Sugar Substrates Research Articles

Utility of bacterial peptidoglycan recycling enzymes in the chemoenzymatic synthesis of valuable UDP sugar substrates.

Uridine diphosphate (UDP) sugars are essential precursors for glycosylation reactions in all forms of life. Reactions that transfer the carbohydrate from the UDP donor are catalyzed by glycosyltransferases (Gtfs). While the stereochemistry and negative physiological charge of UDP-sugars are essential for their biochemical function in the cell, these characteristics make them challenging molecules to synthesize and purify on scale in the laboratory. This chapter focuses on the utilization of a chemoenzymatic synthesis of muramyl UDP-sugars, key building blocks in the bacterial cell peptidoglycan. A scalable strategy to obtain UDP-N-acetyl muramic acid derivatives (UDP-NAM), the first committed intermediate used solely in peptidoglycan biosynthesis, is described herein. This methodology utilizes two enzymes involving the cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1-phosphate uridylyl transferase (MurU), respectively. The promiscuity of these enzymes allows for the unique chemical functionality to be embedded in bacterial peptidoglycan both in vitro and in whole bacterial cells for subsequent structural and functional studies of this important biopolymer.

Identification of the complete coding cDNAs and expression analysis of B4GALT1, LALBA, ST3GAL5, ST6GAL1 in the colostrum and milk of the Garganica and Maltese goat breeds to reveal possible implications for oligosaccharide biosynthesis

BackgroundMilk sialylated oligosaccharides (SOS) play crucial roles in many biological processes. The most abundant free SOS in goat’s milk are 3’sialyllactose (3′-SL), 6’sialyllactose (6′-SL) and disialyllactose (DSL). The production of these molecules is determined genetically by the expression of glycosyltransferases and by the availability of nucleotide sugar substrates, but the precise mechanisms regulating the differential patterns of milk oligosaccharides are not known. We aimed to identify the complete cDNAs of candidate genes implicated in SOS biosynthesis (B4GALT1, LALBA, ST3GAL5, ST6GAL1) and to analyse their expression during lactation in the Garganica and Maltese goat breeds. Moreover, we analysed the colostrum and milk contents of 3′-SL, 6′-SL and disialyllactose (DSL) and the possible correlations between expressed genes and SOS.ResultsWe identified the complete coding cDNAs of B4GALT1 (HQ700335.1), ST3GAL5 (KF055858.2), and ST6GAL1 (HQ709167.1), the single nucleotide polymorphism (SNPs) of these genes and 2 splicing variants of the ST6GAL1 cDNA. RT-qPCR analysis showed that LALBA and ST6GAL1 were the genes with the highest and lowest expression in both breeds, respectively. The interaction effects of the breeds and sampling times were associated with higher levels of B4GALT1 and ST3GAL5 gene expression in Garganica than in Maltese goats at kidding. B4GALT1, LALBA, and ST3GAL5 gene expression changed from kidding to 60 and 120 days in Maltese goats, while in Garganica goats, a difference was observed only for the LALBA gene. Breed and lactation effects were also found for SOS contents. Positive correlations of B4GALT1, LALBA, ST3GAL5, and ST6GAL1 with 3′-SL/6′SL and DSL were found.ConclusionsThe genetic effect on the oligosaccharide content of milk was previously highlighted in bovines, and this study is the first to investigate this effect in two goat breeds (Garganica and Maltese) during lactation. The genetic variability of candidate genes involved in SOS biosynthesis highlights their potential role in affecting gene expression and ultimately biological function. The investigation of gene regulatory regions as well as the examination of other sialyltransferase genes will be needed to identify the genetic pattern leading to a higher SOS content in the autochtonous Garganica breed and to protect it using a focused breeding strategy.

Protein:Protein interactions in the cytoplasmic membrane apparently influencing sugar transport and phosphorylation activities of the e. coli phosphotransferase system.

The multicomponent phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates from the medium and concomitantly phosphorylates them, releasing sugar phosphates into the cytoplasm. We have recently provided evidence that many of the integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the biochemical and physiological significance of this finding was not known. We have carried out molecular genetic/biochemical/physiological studies that show that interactions of the fructose PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, depending on the target PTS system under study. Thus, the glucose (Glc), mannose (Man), mannitol (Mtl) and N-acetylglucosamine (NAG) permeases exhibit enhanced in vivo sugar transport and sometimes in vitro PEP-dependent sugar phosphorylation activities while the galactitol (Gat) and trehalose (Tre) systems show inhibited activities. This is observed when the fructose system is induced to high levels and prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in the absence of fructose induction during growth also enhances the rates of uptake of other hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the sugar-specific transphosphorylation activities of these enzyme transporters were not affected either by frustose induction or by fruAB overexpression, showing that the rates of synthesis of the target PTS permeases were not altered. We thus suggest that specific protein-protein interactions within the cytoplasmic membrane regulate transport in vivo (and sometimes the PEP-dependent phosphorylation activities in vitro) of PTS permeases in a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars. These observations appear to be applicable in principle to other types of transport systems as well.

