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BMC Microbiology Oct 2023Agitation speed influenced the production rate of laccase. Orbital speed not only influenced the enzyme production, but was also effective to dissolve the oxygen during...
BACKGROUND
Agitation speed influenced the production rate of laccase. Orbital speed not only influenced the enzyme production, but was also effective to dissolve the oxygen during growth of mycelium, spores, and chlamydospores. Shear effects of speed greatly influenced the morphology of mycelium.
METHODS
Ganoderma multistipitatum was identified by ITS marker. Phylogenetic tree was constructed for species identification. Qualitatively by plate method contained guaiacol indicator, while quantitatively by submerged fermentation and Central Composite Design applied on agitation parameter for maximum laccase potential of this species. The effects of agitation speed on mycelium morphology were observed under compound and scanning electron microscope.
RESULTS
Statistical optimization of agitation conditions were performed by using response surface methodology to enhance the production of laccase from Ganoderma multistipitatum sp. nov. Maximum laccase yield (19.44 × 10 ± 0.28 U/L) was obtained at 150 rpm grown culture, which was higher than predicted value of laccase production (19.18 × 10 U/L) under aerobic conditions (150 rpm). The 150 rpm provided the continuous flush of oxygen. The DO (dissolved oxygen) was maximum (65%) for "27 h" incubation at 150 rpm during laccase synthesis. The statistical value of laccase production was minimum under anaerobic or nearly static condition of 50 rpm. The predicted (12.78 × 10 U/L) and obtained (12.82 × 10 U/L) yield was low at 50 rpm. Optimization of orbital shaking for aeration conditions were performed by the use of "Response Surface Methodology". The submerged shaking flasks were utilized as a nutrients growth medium to maximize the production of laccase from G. multistipitatum. The minimum incubation time highly influenced the laccase yield from 7 to 15 days via utilization of less cost-effective medium under a promising and eco-friendly method. The morphological effects of rpm on mycelium were examined under compound and scanning electron microscopy. Higher rpm (200, 230) shear the mycelium, while 150 to 200 rpm exhibited smoother and highly dense branches of mycelia.
CONCLUSION
The shear forces of 200 rpm caused the damages of mycelium and cells autolysis with less laccase production. This study concluded that 150 rpm saved the life of mycelium and enhanced the production rate of enzymes.
Topics: Laccase; Oxygen; Ganoderma; Phylogeny; Fermentation; Mycelium; Bioreactors; Culture Media
PubMed: 37784032
DOI: 10.1186/s12866-023-03009-2 -
International Journal of Food... Dec 2023In the manufacture of rennet-coagulated cheese, autolysis is a rate-limiting step for ripening. Previously, a highly autolytic and thermotolerant Lactococcus lactis...
In the manufacture of rennet-coagulated cheese, autolysis is a rate-limiting step for ripening. Previously, a highly autolytic and thermotolerant Lactococcus lactis strain, RD07, was generated, which in preliminary laboratory cheese trials demonstrated great potential as a cheese ripening accelerant. RD07 is proteinase positive (Prt) and capable of metabolizing citrate (Cit). In this study, we obtained two derivatives of RD07: EC8 lacking the citrate plasmid, and EC2 lacking the proteinase plasmid. EC2 and EC8 retained the autolytic properties of RD07, and autolyzed 20 times faster than Flora Danica (FD) and SD96, where the latter is the parent of RD07. The three strains EC2, EC8 and RD07 were used in a ratio of 90:8:2, to create a simple starter termed ERC. ERC was less sensitive to cooking when cultured in milk and autolyzed well after entering the stationary phase upon facing sugar starvation. The ERC starter was benchmarked against FD and SD96 in laboratory cheese trials. The free amino acid content in cheese prepared using the ERC culture was 31 % and 34 % higher than in cheese prepared using FD and SD96, respectively. Overall, the ERC culture resulted in a more rapid release of free amino acids. A large-scale (5000 L) Gouda cheese trial at a Danish dairy demonstrated that the single strain ERC starter was comparable in performance to FD + an adjunct Lactobacillus helveticus culture. Furthermore, a large-scale Danbo cheese trial demonstrated that ERC could reduce the ripening period by 50 % for long-term ripened (25 weeks) cheese, resulting in better cheese.
