Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

Leptothrix cholodnii Response to Nutrient Limitation

Microorganisms are widely utilized for the treatment of wastewater in activated sludge systems. However, the uncontrolled growth of filamentous bacteria leads to bulking and adversely affects wastewater treatment efficiency. To clarify the nutrient requirements for filament formation, we track the growth of a filamentous bacterium, Leptothrix cholodnii SP-6 in different nutrient-limited conditions using a high aspect-ratio microfluidic chamber to follow cell-chain elongation and sheath formation. We find that limitations in Na+, K+, and Fe2+ yield no observable changes in the elongation of cell chains and sheath formation, whereas limitations of C, N, P, or vitamins lead to more pronounced changes in filament morphology; here we observe the appearance of partially empty filaments with wide intercellular gaps. We observe more dramatic differences when SP-6 cells are transferred to media lacking Mg2+ and Ca2+. Loss of Mg2+ results in cell autolysis, while removal of Ca2+ results in the catastrophic disintegration of the filaments. By simultaneously limiting both carbon and Ca2+ sources, we are able to stimulate planktonic cell generation. These findings paint a detailed picture of the ecophysiology of Leptothrix, which may lead to improved control over the unchecked growth of deleterious filamentous bacteria in water purification systems.

Ability of The Saccharomyces Cerevisiae Y904 to Tolerate and Adapt to High Concentrations of Selenium

The rational use of by-products is essential for the development of a sustainable society. Worldwide, the alcoholic fermentation industry generates a large surplus of yeasts, on the scale of millions of tons. So there is a need for beneficial applications to humanity of this surplus. Yeasts, in turn, have the ability to bioaccumulate minerals and enable their bioavailability after cell autolysis. Among these minerals, we highlight selenium (Se), which participates in the formation of antioxidant enzymes. The objectives of the work were to define the minimum and maximum concentration of Se that yeasts (Saccharomyces cerevisiae – Y904) support and the concentrations that they tolerate once adapted. To this end, a test of tolerance to Se was carried out, using treatments with different concentrations of Se. The adaptive process started at the maximum concentration obtained in the tolerance test of 60 µg mL− 1, with an increasing addition of 6 µg mL− 1, reaching up to 246 µg mL− 1 of Se. The macromorphological characteristics and number of colony forming units were evaluated. It was identified that yeasts without adaptation grew on substrate containing up to 60 µg mL− 1 of Se and those adapted, up to 246 µg mL− 1 of Se. In addition to the reduction in yeast growth speed, from the concentration of 84 µg mL− 1 of Se in the medium, morphological changes in colony color were observed. It is concluded that non-adapted yeasts support up to 60 µg mL− 1 of Se and, after the adaptive process, they support 246 µg mL− 1 of Se in the medium.

A Review on Cetylpyridinium Chloride

Cetylpyridinium chloride (CPC) is a quaternary ammonium surfactant having broad spectrum antimicrobial activity. The hexadecane chain of the CPC disorganizes lipid membrane and produces a complete rupture of the bacterial cell membrane. At low concentration of CPC, it leads to activation of intracellular latent ribonucleases of microbes to promote cell autolysis. At high concentration, CPC forms a vesicle-like structure in the bacterial cell surface and leads to leakage of cytoplasmic content. CPC can be estimated using several methods like HPLC, ion-pair extraction TLC, spectrophotometry, chemiluminescence, and spectrometry. It is a strong antimicrobial, anti plaque, deodorizer and bleeding control agent and is widely used in mouthwashes, tooth paste, sanitizers and disinfecting liquids. Keywords: Cetylpyridinium chloride, antimicrobial agent, deodorizer, anti plaque agent.

The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis

Disruption of the human gut microbiota by antibiotics can lead to Clostridium difficile (CD)-associated diarrhea. CD overgrowth and elevated CD toxins result in gut inflammation. Herein, we report that a gut symbiont, Bacteroides thetaiotaomicron (BT), suppressed CD toxin production. The suppressive components are present in BT culture supernatant and are both heat- and proteinase K-resistant. Transposon-based mutagenesis indicated that the polysaccharide metabolism of BT is involved in the inhibitory effect. Among the genes identified, we focus on the methylerythritol 4-phosphate pathway gene gcpE, which supplies the isoprenoid backbone to produce the undecaprenyl phosphate lipid carrier that transports oligosaccharides across the membrane. Polysaccharide fractions prepared from the BT culture suppressed CD toxin production in vitro; the inhibitory effect of polysaccharide fractions was reduced in the gcpE mutant (ΔgcpE). The inhibitory effect of BT-derived polysaccharide fraction was abrogated by lysozyme treatment, indicating that cellwall-associated glycans are attributable to the inhibitory effect. BT-derived polysaccharide fraction did not affect CD toxin gene expression or intracellular toxin levels. An autolysis assay showed that CD cell autolysis was suppressed by BT-derived polysaccharide fraction, but the effect was reduced with that of ΔgcpE. These results indicate that cell wall-associated glycans of BT suppress CD toxin release by inhibiting cell autolysis.

