Search Results (67)

Background: Iminosugars are natural or synthetic sugar analogues with a very broad spectrum of activities, including those against the most prominent bacterial pathogens, like P. aeruginosa or S. aureus. In a series of studies, we have demonstrated that one of the synthetic iminosugars, PDIA (beta-1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol), possesses the ability to suppress biofilm production by different pathogenic bacteria without inhibiting their growth. Thereby, PDIA is able to influence experimental skin infection caused by S. aureus. Methods: To elucidate molecular mechanisms by which PDIA impedes biofilm formation by S. aureus, a transcriptomic study was performed in which a biofilm-producing S. aureus strain was grown in the presence of PDIA for 24 and 48 h in comparison to a control without the iminosugar. The RNA was then isolated, converted into cDNA, sequenced, and data analysis was performed. Results: It appeared that PDIA caused the down-regulation of many bacteriophage genes responsible for the processes of bacterial cell lysis, and some genes responsible for cell wall degradation were also down-regulated. Among the 25 most upregulated genes were those representing the phosphotransferase system (PTS), which is required for carbohydrate uptake and control of carbon metabolism. The ranking of the most significant down-regulated genes after 24 h exposure to PDIA shows that they predominantly coded for both the synthesis and lysis of the peptidoglycan. Conclusions: We have shown here that the influence of PDIA on the expression of S. aureus genes is broad and affects many genes encoding metabolism and ribosomes. Full article

Xenorhabdus nematophila has excellent potential for application in both medicine and agriculture due to its various active secondary metabolites. The transcriptional regulator OmpR negatively regulates Xenocoumacin 1 (Xcn1), which has wide antimicrobial activity. Here, we expressed and purified OmpR and verified its binding activities to promoters via an electrophoretic mobility shift assay. RNA sequencing was used to analyze the relevance and difference of differentially expressed genes between X. nematophila and its mutant ΔompR. Compared with the WT, 1127 differentially expressed genes were found in ΔompR, while 4150 co-expressed genes were detected. RT-qPCR data validated the RNA-seq results with 20 randomly selected genes. OmpR positively regulates the process of porphyrin metabolism, quorum sensing, β-Lactam resistance and glyoxylate and dicarboxylate metabolism, while negatively regulating the phosphotransferase system, two-component system and bacterial chemotaxis. OmpR indirectly regulates the biosynthesis of Xcn1 by positively regulating the process of glyoxylate metabolism, which consumes energy and precursors, and negatively regulates biomacromolecules biosynthesis, which provides energy and precursors. Overall, this work revealed the indirect effects of OmpR on the biosynthesis of Xcn1, serving as a foundation for future research into the intricate regulatory network of X. nematophila. Full article

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that seriously harms the swine industry and human health. However, its pathogenic mechanisms are largely unknown, and the few virulence factors reported so far are insufficient to systematically explain its infectious and pathogenic mechanisms. In preliminary research, we identified a gene named G22 encoding a hypothetical secreted protein that may be closely associated with the high-level pathogenicity of S. suis. In this study, we constructed deletion and complementation strains of the G22 gene through homologous recombination and explored its roles in the pathogenicity and susceptibility of S. suis to environmental stresses through in vitro and in vivo experiments. The deletion of G22 clearly influenced the typical capsular structure of SS2 and impaired the bacterium’s growth in a medium containing hydrogen peroxide (showing a growth reduction of 32.98% ± 5.23% compared to the wild-type strain SC19, p < 0.001) or with a low pH (with a growth inhibition of 17.44% ± 1.9% relative to the wild-type strain SC19, p < 0.01). ΔG22 also showed reduced survival in whole blood and in RAW 264.7 macrophages (with a survival reduction of 16.44% ± 2.29% compared to the wild-type, p < 0.001). The deletion of G22 also sharply attenuated the virulence of SS2 in a mouse infection model (reducing the mortality rate by 50% ± 0.04%, p < 0.05). We also demonstrated that G22 is required for the adhesion and invasion of SS2 in host cells. An RNA sequencing analysis revealed that 50 genes were differentially expressed in the ΔG22 and wild-type strains: 23 upregulated and 37 downregulated. Many of the genes are involved in carbohydrate metabolism and the synthesis of virulence-associated factors. Several genes associated with the phosphotransferase system were significantly upregulated in strain ΔG22. In summary, G22 plays a role in the morphological development and pathogenesis of the highly virulent SS2 strain SC19. Full article