Topics: Cheese; Lactococcus lactis; Peptide Hydrolases; Citrates
PubMed: 37714070
DOI: 10.1016/j.ijfoodmicro.2023.110398 -
BioRxiv : the Preprint Server For... Aug 2023Infection with the Gram-negative species leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic...
Infection with the Gram-negative species leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic inflammatory disease. During growth these bacteria release significant amounts of peptidoglycan (PG) fragments which elicit inflammatory responses in the human host. To better understand the mechanisms involved in PG synthesis and breakdown in , we characterized the effects of mutation of . MltG has been identified in other bacterial species as a terminase that stops PG strand growth by cleaving the growing glycan. Mutation of in did not affect bacterial growth rate but resulted in increased PG turnover, more cells of large size, decreased autolysis under non-growth conditions, and increased sensitivity to antibiotics that affect PG crosslinking. An mutant released greatly increased amounts of PG monomers, PG dimers, and larger oligomers. In the background, mutation of either or , encoding the lytic transglycosylases responsible for PG monomer liberation, resulted in wild-type levels of PG monomer release. Bacterial two-hybrid assays identified positive interactions of MltG with synthetic penicillin-binding proteins PBP1 and PBP2 and the PG-degrading endopeptidase PBP4 (PbpG). These data are consistent with MltG acting as a terminase in and suggest that absence of MltG activity results in excessive PG growth and extra PG in the sacculus that must be degraded by lytic transglycosylases including LtgA and LtgD. Furthermore, absence of MltG causes a cell wall defect that is manifested as large cell size and antibiotic sensitivity.
PubMed: 37662418
DOI: 10.1101/2023.08.23.554517 -
Journal of Proteomics Oct 2023Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate...
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70-90 °C and pH of 2-4 with rapid digestion kinetics. The extreme HTA-Protease reaction conditions actively denature sample proteins, obviate the use of chaotropes, are largely independent of reduction and alkylation, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests alone. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights. SIGNIFICANCE: Here we introduce new capabilities for bottom-up proteomics applications with hyperthermoacidic archaeal proteases (HTA-Proteases©). HTA-Proteases have novel cleavage specificity, require no chaotropes, and allow simple one-step/five-minute sample preparations that promise to reduce variability between samples and laboratories. HTA-Proteases generate unique sets of observable peptides that are non-overlapping with tryptic peptides and significantly increase sequence coverage and available peptide targets relative to trypsin alone. HTA-Proteases show some bias for the detection and coverage of nucleic acid-binding proteins and membrane proteins relative to trypsin. These new ultra-stable enzymes function optimally in nearly boiling acidic conditions, show no autolysis, and do not require aliquoting as they are stable for years at ambient temperatures. Used independently or in conjunction with tryptic digests, HTA-Proteases offer increased proteome coverage, unique peptide targets, and brief one-step protocols amenable to automation, rapid turnaround, and high-throughput approaches.
Topics: Peptide Hydrolases; Trypsin; Proteome; Proteomics; Workflow; Peptides; Membrane Proteins
PubMed: 37634627
DOI: 10.1016/j.jprot.2023.104992 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Aug 2023Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast...
Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the and genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.
Topics: Humans; Isocitrate Dehydrogenase; NADP; Reactive Oxygen Species; Autolysis; Adenosine Triphosphate
PubMed: 37622372
DOI: 10.13345/j.cjb.220790 -
Cell Sep 2023Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria....
Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (CPP, lipid II, and lipid III). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.
Topics: Anti-Bacterial Agents; Bacteria; Biological Assay; Diphosphates; Soil Microbiology
PubMed: 37611581
DOI: 10.1016/j.cell.2023.07.038 -
BioRxiv : the Preprint Server For... Jul 2023Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and...
UNLABELLED
Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and morphogenesis, and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in , which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a β-lactam antibiotic used in the clinic. and analysis revealed that PA3978 binds to the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. ΔPA3978 mutant phenotypes were suppressed by deleting , consistent with them having been caused by elevated MltF activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a ΔPA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 towards MltF, and we confirmed that activity for PA5502 . Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins Ilt (inhibitor of lytic transglycosylase) and Lii (lytic transglycosylase inhibitor, inhibitor).