Identification and investigation of autolysin genes in Clostridium saccharoperbutylacetonicum N1-4 for enhanced biobutanol production

Biobutanol is a valuable biochemical and one of the most promising biofuels. Clostridium saccharoperbutylacetonicum N1-4 is a hyper-butanol producing strain. However, its strong autolytic behavior leads to poor cell stability especially during continuous fermentation, thus limiting the applicability of the strain for long-term and industrial scale processes. In this study, we aimed to evaluate the role of autolysin genes within C. saccharoperbutylacetonicum genome related to cell autolysis and further develop more stable strains for enhanced butanol production. Firstly, putative autolysin encoding genes were identified in the strain based on comparison of amino acid sequence with homologous genes in other strains. Then, by overexpressing all these putative autolysin genes individually and characterizing the corresponding recombinant strains, four key genes were pinpointed to be responsible for significant cell autolysis activities. Further, these key genes were deleted using CRISPR-Cas9. Fermentation characterization demonstrated enhanced performance of the resultant mutants. Results from this study reveal valuable insights concerning the role of autolysins for cell stability, solvent production, and provide essential reference for developing robust strains for enhanced biofuel and biochemical production. IMPORTANCE: Severe autolytic behavior is a common issue in Clostridium and many other microorganisms. This study revealed the key genes responsible for the cell autolysis within Clostridium saccharoperbutylacetonicum, a prominent platform for biosolvent production from lignocellulosic materials. The knowledge generated in this study provides insights concerning the cell autolysis in relevant microbial systems, and gives essential references for enhancing strain stability through rational genome engineering.

Characterization of a novel endopeptidase Cwl0971 that hydrolyzes peptidoglycan of Clostridioides difficile involved in pleiotropic cellular processes

SummaryClostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from intracell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (endopeptidase, CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary / late phase of cell growth. Moreover, biofilm formation, germination, and sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the depletion of Cwl097 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which can affect several phenotypes of R20291. Our data indicate that the endopeptidase Cwl0971 is an attractive target for C. difficile infection therapeutics and prophylactics.

Processes controlling programmed cell death of root velamen radicum in an epiphytic orchid

Background and Aims Development of the velamen radicum on the outer surface of the root epidermis is an important characteristic for water uptake and retention in some plant families, particularly epiphytic orchids, for survival under water-limited environments. Velamen radicum cells derive from the primary root meristem; however, following this development, velamen radicum cells die by incompletely understood processes of programmed cell death (PCD). Methods We combined the use of transmission electron microscopy, X-ray micro-tomography and transcriptome methods to characterize the major anatomical and molecular changes that occur during the development and death of velamen radicum cells of Cymbidium tracyanum, a typical epiphytic orchid, to determine how PCD occurs. Key Results Typical changes of PCD in anatomy and gene expression were observed in the development of velamen radicum cells. During the initiation of PCD, we found that both cell and vacuole size increased, and several genes involved in brassinosteroid and ethylene pathways were upregulated. In the stage of secondary cell wall formation, significant anatomical changes included DNA degradation, cytoplasm thinning, organelle decrease, vacuole rupture and cell wall thickening. Changes were found in the expression of genes related to the biosynthesis of cellulose and lignin, which are instrumental in the formation of secondary cell walls, and are regulated by cytoskeleton-related factors and phenylalanine ammonia-lyase. In the final stage of PCD, cell autolysis was terminated from the outside to the inside of the velamen radicum. The regulation of genes related to autophagy, vacuolar processing enzyme, cysteine proteases and metacaspase was involved in the final execution of cell death and autolysis. Conclusions Our results showed that the development of the root velamen radicum in an epiphytic orchid was controlled by the process of PCD, which included initiation of PCD, followed by formation of the secondary cell wall, and execution of autolysis following cell death.