of P. zeae. To further confirm reproduction on corn, single corn seeds (cv. Zhengdan 958) were sown in eight 2-liter pots filled with 1.8-liter of sterilized soil in greenhouse at 28°C. About 15 days after sowing, each pot with one corn plant with the same growth status was selected to inoculate with 1,000 mixed stage nematodes of P. zeae , Eight pots of uninoculated corn plants were used as controls. After 60 days, the inoculated plants were harvested and brown lesions were observed on roots. No symptoms and nematodes was detected in the control. An average number of RLN per pot was 3,752 in soil and 1,183 in roots were extracted, the reproduction factor (final population/initial population) was 4.94, indicating that P. zeae infects and reproduces well on this corn cultivar. P. zeae has only been reported on corn in Guangxi Province, southern in China(Fang et al. 1994). To our knowledge, this is the fist report of P. zeae infecting corn in Jiangsu Province, eastern in China. As P. zeae can cause great damage to corn, necessary measures should be taken to prevent the spread of P. zeae to other areas.

Exploring the Potential of a Genome-Reduced Escherichia coli Strain for Plasmid DNA Production.

The global demand for nucleic acid-based vaccines, including plasmid DNA (pDNA) and mRNA vaccines, needs efficient production platforms. However, conventional hosts for plasmid production have encountered challenges related to sequence integrity due to the presence of insertion sequences (ISs). In this study, we explored the potential of a genome-reduced Escherichia coli as a host for pDNA production. This strain had been constructed by removing approximately 23% of the genome which were unessential genes, including the genomic unstable elements. Moreover, the strain exhibits an elevated level of NADPH, a coenzyme known to increase plasmid production according to a mathematical model. We hypothesized that the combination of genome reduction and the abundance of NADPH would significantly enhance pDNA production capabilities. Remarkably, our results confirmed a three-fold increase in pDNA production compared to the widely employed DH5α strain. Furthermore, the genome-reduced strain exhibited heightened sensitivity to various antibiotics, bolstering its potential for large scale industrial pDNA production. These findings suggest the genome-reduced E. coli as an exciting candidate for revolutionizing the pDNA industry, offering unprecedented efficiency and productivity.

Optimisation of transformation in the heterologous fusion gene GAPDH-IFN cloning using DH5α strain of Escherichia coli

DNA vaccines are the most essential tool of the disease prevention strategy. In the present study, cloning of a heterologous fusion gene involving glyceraldehyde 3-phosphate dehydrogenase and interferon gamma (GAPDH-IFN) was conceptualised. Cloning was tried using four distinct transformation techniques viz. InstAclone PCR Cloning kit (Fermentas, USA); CaCl2 transformation protocol; Clontech stellar competent cells protocol and PEG 8000-mediated transformation method, for the heterologous GAPDH-IFN fusion gene using DH5α strain of Escherichia coli. The first three methods were found to be unsuitable, and the PEG 8000-mediated transformation method yielded positive clones. Keywords: DNA vcaccine, Edwardsiella tarda, Fusion genes, Labeo rohita, PEG 8000, Transformation methods

Identification and characterization of a novel aminoglycoside O-nucleotidyltransferase ANT(6)-If from Paenibacillus thiaminolyticus PATH554.

Paenibacillus thiaminolyticus, a species of genus Paenibacillus of the family Paenibacillaceae, exists widely in environments and habitats in various plants and worms, and occasionally causes human infections. This work aimed to characterize the function of a novel aminoglycoside O-nucleotidyltransferase resistance gene, designated ant(6)-If, from a P. thiaminolyticus strain PATH554. Molecular cloning, antimicrobial susceptibility testing, enzyme expression and purification, and kinetic analysis were used to validate the function of the novel gene. Whole-genome sequencing and comparative genomic analysis were performed to investigate the phylogenetic relationship of ANT(6)-If and other aminoglycoside O-nucleotidyltransferases, and the synteny of ant(6)-If related sequences. The recombinant with the cloned ant(6)-If gene (pMD19-ant(6)-If/DH5α) demonstrated a 128-fold increase of minimum inhibitory concentration level against streptomycin, compared with the control strains (DH5α and pMD19/DH5α). The kinetic parameter kcat/Km of ANT(6)-If for streptomycin was 9.01 × 103 M-1·s-1. Among the function-characterized resistance genes, ANT(6)-If shared the highest amino acid sequence identity of 75.35% with AadK. The ant(6)-If gene was located within a relatively conserved genomic region in the chromosome. ant(6)-If conferred resistance to streptomycin. The study of a novel resistance gene in an unusual environmental bacterium in this work contributed to elucidating the resistance mechanisms in the microorganisms.

First report of meadow saffron breaking virus on wild Colchicum autumnale from a stricly protected Natura2000 site at a Hungarian National Park.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants - which are native to Hungary - at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results. The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen's phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5'-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3') primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5'-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3') and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain. The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.

Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) extracts: promising prospects for anticancer and antibacterial potential through in vitro and in silico studies

Neolamarckia cadamba is an Indian traditional medicinal plant having various therapeutic potentials. In the present study, we did solvent-based extraction of Neolamarckia cadamba leaves. The extracted samples were screened against liver cancer cell line (HepG2) and bacteria (Escherichia coli). MTT cytotoxic assay was performed for in vitro analysis of extracted samples against the HepG2 cell lines and the normal human prostate PNT2 cell line. Chloroform extract of Neolamarckia cadamba leaves showed better activity with IC50 value 69μg/ml. DH5α strain of Escherichia coli (E. coli) was cultured in Luria Bertani (LB) broth media and minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) were calculated. Solvent extract chloroform showed better activity in MTT analysis and antibacterial screening and it was taken for characterization of phytocomposition by Fourier transform infrared (FTIR) and gas chromatography mass spectrometry (GC-MS). The identified phytoconstituents were docked with potential targets of liver cancer and E. coli. The phytochemical 1-(5-Hydroxy-6-hydroxymethyl-tetrahydropyran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione shows highest docking score against the targets PDGFRA (PDB ID: 6JOL) and Beta-ketoacyl synthase 1(PDB ID: 1FJ4) and their stability was further confirmed by molecular dynamics simulation studies.

Simple approach for expression and rapid purification of Taq DNA polymerase in three Escherichia coli strains

Background and objectives: One of the most commonly employed bacterial hosts for the synthesis of recombinant proteins is Escherichia coli (E. coli). Choosing an ideal host for the production of the protein of interest is an important step in the large-scale production process. Due to its thermostable characteristic, Taq Pol I, which was first isolated from the bacterium Thermus aquaticus (Taq), is now a typical enzyme found in many laboratories. This study aimed to identify the ideal host for large-scale production of Taq Pol I and purify the enzyme under a simple and rapid purification method. Methods: Taq Pol I gene in pSE420 plasmid was overexpressed in E. coli strains DH5α, TOP10 and BL21(DE3) pLysS. The enzyme was purified using Pluthero’s method and dialyzed using Amicon® Ultra-4 Centrifugal Filter. Results: The host strain E. coli TOP10 produced the highest amounts of Taq Pol I, followed by DH5α and BL21(DE3) pLysS. An estimated 4.5 U/μL of Taq Pol I was produced from a 200 mL culture of TOP10 host. Conclusion: This study provides data on the capacity of the E. coli strains used for Taq Pol I protein production. This information can be used to accelerate future targeted strain selection for the production of a specific protein of interest.

Evaluation of Proteins Released to Medium in Yeast-Bacteria Co-culture System

The natural ecosystem contains an astonishing variety of microbial species. Populations of microorga-nisms exist in a common habitat in terms of access to nutrients and other resources in the environment. Cultivation of microorganisms in ideal laboratory conditions seperates them from their natural ecosystem conditions and isolates them from their microbial world, especially from their competitors. With traditional pure culture-oriented cultuvation techniques used in laboratories, interactions mediated by small molecules are not taken into account, resulting in the precise nature of the interactions being largely unknown. Coculture systems are systems in which two or more different cell populations are grown together. In this way, studies on natural interactions between populations can be made and synthetic interactions that are not observed in nature can be provided. With these systems, natural product discovery, microbial ecology, evolution and pathogenesis studies are carried out. In addition, coculture systems are also used in industrial, environmental and medical studies. In this study, the wild strain of Schizosaccharomyces pombe and the DH5α strain of Escherichia coli were grown in their own specific media, then cultured for 48 hours and 72 hours by cultivating in media containing 0,1% glucose with different cell number, and finally the differentiation in the proteins released by the cells into the medium was observed in SDS polyacrylamide gels. Different from the control conditions, new protein bands that emerged under the co-culture conditions were detected and two of these bands were analyzed by mass spectrometry (MS). While 6 of differentaited proteins were released by S.pombe, 257 proteins matched with E.coli proteom. These proteins are; Various carbohydrate-binding proteins, membrane proteins involved in the identification of various signaling molecules and antibiotics, and other proteins involved in various cellular processes.

Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia.

The intrinsic resistance mechanism plays an essential role in the bacterial resistance to a variety of the antimicrobials. The aim of this study is to find the chromosome-encoded novel antimicrobial resistance gene in the clinical isolate. The function of the predicted resistance gene was verified by gene cloning and antibiotic susceptibility test. Recombinant protein expression and enzyme kinetic studies were performed to explore the in vivo activity of the enzyme. Expression of the resistance gene exposed to antimicrobial was determined by RT-qPCR. Whole genome sequencing and bioinformatic analysis were applied to analyze the genetic context of the resistance gene. The novel aminoglycoside (AG) resistance genes designated aph(9)-Ic and aph(9)-Ic1 confer resistance to spectinomycin, and a recombinant strain harboring aph(9)-Ic (pMD19-T-aph(9)-Ic/DH5α) showed a significantly increased minimum inhibitory concentration (MIC) level against spectinomycin compared with the control strains (DH5α and pMD19-T/DH5α). The result of the kinetic analysis of APH(9)-Ic was consistent with the MIC result for the recombinant pMD19-T-aph(9)-Ic/DH5α, showing the efficient catalytic activity for spectinomycin [kcat/Km ratio = (5.58 ± 0.31) × 104 M-1·s-1]. Whole-genome sequencing demonstrated that the aph(9)-Ic gene was located on the chromosome with a relatively conserved genetic environment, and no mobile genetic element was found in its surrounding region. Among all the function-characterized resistance genes, APH(9)-Ic shares the highest amino acid sequence identity of 33.75% with APH(9)-Ia. We characterized a novel AG resistance gene aph(9)-Ic and its variant aph(9)-Ic1 that mediated spectinomycin resistance from S. maltophilia. The identification of the novel AG resistance genes will assist us in elucidating the complexity of resistance mechanisms in microbial populations.