Background/Objectives: Limosilactobacillus fermentum KUB-D18, a heterofermentative lactic acid bacterium with promising probiotic properties, is known for promoting gut health and nutrient absorption. Originally isolated from chicken intestines, this strain demonstrates versatile metabolic capabilities in diverse gastrointestinal environments. However, the metabolic functions and sugar transport-related genes remain largely unexplored. This study thus aimed to dissect metabolic functions and sugar transports of L. fermentum KUB-D18. Methods: Next-generation and third-generation sequencing techniques using integrative genomic platform towards transportome analysis were performed. Results: The complete genome, sized at 2.12 Mbps with a GC content of 51.36%, revealed 2079 protein-encoding genes, of which 1876 protein functions were annotated and identified in top categories involved in amino acids, nucleotide, energy, and carbohydrate transports and metabolisms. Comparative genes analysis identified 50 core and 12 strain-specific genes linked to probiotic properties, e.g., acid resistances and bile tolerances, antioxidant functions, or anti-inflammatory properties. Further, sugar transportome analysis uncovered 57 transporter genes, demonstrating diverse carbon utilization and phosphotransferase (PTS) systems, corroborated by API 50 CHL test results for carbohydrate metabolism profile. Conclusions: These findings enhance the comprehensive metabolic understanding of L. fermentum KUB-D18, supporting its industrial potential and applications in engineered probiotics. Full article

Background: Coronary artery bypass grafting (CABG) is one of the main treatments for coronary heart disease (CHD). Gut microbiota, including bacteria, fungi, archaea, and virus, has been reported to be associated with CHD. However, the changes in the multi-kingdom gut microbiota after CABG are not yet clear. This study aimed to explore the changes in multi-kingdom gut microbiota during the early postoperative period of CABG. Methods: We collected fecal samples from 40 patients before and 1 week after CABG surgery. Metagenomic sequencing was used to detect the microbial spectrum and gene functions in the patients’ fecal samples. Results: Post-CABG patients exhibited significant changes in the composition of multi-kingdom gut microbiota and gene functions. Among bacteria, beneficial species such as Bifidobacterium, Bacteroides, and Blautia were significantly reduced after CABG, while the harmful species Enterococcus was significantly increased. In fungi, Schizosaccharomyces pombe was significantly decreased in the postoperative group, while Saccharomyces cerevisiae and Aspergillus chevalieri were significantly increased postoperatively. Spearman correlation analysis indicated that Schizosaccharomyces pombe had positive interactions with beneficial bacteria such as Lachnospiraceae, Ruminococcus, and Blautia. Among archaea, the preoperatively enriched Methanomethylovorans-SGB40959 was significantly reduced postoperatively, and Spearman correlation analysis showed a significant positive interaction with probiotics Ruminococcus and Dorea. In viruses, the phage Enterococcus virus EFP01, which infects Enterococcus, was significantly increased postoperatively and showed a significant positive interaction with Enterococcus. Additionally, postoperative dysregulation of gene functions such as the Phosphoenolpyruvate-dependent Sugar Phosphotransferase System (PTS), Transposition, DNA-mediated, and Transposase Activity was observed, and Spearman correlation analysis indicated significant correlations between the dysregulated gene functions and the microbial communities. Conclusions: This study comprehensively revealed the changes in multi-kingdom species post-CABG. The reduction of beneficial microorganisms and the increase of harmful microorganisms after surgery are of significant clinical importance for understanding the overall health status of post-CABG patients and for optimizing postoperative treatment plans. Future research needs to further explore how to improve the prognosis of post-CABG patients by modulating the gut microbiota. Full article

Bacillus microorganisms play an important role in the zearalenone (ZEA) biotransformation process in natural environments. The phosphotransferase pathway in Bacillus is both widespread and relatively well conserved. However, the reaction kinetics of these phosphotransferases remain poorly understood, and their catalytic activities are suboptimal. In this study, a ZEA phosphotransferase, ZPH1101, was identified from Bacillus subtilis 1101 using genome sequencing. The product transformed by ZPH1101 was identified as phosphorylated ZEA (ZEA-P) through LC-TOF-MS/MS analysis. The experiments conducted on MCF-7 cells demonstrated that ZEA-P exhibited a lower level of estrogenic toxicity than ZEA. The optimal reaction conditions for ZPH1101 were determined to be 45 °C and pH 8.0. The maximum velocity (V_max), Michaelis constant (K_m), and catalytic constant (k_cat) were calculated through fitting to be 16.40 μM·s⁻¹·mg⁻¹, 18.18 μM, and 54.69 s⁻¹, respectively. Furthermore, adding 1 mmol/L Fe²⁺ or Fe³⁺ to the reaction system increased the efficiency of ZPH1101 in converting ZEA by 100% relative to the system containing solely 1 mmol/L ATP and 1 mmol/L Mg²⁺, suggesting that low concentrations of Fe²⁺ or Fe³⁺ can improve the ZPH1101-mediated transformation of ZEA. This study contributes to the enzymatic removal of ZEA and broadens the spectrum of strain and enzyme options available to researchers for ZEA detoxification efforts. Full article

Bees play important roles in socio-economic development, biodiversity conservation, and ecosystem stability. However, during the cold season, resources become limited, leading to significant losses in bee colonies. Although many studies have described the characteristics of winter bees and demonstrated that notable changes occur in their gut microflora, the underlying mechanisms remain yet to be fully elucidated. Therefore, this study was conducted to compare the gut microbiota dynamics of overwintering bees. Sample acquisition involved randomly selecting ten colonies each from three bee farms containing Apis cerana (AC) and Apis mellifera (AM), followed by dissection for further analysis. DNA was extracted, and 16S rDNA sequencing, along with various bioinformatics tools, was used to assess microbial diversity, functional differences, and species comparisons between AC and AM gut microbiota. AC exhibited lower β diversity in the gut microbiota than AM during winter. Moreover, Gilliamella and Apibacter were relatively more abundant in AC. Regarding microbial functions, key pathways included the phosphotransferase system, galactose metabolism, the pentose phosphate pathway, and carbohydrate transport and metabolism. These results suggest the presence of microbial diversity differences between AC and AM, with the differential microbial functions mainly enriched in metabolic pathways that facilitate adaptation to cold environmental stress. Full article

The interaction between gut microbiota and the host immune system is a complex and understudied field, with cytokines like TNFα, IL-6, IL-8, and IL-10 playing pivotal roles. Commensal bacteria, including lactobacilli, respond to these cytokines through adaptive mechanisms that support their survival and function within the gut. While the influence of cytokines on pathogenic bacteria is well documented, their impact on commensal bacteria, particularly lactobacilli, remains underexplored. This study investigates the transcriptional responses of Lacticaseibacillus rhamnosus strains K32 and R19-3 to various cytokines using next-generation RNA sequencing (RNA-seq). Our findings reveal that cytokines, especially IL-8 and IL-10, significantly alter the L. rhamnosus transcriptome, affecting genes involved in carbohydrate metabolism, stress response, and transcriptional regulation. Notably, IL-8 and IL-10 induce a significant downregulation of genes related to the phosphotransferase system, suggesting a reduction in metabolic activity in response to inflammatory signals. This study unveils a previously unexplored aspect of L. rhamnosus adaptation, highlighting its intricate response to cytokine signals. By modulating gene expression, L. rhamnosus may mitigate the adverse effects of inflammation and promote gut health. These insights could inform the development of targeted probiotic therapies for inflammatory bowel disease (IBD) and other conditions with altered cytokine levels. Our results suggest that co-evolution between a host and gut microbiota enables bacteria to respond to specific cytokines through gene expression changes, revealing a unique and underexplored facet of the interaction between commensal bacteria and the host organism. Full article

