Genome-Wide Screens Identify Genes Responsible for Intrinsic Boric Acid Resistance in Escherichia coli.

Boric acid (BA) has antimicrobial properties and is used to combat bacterial infections, including Enterobacteria. However, the molecular mechanisms and cellular responses to BA are still unknown. This genomics study aims to provide new information on the genes and molecular mechanisms related to the antimicrobial effect of BA in Escherichia coli. The Keio collection of E. coli was used to screen 3985 single-gene knockout strains in order to identify mutant strains that were sensitive or hypersensitive to BA at certain concentrations. The mutant strains were exposed to different concentrations of BA ranging from 0 to 120 mM in LB media. Through genome-wide screens, 92 mutants were identified that were relatively sensitive to BA at least at one concentration tested. The related biological processes in the particular cellular system were listed. This study demonstrates that intrinsic BA resistance is the result of various mechanisms acting together. Additionally, we identified eighteen out of ninety-two mutant strains (Delta_aceF, aroK, cheZ, dinJ, galS, garP, glxK, nohA, talB, torR, trmU, trpR, yddE, yfeS, ygaV, ylaC, yoaC, yohN) that exhibited sensitivity using other methods. To increase sensitivity to BA, we constructed double and triple knockout mutants of the selected sensitive mutants. In certain instances, engineered double and triple mutants exhibited significantly amplified effects. Overall, our analysis of these findings offers further understanding of the mechanisms behind BA toxicity and intrinsic resistance in E. coli.

Functional groups matter: metabolomics analysis of Escherichia coli exposed to trans-cinnamic acid and its derivatives unveils common and unique targets.

Phenolic acids are derivatives of benzoic and cinnamic acids, which possess important biological activities at certain concentrations. Trans-cinnamic acid (t-CA) and its derivatives, such as p-coumaric acid (p-CA) and ferulic acid (FA) have been shown to have antibacterial activity against various Gram-positive and -negative bacteria. However, there is limited information available concerning the antibacterial mode of action of these phenolic acids. In this study, we aimed to ascertain metabolic alterations associated with exposure to t-CA, p-CA, and FA in Escherichia coli BW25113 using a nuclear magnetic resonance (NMR)-based metabolomics approach. The results showed that t-CA, p-CA, and FA treatments led to significant changes (p < 0.05) in the concentration of 42, 55, and 74% of the identified metabolites in E. coli, respectively. Partial least-squares discriminant analysis (PLS-DA) revealed a clear separation between control and phenolic acid groups with regard to metabolic response. Moreover, it was found that FA and p-CA treatment groups were clustered closely together but separated from the t-CA treatment group. Arginine, putrescine, cadaverine, galactose, and sucrose had the greatest impact on group differentiation. Quantitative pathway analysis demonstrated that arginine and proline, pyrimidine, glutathione, and galactose metabolisms, as well as aminoacyl-tRNA and arginine biosyntheses, were markedly affected by all phenolic acids. Finally, the H2O2 content of E. coli cells was significantly increased in response to t-CA and p-CA whereas all phenolic acids caused a dramatic increase in the number of apurinic/apyrimidinic sites. Overall, this study suggests that the metabolic response of E. coli cells to t-CA is relatively different from that to p-CA and FA. However, all phenolic acids had a certain impact on oxidative/antioxidant status, genomic stability, arginine-related pathways, and nucleic acid metabolism.

NMR metabolomics analysis of Escherichia coli cells treated with Turkish propolis water extract reveals nucleic acid metabolism as the major target.

AIMS: Propolis is a resinous bee product containing several hundred biologically active compounds. Although the antibacterial activity of propolis has been demonstrated in many in vitro studies, less is known about its mode of action. In this study, we aimed to shed some light on the antibacterial mechanism of action of propolis against Escherichia coli BW25113 using a nuclear magnetic resonance (NMR) based metabolomics approach. METHODS: E. coli BW25113 cells were subjected to different sub-lethal concentrations (0, 2, 4, and 6 mg/mL) of Turkish propolis water extract (PWE). The 500-MHz 1H NMR spectroscopy was then employed to ascertain the metabolic profiles of E. coli extracts. RESULTS: A total of 52 metabolites were identified from the NMR spectra, belonging to 17 main classes, such as amino acids and peptides, purines, and fatty acids. Twelve out of these 52 metabolites displayed remarkable changes at all tested PWE concentrations when compared to control conditions (P < .05). Levels of 28 more metabolites were significantly altered in at least one of the three PWE treatments. The results of partial least squares discriminant analysis showed that there was a clear separation between control and propolis-treated cells and that putrescine, adenine, adenosine, guanosine, glucose, N6-acetyllysine, and acetamide had the highest effect on group differentiation. Finally, quantitative pathway analysis revealed that purine metabolism was significantly affected by PWE treatments. CONCLUSIONS: Our results suggest that PWE inhibits the growth of E. coli BW25113 by affecting nucleic acid metabolism to a great extent. To the best of our knowledge, this is the first study to evaluate the global metabolic response of a bacterium to propolis.

Synovial fluid niche promoted differentiation of dental follicle mesenchymal stem cells toward chondrogenesis in rheumatoid arthritis.