IMPORTANCE
A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division, and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases, but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-nhibitor system that controls the activity of a cell wall hydrolase in the human pathogen , and which also affects resistance to an antibiotic used in the clinic.
PubMed: 37546783
DOI: 10.1101/2023.07.28.551027 -
Frontiers in Microbiology 2023is a genus of lactic acid bacteria used in the dairy industry as a starter. Lactococci have been found to produce altogether more than 40 different bacteriocins,...
INTRODUCTION
is a genus of lactic acid bacteria used in the dairy industry as a starter. Lactococci have been found to produce altogether more than 40 different bacteriocins, ribosomally synthesized antimicrobial proteins. All known spp. bacteriocins belong to classes I and II, which are mainly heat-resistant peptides. No class III bacteriocins, bigger heat-sensitive proteins, including phage tail-like bacteriocins, have been found from the spp. Unlike phage tail-like bacteriocins, prophage lysins have not been regarded as bacteriocins, possibly because phage lysins contribute to autolysis, degrading the host's own cell wall.
METHODS
Wild-type strain LAC460, isolated from spontaneously fermented idli batter, was examined for its antimicrobial activity. We sequenced the genome, searched phage lysins from the culture supernatant, and created knock-out mutants to find out the source of the antimicrobial activity.
RESULTS AND DISCUSSION
The strain LAC460 was shown to kill other strains with protease- and heat-sensitive lytic activity. Three phage lysins were identified in the culture supernatant. The genes encoding the three lysins were localized in different prophage regions in the chromosome. By knock-out mutants, two of the lysins, namely LysL and LysP, were demonstrated to be responsible for the antimicrobial activity. The strain LAC460 was found to be resistant to the lytic action of its own culture supernatant, and as a consequence, the phage lysins could behave like bacteriocins targeting and killing other closely related bacteria. Hence, similar to phage tail-like bacteriocins, phage lysin-like bacteriocins could be regarded as a novel type of class III bacteriocins.
PubMed: 37520360
DOI: 10.3389/fmicb.2023.1219723 -
Nature Communications Jul 2023Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their...
Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.
Topics: Humans; N-Acetylmuramoyl-L-alanine Amidase; Caulobacter crescentus; Feedback; Constriction; Autolysis
PubMed: 37433794
DOI: 10.1038/s41467-023-39783-w -
International Journal of Antimicrobial... Oct 2023The pathogenicity of Staphylococcus epidermidis is largely attributed to its exceptional ability to form biofilms. Here, we report that mupirocin, an antimicrobial agent...
The pathogenicity of Staphylococcus epidermidis is largely attributed to its exceptional ability to form biofilms. Here, we report that mupirocin, an antimicrobial agent widely used for staphylococcal decolonization and anti-infection, strongly stimulates the biofilm formation of S. epidermidis. Although the polysaccharide intercellular adhesin (PIA) production was unaffected, mupirocin significantly facilitated extracellular DNA (eDNA) release by accelerating autolysis, thereby positively triggering cell surface attachment and intercellular agglomeration during biofilm development. Mechanistically, mupirocin regulated the expression of genes encoding for the autolysin AtlE as well as the programmed cell death system CidA-LrgAB. Critically, through gene knockout, we found out that deletion of atlE, but not cidA or lrgA, abolished the enhancement of biofilm formation and eDNA release in response to mupirocin treatment, indicating that atlE is required for this effect. In Triton X-100 induced autolysis assay, mupirocin treated atlE mutant displayed a slower autolysis rate compared with the wild-type strain and complementary strain. Therefore, we concluded that subinhibitory concentrations of mupirocin enhance the biofilm formation of S. epidermidis in an atlE dependent manner. This induction effect could conceivably be responsible for some of the more unfavourable outcomes of infectious diseases.
Topics: Staphylococcus epidermidis; Mupirocin; Biofilms; Staphylococcus; Virulence; Bacterial Proteins
PubMed: 37385560
DOI: 10.1016/j.ijantimicag.2023.106904