Klebsiella pneumoniae (K. pneumoniae), a kind of zoonotic bacteria, is among the most common antibiotic-resistant pathogens, and it causes nosocomial infections that pose a threat to public health. In this study, the roles of synthetic bovine neutrophil β-defensin-5 (B5) in regulating inflammatory response and metabolic response against multidrug-resistant K. pneumoniae infection in a mouse model were investigated. Mice were administrated intranasally with 20 μg of B5 twice and challenged with K. pneumoniae three days after B5 pretreatment. Results showed that B5 failed to directly kill K. pneumoniae in vitro, but it provided effective protection against multidrug-resistant K. pneumoniae via decreasing the bacterial load in the lungs and spleen, and by alleviating K. pneumoniae-induced histopathological damage in the lungs. Furthermore, B5 significantly enhanced the mRNA expression of TNF-α, IL-1β, Cxcl1, Cxcl5, Ccl17, and Ccl22 and obviously enhanced the rapid recruitment of macrophages and dendritic cells in the lungs in the early infection phase, but significantly down-regulated the levels of TNF-α, IL-1β, and IL-17 in the lungs in the later infection phase. Moreover, RNA-seq results showed that K. pneumoniae infection activated signaling pathways related to natural killer cell-mediated cytotoxicity, IL-17 signaling pathway, inflammatory response, apoptosis, and necroptosis in the lungs, while B5 inhibited these signaling pathways. Additionally, K. pneumoniae challenge led to the suppression of glycerophospholipid metabolism, the phosphotransferase system, the activation of microbial metabolism in diverse environments, and metabolic pathways in the lungs. However, B5 significantly reversed these metabolic responses. Collectively, B5 can effectively regulate the inflammatory response caused by K. pneumoniae and offer protection against K. pneumoniae. B5 may be applied as an adjuvant to the existing antimicrobial therapy to control multidrug-resistant K. pneumoniae infection. Our study highlights the potential of B5 in enhancing pulmonary bacterial clearance and alleviating K. pneumoniae-caused inflammatory damage. Full article

Streptococcus mutans is a major pathogenic habitant of oral caries. Owing to its physiological and biochemical features, it prevails in the form of plaque biofilm together with another important mutans streptococci species, Streptococcus sobrinus. Both species are considered as initiators of cavity lesions, and biofilm is essential to the dental caries process. Compared with the planktonic populations, the biofilm form has higher resistance to environmental conditions and antibiotics. Dental plaques also secure the long-term survival of microorganisms and protection from any stress conditions. To address the need for new antibiofilm agents, we have focused on L-carnitine-fumarate, a fumarate-conjugated quaternary ammonium compound. Using the macro-broth susceptibility testing method, we established its MIC value as 6.0 mg/mL. The MBC value, determined from the broth dilution minimum inhibitory concentration test by sub-culturing it to BHI agar plates, was established as 7.0 mg/mL. Antibiofilm efficacy was tested in 96-well plates coated with saliva using BHI broth supplemented with 1% sucrose as a standard approach. The obtained results allowed us to assess the MIBC as 7.5 mg/mL and the MBBC value as 10.0 mg/mL. The latter concentration also caused approximately 20% eradication of pre-existing biofilm. EPS-rich matrix, forming the core of the biofilm and enabling a confined acidic microenvironment, was also examined and confirmed the effectiveness of 10.0 mg/mL L-carnitine-fumarate concentration in inhibiting EPS formation. Furthermore, the anti-adherent and anti-aciduric impacts of L-carnitine-fumarate were investigated and revealed significant inhibitory effects at sub-MIC concentrations. The influence of L-carnitine-fumarate on the phosphotransferase system was investigated as well. Our results provide a new insight into the antibacterial potential of L-carnitine-fumarate as a valuable compound to be considered for alternative or adjunct anti-caries and antibiofilm preventive approaches. Full article