Objectives: In this study, we aimed to investigate the differentiation potential of dental follicle mesenchymal stem cells (MSCs) in the synovial fluid (SF) niche of early-onset or end-stage rheumatoid arthritis (RA). Patients and methods: Between May 2020 and January 2021, six patients (1 male, 5 females; mean age: 57.5±11.2 years; range, 49 to 65 years) who were diagnosed with RA with the indication of SF aspiration were included in the study. The third passage dental follicle stem cells (DFSCs) were cocultured with fresh SF samples of end-stage or early-onset RA patients in micromass culture system for 21 days. SF samples were analyzed for secreted cytokines. Chondrogenic markers (CD49e, CD49f) were analyzed in DFSCs, gene expression analysis was performed for the expressions of Col I, Col II, Aggrecan and Sox-9, and histochemical analysis was performed by staining three-dimensional pellets with anti-collagen II antibody. The neutralization assay was performed with anti-interleukin (IL)-6, anti-interferon-gamma (IFN-g), and anti-IL-1beta(b). Results: The high levels of IL-1b and IL-6 were observed in end-stage RA patients' SF samples compared to the early-onset patients (p<0.05). The CD49e and CD49f expressions in DFSCs were significantly higher in the SF samples of end-stage RA patients (p<0.05). Also, the Col II, Sox-9 and Aggrecan messenger ribonucleic acid (mRNA) expressions increased in the DFSCs, when cultured with end-stage RA patients' SF samples (p<0.01). Collagen-II expression in histochemical analysis of micromass pellets was higher in the DFSCs cultured with end-stage RA patients' SF samples. The neutralization of IL-6 significantly decreased the CD49e and CD49f expressions (p<0.05). Conclusion: The high levels of IL-6 in SF niche of end-stage RA patients were found to differentiate DFSCs toward chondrogenesis. Based on these findings, DFSCs can be used as a new cell-based treatment in RA patients for the cartilage damage.

Analysis of the fluid biochemistry in patients with prolonged wound drainage after hip hemiarthroplasty.

INTRODUCTION: The origin and content of prolonged wound drainage (PWD) after arthroplasty remain uncertain. In this study, we performed the biochemical, biological and advanced proteomic analysis of the drainage fluid collected from PWD patients following hip hemiarthroplasty (HA). METHODS: Data of 28 patients who developed PWD after HA were prospectively analyzed. After examining the biochemical content of the drainage fluid collected on postoperative day 6, to find out if the drainage fluid was transudate or exudate, it was compared with the patient's serum values according to the Light criteria. Subsequently, biological and proteomic analyzes of both drainage fluid and serum were performed. The similarities and differences in terms of protein concentrations, protein identities were examined. In the drainage fluid, we analyzed lymph-specific proteins. RESULTS: 16 patients with PWD were male (61.1%), 12 were female (38.9%), and the mean age of all patients was 79.64 ± 8.44 (65-95). Biochemical test results of the drainage fluid / serum were as follows: Total protein: 2.1 / 5.2 g/dl, albumin: 1.3 / 3.1 g/dl, lactate dehydrogenase (LDH): 121/324 U/l, cholesterol: 28/160 mg/dl, triglyceride: 37/122 mg/dl, sodium (Na): 140/ 140mg/dl, potassium (K): 4.1/ 4.1 mg/dl. pH of the drainage fluid was 7.6. According to these biochemical values, drainage fluid was classified as transudate. As a result of protein identification, fibrinogen beta chain, keratin type 1, creatine kinase M-type protein were detected in drainage fluid. Subsequent western analysis revealed that, gliseraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin antibody were detected in the drainage fluid but not in serum. CONCLUSION: Despite the similarity in serum and transudative PWD fluid in terms of biochemical content, we found that when we carried out further proteomic analysis, PWD contains lymph-specific proteins. Unlike PWD, these proteins were not determined in serum. PWD fluid can be also called as lymphorrhea. PWD fluid with abundant proteins may also provide an appropriate environment for the growth of microorganisms.

Genome Sequence of the Boron-Tolerant and -Requiring Bacterium Bacillus boroniphilus.

Bacillus boroniphilus is a highly boron-tolerant bacterium that also requires this element for its growth. The complete genome sequence of B. boroniphilus was determined by a combination of shotgun sequencing and paired-end sequencing using 454 pyrosequencing technology. A total of 84,872,624 reads from shotgun sequencing and a total of 194,092,510 reads from paired-end sequencing were assembled using Newbler 2.3. The estimated size of the draft genome is 5.2 Mb.

Boron contents of some wild-growing mushrooms collected from the vicinity of boron mines in Balikesir, Turkey.

The aim of the study was to determine the boron contents of some wild-growing mushrooms collected from the vicinity of several boron mines located in Balikesir, Turkey and compare the boron contents of some mushroom and soil samples. The locations of the mushroom samples collected were within the distance of 0-100, 100-500, and 500-1,000 m to the mines. Soil samples were taken from beneath randomly selected fungal fruit bodies. A total of 40 mushroom samples were found in the study area and 37 different species were identified. Among the 40 samples analyzed, the highest boron content was detected to be 273 mg kg(-1) in Suillus collinitus. Also, Tricholoma terreum (69.52 mg kg(-1)), Myxomphalia maura (36.52 mg kg(-1)), Otidea concinna (30.70 mg kg(-1)), Sepultaria arenosa (28.94 mg kg(-1)), Melanoleuca paedida (28.33 mg kg(-1)), and Lycoperdon lividum (28.31 mg kg(-1)) relatively have high levels of boron and these species are proposed as candidates for boron accumulation. The boron contents of the other mushrooms varied and a pattern was observed where the boron content was found to be decreased as the distance from the mines increased.

Translational regulation of human methionine synthase by upstream open reading frames.

Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5' leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence.