Cell Wall Synthesis Research Articles

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

Vegetative cell wall protein OsGP1 regulates cell wall mediated soda saline-alkali stress in rice.

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.

Integrated Metabolome and Transcriptome Analysis of Gibberellins Mediated the Circadian Rhythm of Leaf Elongation by Regulating Lignin Synthesis in Maize.

Plant growth exhibits rhythmic characteristics, and gibberellins (GAs) are involved in regulating cell growth, but it is still unclear how GAs crosstalk with circadian rhythm to regulate cell elongation. The study analyzed growth characteristics of wild-type (WT), zmga3ox and zmga3ox with GA3 seedlings. We integrated metabolomes and transcriptomes to study the interaction between GAs and circadian rhythm in mediating leaf elongation. The rates of leaf growth were higher in WT than zmga3ox, and zmga3ox cell length was shorter when proliferated in darkness than light, and GA3 restored zmga3ox leaf growth. The differentially expressed genes (DEGs) between WT and zmga3ox were mainly enriched in hormone signaling and cell wall synthesis, while DEGs in zmga3ox were restored to WT by GA3. Moreover, the number of circadian DEGs that reached the peak expression in darkness was more than light, and the upregulated circadian DEGs were mainly enriched in cell wall synthesis. The differentially accumulated metabolites (DAMs) were mainly attributed to flavonoids and phenolic acid. Twenty-two DAMs showed rhythmic accumulation, especially enriched in lignin synthesis. The circadian DEGs ZmMYBr41/87 and ZmHB34/70 were identified as regulators of ZmHCT8 and ZmBM1, which were enzymes in lignin synthesis. Furthermore, GAs regulated ZmMYBr41/87 and ZmHB34/70 to modulate lignin biosynthesis for mediating leaf rhythmic growth.

Dynamics of cell wall-binding proteins at a single molecule level: B. subtilis autolysins show different kinds of motion.

The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of Bacillus subtilis autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.

Identification of Genes Associated with Resistance to Persulcatusin, a Tick Defensin from Ixodes persulcatus.

Antimicrobial peptides (AMPs) are present in a wide range of plants, animals, and microorganisms. Since AMPs are characterized by their effectiveness against emergent antibiotic-resistant bacteria, they are attracting attention as next-generation antimicrobial compounds that could solve the problem of drug-resistant bacteria. Persulcatusin (IP), an antibacterial peptide derived from the hard tick Ixodes persulcatus, shows high antibacterial activity against various Gram- positive bacteria as well as multidrug-resistant bacteria. However, reports on the antibacterial action and resistance mechanisms of IP are scarce. In this study, we spontaneously generated mutants showing increased a minimum inhibitory concentration (MIC) of IP and analyzed their cross-resistance to other AMPs and antibiotics. We also used fluorescent probes to investigate the target of IP activity by evaluating IP-induced damage to the bacterial cytoplasmic membrane. Our findings suggest that the antimicrobial activity of IP on bacterial cytoplasmic membranes occurs via a mechanism of action different from that of known AMPs. Furthermore, we screened for mutants with high susceptibility to IP using a transposon mutant library and identified 16 genes involved in IP resistance. Our results indicate that IP, like other AMPs, depolarizes the bacterial cytoplasmic membrane, but it may also alter membrane structure and inhibit cell-wall synthesis.

Tip Growth Defective1 interacts with the cellulose synthase complex to regulate cellulose synthesis in Arabidopsis thaliana.

Plant cells possess robust and flexible cell walls composed primarily of cellulose, a polysaccharide that provides structural support and enables cell expansion. Cellulose is synthesised by the Cellulose Synthase A (CESA) catalytic subunits, which form cellulose synthase complexes (CSCs). While significant progress has been made in unravelling CSC function, the trafficking of CSCs and the involvement of post-translational modifications in cellulose synthesis remain poorly understood. In order to deepen our understanding of cellulose biosynthesis, this study utilised immunoprecipitation techniques with CESA6 as the bait protein to explore the CSC and its interactors. We have successfully identified the essential components of the CSC complex and, notably, uncovered novel interactors associated with CSC trafficking, post-translational modifications, and the coordination of cell wall synthesis. Moreover, we identified TIP GROWTH DEFECTIVE 1 (TIP1) protein S-acyl transferases (PATs) as an interactor of the CSC complex. We confirmed the interaction between TIP1 and the CSC complex through multiple independent approaches. Further analysis revealed that tip1 mutants exhibited stunted growth and reduced levels of crystalline cellulose in leaves. These findings suggest that TIP1 positively influences cellulose biosynthesis, potentially mediated by its role in the S-acylation of the CSC complex.

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

The Effects of Mycovirus BmPV36 on the Cell Structure and Transcription of Bipolaris maydis.

Bipolaris maydis partitivirus 36 (BmPV36) is a mycovirus that can significantly reduce the virulence of the host Bipolaris maydis, but its hypovirulence mechanism is not clear. To investigate the response of B. maydis to BmPV36, the effects of BmPV36 on host cell structure and gene expression were studied via transmission electron microscopy and transcriptome sequencing using BmPV36-carrying and virus-free mycelium on the second and fifth culture. The results of transmission electron microscopy showed that the cell wall microfibrils of B. maydis were shortened, the cell membrane was broken, and membrane-bound vesicles and vacuoles appeared in the cells after carrying BmPV36. Transcriptome sequencing results showed that after carrying BmPV36, B. maydis membrane-related genes were significantly up-regulated, but membrane transport-related genes were significantly down-regulated. Genes related to carbohydrate macromolecule polysaccharide metabolic and catabolic processes were significantly down-regulated, as were genes related to the synthesis of toxins and cell wall degrading enzymes. Therefore, we speculated that BmPV36 reduces the virulence of B. maydis by destroying the host's cell structure, inhibiting the synthesis of toxins and cell wall degrading enzymes, and reducing cell metabolism. Gaining insights into the hypovirulence mechanism of mycoviruses will provide environmentally friendly strategies for the control of fungal diseases.

Optimizations of lipid II synthesis: an essential glycolipid precursor in bacterial cell wall synthesis and a validated antibiotic target.

Lipid II is an essential glycolipid found in bacteria. Accessing this valuable cell wall precursor is important both for studying cell wall synthesis and for studying/identifying novel antimicrobial compounds. Herein, we describe optimizations to the modular chemical synthesis of lipid II and unnatural analogues. In particular, the glycosylation step, a critical step in the formation of the central disaccharide unit (GlcNAc-MurNAc), was optimized. This was achieved by employing the use of glycosyl donors with diverse leaving groups. The key advantage of this approach lies in its adaptability, allowing for the generation of a wide array of analogues through the incorporation of alternative building blocks at different stages of synthesis.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1β (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

Phytochemical screening and antimicrobial activity of Polygala sadebeckiana Gürke extracts on bacterial isolates from Wound samples of patients with “Shimetere”

BackgroundModern medicine is not the choice of patients with “shimetere” in the Gurage community owing to their perception of ‘parenteral medication use severely aggravates the disease’. For this reason, the root part of Polygala sadebeckiana Gürke is commonly utilized as traditional medicine in the management of the disease. The aim of this study was to evaluate the antimicrobial activity of Polygala sadebeckiana Gürke extract on bacterial isolates from wound samples of patients with “Shimetere”.MethodsThe agar well diffusion method was used to evaluate antibacterial activity, and the agar dilution method was utilized to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MICs). The crude extract was tested against isolated bacteria at concentrations of 25, 50, 75 and 100 mg/mL in triplicate (3x). The positive controls were azithromycin (15 µg) and cloxacillin disk (5 µg), and the negative control was dimethylsulfoxide (5%). The group mean comparisons were made using one-way ANOVA at a significance level of p < 0.05, and the results are presented as the mean ± standard deviation. The presence of secondary metabolites from crude extract was checked by standard testing procedures.ResultsS. aureus and S. pyrogen were the two identified bacteria from 9 (60%) and 3 (20%) wound samples, respectively. All identified bacterial strains were susceptible to the reference antibiotics. Tannins and saponins were the most abundant secondary metabolites found in the crude extracts. The average inhibition zones of the plant extracts with 100, 75, 50 and 25 mg/mL concentrations were 27, 20.33, 15.25, and 11.96 mm (p < 0.000) for S. aureus and 30.02, 24.50, 19.07, and 15.77 mm (p < 0.000) for S. pyrogen bacteria, respectively. The MIC and MBC of the crude extract were 1.67 and 10 mg/mL for S. aureus and 0.98 and 4 mg/mL for S. pyrogen.ConclusionPolygala sadebeckiana Gürke contained significant tannins and saponins as secondary metabolites and had antibacterial activities against isolated bacteria (S. aureus and S. pyrogen) from “Shimetere”. The potential mechanism of antibacterial action of the plant extract was cell wall synthesis inhibition.

Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit.

Phenotypic analysis assays such as bacterial cytological profiling (BCP) have become increasingly popular for antibiotic mode of action analysis. A plethora of dyes, protein fusions, and reporter strains are available and have been used for this purpose, enabling both rapid mode of action categorization and in-depth analysis of antibiotic mechanisms. However, non-expert researchers may struggle choosing suitable assays and interpreting results. This is a particular problem for antibiotics that have multiple or complex targets, such as the bacterial cell envelope. Here, we set out to curate a minimal set of accessible and affordable phenotypic assays that allow distinction between membrane and cell wall targets, can identify dual-action inhibitors, and can be implemented in most research environments. To this end, we employed BCP, membrane potential, fluidity, and cell wall synthesis assays. To assess specificity and ease of interpretation, we tested three well-characterized and commercially available reference antibiotics: the potassium ionophore valinomycin, the lipid II-binding glycopeptide vancomycin, and the dual-action lantibiotic nisin, which binds lipid II and forms a membrane pore. Based on our experiments, we suggest a minimal set of BCP, a membrane-potentiometric probe, and fluorescent protein fusions to MinD and MreB as basic assay set and recommend complementing these assays with Laurdan-based fluidity measurements and a PliaI reporter fusion, where indicated. We believe that our results can provide guidance for researchers who wish to use phenotypic analysis for mode of action studies but do not possess the specialized equipment or expert knowledge to employ the full breadth of possible techniques.IMPORTANCEPhenotypic analysis assays using specialized fluorescence fusions and dyes have become increasingly popular in antibiotic mode of action analysis. However, it can be difficult to implement these methods due to the need for specialized equipment and/or the complexity of bacterial cell biology and physiology, making the interpretation of results difficult for non-experts. This is especially problematic for compounds that have multiple or pleiotropic effects, such as inhibitors of the bacterial cell envelope. In order to make phenotypic analysis assays accessible to labs, whose primary expertise is not bacterial cell biology, or with limited equipment and resources, a set of simple and broadly accessible assays is needed that is easy to implement, execute, and interpret. Here, we have curated a set of assays and strains that does not need highly specialized equipment, can be performed in most labs, and is straightforward to interpret without knowing the intricacies of bacterial cell biology.

Metabolic adaptations of Microbacterium sediminis YLB-01 in deep-sea high-pressure environments.

The deep-sea environment is an extremely difficult habitat for microorganisms to survive in due to its intense hydrostatic pressure. However, the mechanisms by which these organisms adapt to such extreme conditions remain poorly understood. In this study, we investigated the metabolic adaptations of Microbacterium sediminis YLB-01, a cold and stress-tolerant microorganism isolated from deep-sea sediments, in response to high-pressure conditions. YLB-01 cells were cultured at normal atmospheric pressure and 28 ℃ until they reached the stationary growth phase. Subsequently, the cells were exposed to either normal pressure or high pressure (30MPa) at 4 ℃ for 7days. Using NMR-based metabolomic and proteomic analyses of YLB-01 cells exposed to high-pressure conditions, we observed significant metabolic changes in several metabolic pathways, including amino acid, carbohydrate, and lipid metabolism. In particular, the high-pressure treatment stimulates cell division and triggers the accumulation of UDP-glucose, a critical factor in cell wall formation. This finding highlights the adaptive strategies used by YLB-01 cells to survive in the challenging high-pressure environments of the deep sea. Specifically, we discovered that YLB-01 cells regulate amino acid metabolism, promote carbohydrate metabolism, enhance cell wall synthesis, and improve cell membrane fluidity in response to high pressure. These adaptive mechanisms play essential roles in supporting the survival and growth of YLB-01 in high-pressure conditions. Our study offers valuable insights into the molecular mechanisms underlying the metabolic adaptation of deep-sea microorganisms to high-pressure environments. KEY POINTS: • NMR-based metabolomic and proteomic analyses were conducted on Microbacterium sediminis YLB-01 to investigate the significant alterations in several metabolic pathways in response to high-pressure treatment. • YLB-01 cells used adaptive strategies (such as regulated amino acid metabolism, promoted carbohydrate metabolism, enhanced cell wall synthesis, and improved cell membrane fluidity) to survive in the challenging high-pressure environment of the deep sea. • High-pressure treatment stimulated cell division and triggered the accumulation of UDP-glucose, a critical factor in cell wall formation, in Microbacterium sediminis YLB-01 cells.

Simultaneous volatile fatty acids promotion and antibiotic resistance genes reduction in fluoranthene-induced sludge alkaline fermentation: Regulation of microbial consortia and cell functions

The impact and mechanism of fluoranthene (Flr), a typical polycyclic aromatic hydrocarbon highly detected in sludge, on alkaline fermentation for volatile fatty acids (VFAs) recovery and antibiotic resistance genes (ARGs) transfer were studied. The results demonstrated that VFAs production increased from 2189 to 4272 mg COD/L with a simultaneous reduction of ARGs with Flr. The hydrolytic enzymes and genes related to glucose and amino acid metabolism were provoked. Also, Flr benefited for the enrichment of hydrolytic-acidifying consortia (i.e., Parabacteroides and Alkalibaculum) while reduced VFAs consumers (i.e., Rubrivivax) and ARGs potential hosts (i.e., Rubrivivax and Pseudomonas). Metagenomic analysis indicated that the genes related to cell wall synthesis, biofilm formation and substrate transporters to maintain high VFAs-producer activities were upregulated. Moreover, cell functions of efflux pump and Type IV secretion system were suppressed to inhibit ARGs proliferation. This study provided intrinsic mechanisms of Flr-induced VFAs promotion and ARGs reduction during alkaline fermentation.

A Baeyer-Villiger monooxygenase CgBVMO1 is involved in superoxide anion metabolism, cell wall synthesis, and pathogenicity of Colletotrichum gloeosporioides

Baeyer-Villiger monooxygenases (BVMOs) represent a subset of flavin-dependent monooxygenases that facilitate the oxidation of a diverse range of substrates. Despite their significance, the functional aspects of BVMOs within fungal pathogens have remained relatively uncharted until now. Therefore, the objective of this study is to investigate the role of CgBVMO1 in the causal agent of anthracnose disease, Colletotrichum gloeosporioides. The findings revealed a substantial upregulation of CgBVMO1 transcription in the pathogen during both appressorium formation and the initial invasion to the plant host. Subsequent knockout of CgBVMO1 and related analyses elucidated the enzyme's involvement in various vital processes of C. gloeosporioides, including vegetative growth, conidiation, and the pathogenicity. It is noteworthy that the knockout of CgBVMO1 had no impact on the pathogen’s ability to penetrate the plant epidermis. Furthermore, this research uncovered CgBVMO1's essential role in superoxide anion production. Stress sensitivity assessments, on the other hand, established that CgBVMO1 does not participate in osmotic regulation or cell membrane biosynthesis, but rather actively contributes to cell wall synthesis by regulating chitin synthase. In conclusion, this study provides invaluable insights into the pivotal role played by CgBVMO1 in the pathogenicity of C. gloeosporioides.

Isobavachalcone exhibits antifungal and antibiofilm effects against C. albicans by disrupting cell wall/membrane integrity and inducing apoptosis and autophagy.

Isobavachalcone (IBC) is a natural flavonoid with multiple pharmacological properties. This study aimed to evaluate the efficacy of IBC against planktonic growth and biofilms of Candida albicans (C. albicans) and the mechanisms underlying its antifungal action. The cell membrane integrity, cell metabolic viability, and cell morphology of C. albicans treated with IBC were evaluated using CLSM and FESEM analyses. Crystal violet staining, CLSM, and FESEM were used to assess the inhibition of biofilm formation, as well as dispersal and killing effects of IBC on mature biofilms. RNA-seq combined with apoptosis and autophagy assays was used to examine the mechanisms underlying the antifungal action of IBC. IBC exhibited excellent antifungal activity with 8 μg/mL of MIC for C. albicans. IBC disrupted the cell membrane integrity, and inhibited biofilm formation. IBC dispersed mature biofilms and damaged biofilm cells of C. albicans at 32 μg/mL. Moreover, IBC induced apoptosis and autophagy-associated cell death of C. albicans. The RNA-seq analysis revealed upregulation or downregulation of key genes involved in cell wall synthesis (Wsc1 and Fks1), ergosterol biosynthesis (Erg3, and Erg11), apoptisis (Hsp90 and Aif1), as well as autophagy pathways (Atg8, Atg13, and Atg17), and so forth, in response to IBC, as evidenced by the experiment-based phenotypic analysis. These results suggest that IBC inhibits C. albicans growth by disrupting the cell wall/membrane, caused by the altered expression of genes associated with β-1,3-glucan and ergosterol biosynthesis. IBC induces apoptosis and autophagy-associated cell death by upregulating the expression of Hsp90, and altering autophagy-related genes involved in the formation of the Atg1 complex and the pre-autophagosomal structure. Together, our findings provide important insights into the potential multifunctional mechanism of action of IBC.

Transcriptomic analysis of cell envelope inhibition by prodigiosin in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading threat to public health as it is resistant to most currently available antibiotics. Prodigiosin is a secondary metabolite of microorganisms with broad-spectrum antibacterial activity. This study identified a significant antibacterial effect of prodigiosin against MRSA with a minimum inhibitory concentration as low as 2.5 mg/L. The results of scanning electron microscopy, crystal violet staining, and confocal laser scanning microscopy indicated that prodigiosin inhibited biofilm formation in S. aureus USA300, while also destroying the structure of the cell wall and cell membrane, which was confirmed by transmission electron microscopy. At a prodigiosin concentration of 1.25 mg/L, biofilm formation was inhibited by 76.24%, while 2.5 mg/L prodigiosin significantly reduced the vitality of MRSA cells in the biofilm. Furthermore, the transcriptomic results obtained at 1/8 MIC of prodigiosin indicated that 235and 387 genes of S. aureus USA300 were significantly up- and downregulated, respectively. The downregulated genes were related to two-component systems, including the transcriptional regulator LytS, quorum sensing histidine kinases SrrB, NreA and NreB, peptidoglycan biosynthesis enzymes (MurQ and GlmU), iron-sulfur cluster repair protein ScdA, microbial surface components recognizing adaptive matrix molecules, as well as the key arginine synthesis enzymes ArcC and ArgF. The upregulated genes were mainly related to cell wall biosynthesis, as well as two-component systems including vancomycin resistance-associated regulator, lipoteichoic acid biosynthesis related proteins DltD and DltB, as well as the 9 capsular polysaccharide biosynthesis proteins. This study elucidated the molecular mechanisms through which prodigiosin affects the cell envelope of MRSA from the perspectives of cell wall synthesis, cell membrane and biofilm formation, providing new potential targets for the development of antimicrobials for the treatment of MRSA.