Glutamate Positively Regulates Chitinase Activity and the Biocontrol Efficacy of Pseudomonas protegens.

Broad-spectrum biocontrol by Pseudomonas protegens CHA0 and other fluorescent pseudomonads is achieved through the generation of various secondary metabolites with antibiotic activities against not only other microbes but, also, nematodes and insects present in the rhizosphere. A previous metabolomic study demonstrated that intracellular low-molecular weight effectors, such as guanosine tetraphosphate and γ-aminobutyrate, function as important signals in niche adaptation by strain CHA0 to plant roots. We investigated the role of amino acids in the biocontrol trait of P. protegens Cab57 towards Pythium damping off and root rot in cucumber. Among the 11 amino acids tested, only glutamate markedly enhanced the efficacy of biocontrol. An RNA-Seq analysis revealed that glutamate upregulated the expression of a chitinase gene cluster (c21370-c21380, in which the c21370 gene was annotated as a gene encoding the chitin-binding protein cbp and the c21380 gene encoded chitinase chiC) in strain CHA0. Glutamate upregulated the expression of the regulatory small RNA rsmZ but reduced the production levels of other Gac/Rsm-regulated biocontrol factors, such as 2,4-diacetylphloroglucinol and pyoluteorin. The promoter activity of cbp and chitinase activity were characterized in detail; their activities were up-regulated in response to glutamate and their expression was under the control of GacA. Therefore, glutamate appears to be essential for biocontrol activity in which chitinase production is regulated in response to glutamate. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.

Carbohydrate immobilized on a dendrimer-coated colloidal gold surface for fabrication of a lectin-sensing device based on localized surface plasmon resonance spectroscopy.

Carbohydrate-mediated functions in biological systems have generated considerable interest in recent years. We have developed a device bearing immobilized carbohydrates on a colloidal gold surface and applied this device to the detection of carbohydrate-binding molecules by using localized surface plasmon resonance (LSPR) spectroscopy. The sensing device was constructed by using cyanuric chloride as an amine-linker between an amino residue of a polyamidoamine (PAMAM) dendrimer-coated colloidal gold surface and the amino residue of a 12-aminododecyl glycoside. After optimizing the construction of the device, we characterized its LSPR-based sensing capability. Binding specificity with lectins and linear range responses were obtained with the device. Our LSPR-based sensing device thus provides a label-free, low-cost detection method for use as a laboratory research tool or in medical glycan arrays.

Immobilization of carbohydrate clusters on a quartz crystal microbalance sensor surface.

The immobilization of carbohydrates on gold surfaces is a prerequisite technology for carbohydrate-related studies, including those of carbohydrate-biomolecule interactions. Glycolipid domains in cell membranes, such as lipid rafts, are thought to play an important role in cell biology through their carbohydrate portions. To understand the recognition of glycolipid domains such as receptors for bacterial toxins and viruses, we immobilized clusters of carbohydrates on a gold surface by using polyamidoamine (PAMAM) dendrimers as a scaffold. The PAMAM dendrimers were adsorbed on the gold-coated surface of a quartz crystal microbalance (QCM) sensor and were observed by means of QCM with dissipation (QCM-D). After adsorption of the PAMAM dendrimers, lysoganglioside-GM(1) and 12-aminododecyl-N-acetylglucosaminide (GlcNAc-C12-NH(2)) were immobilized on the amino groups of PAMAM dendrimers by means of an NH(2) cross-linker. Immobilization of the carbohydrates was confirmed by observation of their specific interaction with anti-ganglioside GM(1) antibody or wheat germ agglutinin (WGA). Surfaces with different GlcNAc-C12-NH(2) cluster sizes and densities were prepared by varying the size of the PAMAM dendrimers or the concentration of GlcNAc-C12-NH(2) immobilized on the dendrimers, respectively. Analysis of the binding between the GlcNAc-C12-NH(2)-immobilized surface and WGA revealed that the size of the PAMAM dendrimers influenced the GlcNAc-C12-NH(2)-WGA interaction, with larger dendrimers resulting in higher WGA binding constants.

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis.

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.

Detection of a specific DNA sequence by electrophoresis through a molecularly imprinted polymer.

To develop a simple and inexpensive DNA detection method, we prepared a molecularly imprinted polymer (MIP) gel for recognizing a specific double-stranded DNA (dsDNA) target sequence in MIP gel electrophoresis (MIPGE). During MIPGE, migration of the target sequence of dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This migration hindrance of target dsDNA was determined by plotting the relationship between the migration distance in the MIP gel and that in polyacrylamide gel, commonly used in gel electrophoresis. Using this plot, detection of a target dsDNA from a mixture of different-sized dsDNA fragments was achieved. Moreover, we found the detection method successfully distinguished between a target and its base-pair substitutes. These results suggest that MIPGE could be employed for detection of a target dsDNA sequence.

Imprinted polymer layer for recognizing double-stranded DNA.

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2.

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.

Enzymatic degradation behavior of porous silk fibroin sheets.

We investigated the degradation behavior of porous silk fibroin sheets by in vitro enzymatic experiments with alpha-chymotrypsin, collagenase IA, and protease XIV. With 1.0 U/ml protease XIV, 70% of a silk fibroin sheet was degraded within 15 days at 37 degrees C. When the fibroin sheet was exposed to collagenase IA, the amount of Silk II crystalline structure in the sheets decreased slightly, and a small amount of Silk I crystalline structure was formed. When protease XIV was used, almost all Silk II disappeared, but the crystallinity increased overall because the amount of Silk I increased. During digestion with protease XIV, the pore size of the fibroin sheets increased with increasing degradation time, until the sheets finally collapsed and became totally shapeless. The average molecular weight of the products after degradation with the three enzymes followed the order protease XIV < collagenase IA < alpha-chymotrypsin. More than 50% of the products resulting from degradation with protease XIV were free amino acids.