Melibiose

First Report of Dickeya dadantii Causing Potato Blackleg in Fujian Province China.[Pubmed:38687907]

Plant Dis. 2024 Apr 30.

Blackleg and soft rot are harmful diseases in potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. (Czajkowski et al. 2015). The occurrence of potato blackleg was serious in potato-producing areas around Xiapu County in Fujian Province, China, in 2021 (6 ha) and 2022 (7 ha), with an incidence of approximately 5%, which reached nearly 23%. Three diseased plants were collected to isolate the pathogen. Single colonies from each sampled plant were isolated and streaked onto fresh plates. DNA from three colonies from different plants was PCR amplified with primer pair 27F/1492R (Lane 1991) for the 16S rRNA gene. Since the sequences were identical, we selected strain M2-3 for further analysis. The strain M2-3 was gram-negative, pectolytic on CVP, grew at 37 degrees C and 5% NaCl. The bacterium was positive for phosphatase activity, erythromycin sensitivity, indole production, gelatin liquefaction, malonic utilization, and acid production from, Melibiose, raffinose, and arabinose. The bacterium was negative for sucrose, alpha-methyl glucoside, sorbitol, trehalose, lactose, and sodium citrate (Fujimoto et al. 2018;),although sucrose and lactose did not provide the expected results, there are exception in all species. The genome of strain M2-3 was sequenced and deposited in the NCBI database under accession numbers: CP077422. An Average Nucleotide Identity (ANI) analysis showed that M2-3 clustered with other D. dadantii strains and has a 98.39% identity with D. dadantii strain DSM 18020 (CP023467). The housekeeping genes (recA, dnaX, acnA, gapA, icd, mdh, mtlD and pgi) were amplified with primer pairs designed previously(Fujimoto et al. 2018; Ma et al. 2007) and sequenced. A multilocus sequence analysis (MLSA) was performed by concatenating the 8 gene sequences and constructing a maximum likelihood phylogenetic tree using PhyloSuite version 1.2.1 (Zhang et al. 2020) and IQ-tree version 1.6.8 (Nguyen et al. 2015) software. Strain M2-3 was clustered together with Dickeya dadantii. For the pathogenicity test, three plants per treatment, totaling nine plants, were used. Bacterial suspensions (1x10;8 CFU/mL) were made in a 10mM PBS buffer. 10 muL of M2-3, D. dadantii type strain 18020 (positive control), and buffer (negative control) were injected into the plant stems near the base. Water stains appeared at the site of inoculation after 2 days and they gradually became black and rotten. The leaves became yellow and wilted, and the petiole base rotted within 5 days of inoculation completing the Koch postulate. According to average nucleotide identity and housekeeping gene sequence analysis, strain M2-3 was identified as Dickeya dadantii. Previous studies have reported several pathogens that cause potato blackleg in China, including P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense (Li-ping et al. 2020; Wang et al. 2021). To the best of our knowledge, this study is the first to report potato blackleg disease caused by Dickeya dadantii in Fujian Province, China. This finding suggests that this pathogen may cause a threat to potato production in Fujian Province.

Systematic Chemical Analysis of Crude Glycan Isolates from the Seven‑herb Decoction Quanzhenyiqitang with Anti-COPD Activity.[Pubmed:38686912]

Chem Biodivers. 2024 Apr 30:e202400277.

The classical Chinese Medicine prescription, Quanzhenyiqitang (QZYQT), containing seven tonic herbs (Shudi, Dangshen, Maidong, Baizhu, Niuxi, Fuzi, and Wuweizi) is clinically used to treat chronic obstructive pulmonary disease (COPD). Although there are studies on the pharmacological effects of QZYQT, little attention has been paid to its active carbohydrate ingredients. We performed a systematic chemical analysis of the crude glycan isolates from the seven-herb decoction (GI-QZYQT) after confirming its anti-COPD activity. GI-QZYQT could enhance lung function, reduce lung damage, and alleviate inflammatory response in mice with COPD. Moreover, two monosaccharides (fructose and glucose) and six oligosaccharides (sucrose, Melibiose, 1-kestose, raffinose, mannotriose, and stachyose), accounting for 40.23% of GI-QZYQT, were discovered using hydrophilic interaction liquid chromatography-evaporative light-scattering detection. Inulin-type fructan with an average molecular weight of 2112 Da was identified using high-performance gel-permeation chromatography in combination with monosaccharide mapping analysis, accounting for 20.10% of GI-QZYQT in mass. The comparison study showed that the identified monosaccharides, oligosaccharides, and the inulin-type fructan of GI-QZYQT were mainly derived from herbs of Shudi, Dangshen, Maidong, Baizhu, and Niuxi. These findings provide crucial information on the chemical composition of GI-QZYQT, which is vital for the in-depth understanding of its bioactivity, mechanism, and product development.

Impact of essential and optional ingredients on microbial and metabolic profiles of kimchi.[Pubmed:38623504]

Food Chem X. 2024 Apr 2;22:101348.

This study aimed to examine the impacts of essential and optional ingredients on the microbial and metabolic profiles of kimchi during 100 days of fermentation, using a mix-omics approach. Kimchi manufactured without essential ingredients (e.g., red pepper, garlic, ginger, green onion, and radish) had lower lactic acid content. The absence of garlic was associated with a higher proportion of Latilactobacillus and Lactococcus, while the absence of red pepper was associated with a greater proportion of Leuconostoc than the control group. In addition, red pepper and garlic served as primary determinants of the levels of organic acids and biogenic amines. Sugar was positively correlated with the levels of Melibiose, and anchovy sauce was positively correlated with the levels of amino acids such as methionine, leucine, and glycine. These findings contribute to a fundamental understanding of how ingredients influence kimchi fermentation, offering valuable insights for optimizing kimchi production to meet various preferences.

Distinct roles of the major binding residues in the cation-binding pocket of MelB.[Pubmed:38464317]

bioRxiv [Preprint]. 2024 Mar 2:2024.02.27.582382.

Salmonella enterica serovar Typhimurium Melibiose permease (MelB(St)) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelB(St) catalyzed the symport of galactosides with either H(+), Li(+), or Na(+), but prefers the coupling with Na(+). Previously, we determined the structures of the inward- and outward-facing conformation of MelB(St), as well as the molecular recognition for galactoside and Na(+). However, the molecular mechanisms for H(+)- and Na(+)-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelB(St) cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na(+)-binding residues in cation selectivity for Na(+) and H(+) by the free energy simulations. The D55C mutant converted MelB(St) to a solely H(+)-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelB(St). Unexpectedly, the H(+)-coupled Melibiose transport with poor activities at higher DeltapH and better activities at reversal DeltapH was observed, supporting that the membrane potential is the primary driving force for the H(+)-coupled symport mediated by MelB(St). This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.

Sugar Utilization-Associated Food-Grade Selection Markers in Lactic Acid Bacteria and Yeast.[Pubmed:38437472]

Pol J Microbiol. 2024 Mar 4;73(1):3-10.

This comprehensive review explores the development of food-grade selection markers in lactic acid bacteria and yeast; some of their strains are precisely defined as safe microorganisms and are crucial in the food industry. Lactic acid bacteria, known for their ability to ferment carbohydrates into lactic acid, provide essential nutrients and contribute to immune responses. With its strong fermentation capabilities and rich nutritional profile, yeast finds use in various food products. Genetic engineering in these microorganisms has grown rapidly, enabling the expression of enzymes and secondary products for food production. However, the focus is on ensuring safety, necessitating food-grade selection markers. Traditional antibiotic and heavy metal resistance selection markers pose environmental and health risks, prompting the search for safer alternatives. Complementary selection markers, such as sugar utilization markers, offer a promising solution. These markers use carbohydrates as carbon sources for growth and are associated with the natural metabolism of lactic acid bacteria and yeast. This review discusses the use of specific sugars, such as lactose, Melibiose, sucrose, D-xylose, glucosamine, and N-acetylglucosamine, as selection markers, highlighting their advantages and limitations. In summary, this review underscores the importance of food-grade selection markers in genetic engineering and offers insights into their applications, benefits, and challenges, providing valuable information for researchers in the field of food microbiology and biotechnology.

Purification and Biochemical Characterization of Novel Galectin from the Black Poplar Medicinal Mushroom Cyclocybe cylindracea (Agaricomycetes) Strain MEST42 from Algeria.[Pubmed:38421696]

Int J Med Mushrooms. 2024;26(2):57-70.

In the present study, a new galectin designated Cyclocybe cylindracea lectin (CCL) was extracted from the fruiting bodies of the wild black popular mushroom C. cylindracea grown in Algeria. The protein was isolated using sepharose 4B as affinity chromatography matrix, and galactose as elutant. The purified galectin was composed of two subunits of 17.873 kDa each, with a total molecular mass of 35.6 kDa. Its agglutinant activity was impeded by galactose and its derivatives, as well as Melibiose. Lactose showed the highest affinity, with a minimal inhibitory concentration of 0.0781 mM. CCL was sensitive to extreme pH conditions, and its binding function decreased when incubated with 10 mM EDTA, and it could be restored by metallic cations such as Ca2+, Mg2+, and Zn2+. CCL agglutinated human red blood cells, without any discernible specificity. Circular dichroism spectra demonstrated that its secondary structure contained beta-sheet as dominant fold. In addition, bioinformatics investigation on their peptide fingerprint obtained after MALDI-TOF/TOF ionization using mascot software confirmed that CCL was not like any previous purified lectin from mushroom: instead, it possessed an amino acid composition with high similarity to that of the putative urea carboxylase of Emericella nidulans (strain FGSC A4/ATCC 38163/CBS 112.46/NRRL 194/M139) with 44% of similarity score.

Polycladomyces zharkentensis sp. nov., a novel thermophilic cellulose- and starch-degrading member of the Bacillota from a geothermal aquifer in Kazakhstan.[Pubmed:38407242]

Int J Syst Evol Microbiol. 2024 Feb;74(2).

A thermophilic, aerobic and heterotrophic filamentous bacterium, designated strain ZKZ2(T), was isolated from a pipeline producing hydrothermal water originating from a >2.3 km deep subsurface geothermal source in Zharkent, Almaty region, Kazakhstan. The isolate was Gram-stain-positive, non-motile, heat-resistant and capable of producing a variety of extracellular hydrolases. Growth occurred at temperatures between 55 and 75 degrees C, with an optimum around 70 degrees C, and at pH values between 5.5 and 9.0, with an optimum at pH 7.0-7.5 with the formation of aerial mycelia; endospores were produced along the aerial mycelium. The isolate was able to utilize the following substrates for growth: glycerol, l-arabinose, ribose, d-xylose, d-glucose, d-fructose, d-mannose, rhamnose, d-mannitol, methyl-d-glucopyranoside, aesculin, salicin, cellobiose, maltose, Melibiose, sucrose, trehalose, melezitose, raffinose, starch, turanose and 5-keto-gluconate. Furthermore, it was able to hydrolyse carboxymethylcellulose, starch, skimmed milk, Tween 60 and Tween 80. The major cellular fatty acids were iso-C(15 : 0), iso-C(17 : 0), iso-C(16 : 0) and C(16 : 0). Our 16S rRNA gene sequence analysis placed ZKZ2(T) within the genus Polycladomyces, family Thermoactinomycetaceae, with the highest similarity to the type species Polycladomyces abyssicola JIR-001(T) (99.18 % sequence identity). Our draft genome sequence analysis revealed a genome size of 3.3 Mbp with a G+C value of 52.5 mol%. The orthologous average nucleotide identity value as compared to that of its closest relative, P. abyssicola JIR-001(T), was 90.23 %, with an in silico DNA-DNA hybridization value of 40.7 %, indicating that ZKZ2(T) represents a separate genome species. Based on the phenotypic and genome sequence differences from the other two Polycladomyces species, we propose that strain ZKZ2(T) represents a novel species, for which we propose the name Polycladomyces zharkentensis sp. nov. The type strain is ZKZ2(T) (=CECT 30708(T)=KCTC 43421(T)).

Mobile barrier mechanisms for Na(+)-coupled symport in an MFS sugar transporter.[Pubmed:38381130]

Elife. 2024 Feb 21;12:RP92462.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial Melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na(+)-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Enhancement of alpha-galactosidase production using novel Actinoplanes utahensis B1 strain: sequential optimization and purification of enzyme.[Pubmed:38345638]

World J Microbiol Biotechnol. 2024 Feb 12;40(3):91.

alpha-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzymes, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing alpha-1,6 linked terminal galacto-oligosaccharide residues such as Melibiose, raffinose, and branched polysaccharides such as galacto-glucomannans and galactomannans. In this study, Actinoplanes utahensis B1 was explored for alpha-galactosidase production, yield improvement, and activity enhancement by purification. Initially, nine media components were screened using the Plackett-Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as the best-supporting nutrients for the highest enzyme secretion by A. Utahensis B1. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in alpha-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72 kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified alpha-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the isolation of Actinoplanes utahensis B1 and optimization of the process for the alpha-galactosidase production as well as single-step purification.

Phytochemistry and pharmacological activities of five species of Bauhinia genus: A review.[Pubmed:38286316]

Fitoterapia. 2024 Apr;174:105830.

Genus Bauhinia has been widely used in the treatment of diabetes, malaria, cough, headache, fever, piles, dysentery, flatulence, diarrhoea, ulcer and cardiovascular problems. Among 600 species of this genus, Bauhinia variegata, B. purpurea, B. championii, B. racemosa, and B. forficata are commonly used in the traditional medicine and found to be throughly investigated for their medicinal properties. They possess diverse pharmacological activities such as anti-diabetic, anti-microbial, antioxidant, anti-arthritic, cardioprotective, hepatoprotective, nephroprotective, fibrinolytic, and wound healing properties and most of the biological activities are corelating with traditional knowledge. Phytochemical analysis indicated that steroids, terpenoids, and flavonoids are prominent in the selected species, whereas bauhiniastatins, bauhinoxepins, racemosols, roseosides, and bauhichamines are found unique to the genus. This review aims to decipher active molecules from the aforementioned species of Bauhinia covering comprehensive analysis of phytochemistry, pharmacological activities and traditional uses. The data has been carefully analyzed to find compounds or fractions with a translational value. In most of the cases, the pharmacological activities have been established, however, further studies are needed such as safety evaluations, target identification, bioavailability, metabolite identification, and pharmacokinetic properties. In conclusion, the pharmacological potential of Bauhinia plants show promise with various leads such as insulin-like protein, roseoside, bauhiniastatin, and Melibiose binding lectin. However, further investigations are required to address existing gaps and advance them towards product development. This review will lay the groundwork for future research initiatives aimed at fully realizing the therapeutic potential of Bauhinia plants.