Salt stress is detrimental to the survival of microorganisms, and only a few bacterial species produce hydrolytic enzymes. In this study, we investigated the expression of salt stress-related genes in the salt-tolerant bacterial strain Bacillus subtilis ACP81, isolated from bamboo shoot processing waste, at the transcription level. The results indicate that the strain could grow in 20% NaCl, and the sub-lethal concentration was 6% NaCl. Less neutral protease and higher cellulase and β-amylase activities were observed for B. subtilis ACP81 under sub-lethal concentrations than under the control concentration (0% NaCl). Transcriptome analysis showed that the strain adapted to high-salt conditions by upregulating the expression of genes involved in cellular processes (membrane synthesis) and defense systems (flagellar assembly, compatible solute transport, glucose metabolism, and the phosphotransferase system). Interestingly, genes encoding cellulase and β-amylase-related (malL, celB, and celC) were significantly upregulated and were involved in starch and sucrose metabolic pathways, and the accumulated glucose was effective in mitigating salt stress. RT-qPCR was performed to confirm the sequencing data. This study emphasizes that, under salt stress conditions, ACP81 exhibits enhanced cellulase and β-amylase activities, providing an important germplasm resource for saline soil reclamation and enzyme development. Full article

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality. Full article

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an energetic and persistent explosive with long-lasting properties. Rhodococcus sp. strain DN22 has been discovered to be a microbe capable of degrading RDX. Herein, the complete genome of Rhodococcus sp. strain DN22 was sequenced and analyzed. The entire sequences of genes that encoded the two proteins participating in RDX degradation in Rhodococcus sp. strain DN22 were obtained, and were validated through proteomic data. In addition, few studies have investigated the physiological changes and metabolic pathways occurring within Rhodococcus sp. cells when treated with RDX, particularly through mass spectrometry-based omics. Hence, proteomic and metabolomic analyses were carried out on Rhodococcus sp. strain DN22 with the existence or lack of RDX in the medium. A total of 3186 proteins were identified between the two groups, with 115 proteins being significantly differentially expressed proteins. There were 1056 metabolites identified in total, among which 130 metabolites were significantly different. Through the combined analysis of differential proteomics and metabolomics, KEGG pathways including two-component system, ABC transporters, alanine, aspartate and glutamate metabolism, arginine biosynthesis, purine metabolism, nitrogen metabolism, and phosphotransferase system (PTS), were observed to be significantly enriched. These findings provided ponderable perspectives on the physiological alterations and metabolic pathways in Rhodococcus sp. strain DN22, responding to the existence or lack of RDX. This study is anticipated to expand the knowledge of Rhodococcus sp. strain DN22, as well as advancing understanding of microbial degradation. Full article

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes’ integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum. Full article

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing annually, and emerging evidence suggests that the gut microbiota plays a causative role in the development of NAFLD. However, the role of gut microbiota in the development of NAFLD remains unclear and warrants further investigation. Thus, C57BL/6J mice were fed a high-fat diet (HFD), and we found that the HFD significantly induced obesity and increased the accumulation of intrahepatic lipids, along with alterations in serum biochemical parameters. Moreover, it was observed that the HFD also impaired gut barrier integrity. It was revealed via 16S rRNA gene sequencing that the HFD increased gut microbial diversity, which enriched Colidextribacter, Lachnospiraceae-NK4A136-group, Acetatifactor, and Erysipelatoclostridium. Meanwhile, it reduced the abundance of Faecalibaculum, Muribaculaceae, and Coriobacteriaceae-UCG-002. The predicted metabolic pathways suggest that HFD enhances the chemotaxis and functional activity of gut microbiota pathways associated with flagellar assembly, while also increasing the risk of intestinal pathogen colonization and inflammation. And the phosphotransferase system, streptomycin biosynthesis, and starch/sucrose metabolism exhibited decreases. These findings reveal the composition and predictive functions of the intestinal microbiome in NAFLD, further corroborating the association between gut microbiota and NAFLD while providing novel insights into its potential application in gut microbiome research for NAFLD patients. Full article