Nakazawaea tricholomae f.a., sp. nov., a Novel Ascomycetous Yeast Species Isolated from Two Mushroom Species in China.[Pubmed:38281277]

Curr Microbiol. 2024 Jan 28;81(3):78.

Two yeast strains designated as 20-27-1 and 20-28 were isolated from the fruiting bodies of Tricholoma gambosum and Marasmius maximus, respectively, which were collected in Wudaogou, Weichang county, Chengde area, Hebei Province, China. The multi-locus analysis of the sequences of the rDNA ITS, D1/D2 LSU, and SSU regions, together with partial sequences of two protein-coding genes RPB1 and TEF1 indicates that the two strains are closely related to Nakazawaea ernobii and Nakazawaea holstii, showing the similarity values of 99.3-98.7%, 97.2-97.1%, 91.9-92.5%, and 84.6% in D1/D2 LSU, ITS, TEF1, and RPB1, respectively. Physiologically, the two strains are different from N. ernobii and N. holstii in the assimilation of Melibiose, inulin, and DL-lactic acid. Both the phenotypic and phylogenetic analyses indicate that those two strains represent a novel species in the genus Nakazawaea, for which the name Nakazawaea tricholomae f.a., sp. nov. (Fungal Names: FN 571492) is proposed.

Exploring terahertz spectral characteristics of L-sorbose and D-melibiose in solid and liquid states.[Pubmed:38161414]

iScience. 2023 Dec 1;27(1):108602.

Saccharides are essential organic compounds that perform critical functions in sustaining life processes. As biomolecules, their vibrational frequencies predominantly fall in the terahertz (THz) range, making them amenable to analysis using THz techniques. In this study, L-sorbose and D-Melibiose were measured using a THz time-domain spectroscopy system covering a frequency range of 0.1-2.0 THz, and their crystal structures were simulated using density functional theory. The experimental results demonstrated significant agreement with the simulation findings. In addition, the spectral properties of the two saccharides in solution were determined using microfluidic chip technology, thereby facilitating a comparison between the solid and aqueous states. The results demonstrate that the intramolecular and intermolecular interactions of the saccharides were weakened by the presence of water molecules, and the THz absorption spectrum of the same substance solution was found to be correlated with its concentration and temperature.

Chaperoning the major facilitator superfamily at single-molecule level.[Pubmed:37922865]

Structure. 2023 Nov 2;31(11):1291-1294.

In this issue of Structure, Blaimschein et al. elucidate the chaperoning function of the insertase YidC during the insertion and folding of the Melibiose permease MelB. Their single-molecule forced unfolding approach reveals that YidC significantly reduces the misfolding and enhances the folding of helices near the interface of two folding cores.

Expression and characterization of a protease-resistant beta-d-fructofuranosidase BbFFase9 gene suitable for preparing invert sugars from soybean meal.[Pubmed:37809427]

Heliyon. 2023 Sep 6;9(9):e19889.

A novel gene (BbFFase9), with an ORF of 1557 bp that encodes beta-d-fructofuranosidase from Bifidobacteriaceae bacterium, was cloned and expressed in Escherichia coli. The recombinant protein (BbFFase9) was successfully purified and showed a single band with a molecular mass of 66.2 kDa. This was confirmed as a beta-d-fructofuranosidase and exhibited a high specific activity of 209.2 U/mg. Although BbFFase9 was a soluble protein, it exhibited excellent tolerance to proteases such as pepsin, trypsin, acidic protease, neutral protease and Flavourzyme(R), indicating its potential applicability in different fields. BbFFase9 exhibited typical invertase activity, and highly catalyzed the hydrolysis of the alpha1<-->2beta glycosidic linkage in molecules containing fructosyl moieties but with no detectable fructosyltransferase activity. It was optimally active at pH 6.5 and 50 degrees C and stable between pH 6.0 and 9.0 at a temperature of up to 45 degrees C for 30 min BbFFase9 could also effectively hydrolyze galacto-oligosaccharides, which are a flatulence factor in soybean meal, thus releasing new types of product such as Melibiose and mannotriose, or degrading them into invert sugars, the sweeter fructose and glucose. This study is the first to report the application of this type of beta-d-fructofuranosidase.