An integrated enrichment-detection platform for identification of contamination of Vibrio parahaemolyticus in food samples

A novel and rapid method to identify contamination of Vibrio parahaemolyticus during an enrichment was developed. The principle was based on the bacteria's capability of carbohydrate fermentation and amino acid decarboxylation. Six different broths were developed with four sugar substrates, namely, dextrose (DEX), arabinose (ARA), mannose (MAO), mannitol (MAI), and two amino acids, namely, lysine (LYS) and ornithine (ODB). Bromocresol purple (BP) and phenol red (PR) were added to detect pH changes of the broths. The absorbance of the color change was determined for all sugar broths at 600 nm and both amino acids at 550 nm. Salinity and pH were adjusted to suppress competitive bacteria. The optimal conditions for the sugar substrates and the amino acids were 8% NaCl/pH = 9.0 and 6% NaCl/pH = 6.0, respectively. In real samples, this method was capable of detecting V. parahaemolyticus with a limit of detection of 0.1–1.0 log CFU/mL (measured as an initial cell concentration in the samples). At 24 h, this method also yielded a 100% sensitivity and improved a specificity to 95%–100%, compared to the 88% specificity as obtained in the conventional method. Integrating the detection of V. parahaemolyticus provided a quick and effective screening for the presence of V. parahaemolyticus.

Preparation of 4'-Spirocyclobutyl Nucleoside Analogues as Novel and Versatile Adenosine Scaffolds.

Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5.

An Insight into Storage Lipid Synthesis by Yarrowia lipolytica Yeast Relating to Lipid and Sugar Substrates Metabolism.

Single cell oil (SCO) is the lipid accumulated in the cells of oleaginous microorganisms. Cellular lipids can be synthesized in two different pathways: de novo by metabolizing hydrophilic substrates and ex novo by fermenting hydrophobic substrates. The aim of the study was to evaluate the effect of carbon source (glucose and olive oil) in the culture medium on the course of microbial oil accumulation in Y. lipolytica cells. The level of selected gene expression by real time quantitative PCR method was investigated. The significant increase in expression of the POX2 gene encoding acyl-CoA oxidase II, which preferentially oxidizes long-chain acyl-CoAs formed from substrate fatty acids incorporated inside the microbial cell, was observed in medium with olive oil in relation to glucose containing medium. Noteworthily, the presence of lipid carbon substrate did not inhibit the level of ACL gene transcription coding for ATP-citrate lyase, the key enzyme of the lipid de novo accumulation process. The present study indicated that de novo lipid biosynthesis could occur despite the presence of fatty acids in the medium, and the synthesis of storage lipids in the presence of lipid carbon substrates could be carried out with the use of both pathways (de novo and ex novo).

Inhibiting the Hexosamine Biosynthetic Pathway Lowers O-GlcNAcylation Levels and Sensitizes Cancer to Environmental Stress.

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

The Relationship Between Wort Sugar Concentration and Yeast Carbon Partitioning During Brewing Fermentations

High gravity (HG) and very high gravity (VHG) fermentations are increasingly attractive within the brewing industry as a means of optimizing process efficiency and energy-saving. However, the use of highly concentrated worts is concomitant with a number of biological stress factors that can impact on yeast quality and fermentation performance. In order to eliminate or reduce potentially detrimental effects, brewing yeast respond to their environment by shunting carbon into different metabolic end products, which assist in the protection of cells, but also impact on final ethanol yield. The purpose of this research was to investigate the impact of substrate sugar concentration on carbon partitioning in brewing fermentations. This was conducted using a series of laboratory-scale fermentations with worts of 13°P, 18°P and 24°P, pitched using lager and ale yeast strains. Fermentation performance was assessed with respect to the uptake of wort sugars and the production of key carbon-based metabolites, leading to a calculation of yeast central carbon flux. Analysis of carbon assimilation and dissimilation revealed that changes in intracellular trehalose, glycogen, higher alcohols and esters were observed, however the production of yeast biomass acted as the major trade-off with ethanol production. The data presented here shows for the first time the requirements of yeast populations during HG and VHG conditions and the factors that have a major impact on key performance indicators. This data has major significance for fermentation-based industries globally and is especially important for those sectors seeking to maximize yield from existing resources through high gravity fermentations.

Pretreatment Ethanol From Cellulosic

<p><strong>Abstract.</strong> Pre-treatment is an important tool for practical cellulose conversion processes and can be carried out in different ways such as mechanical pre-treatment, steam explosion, ammonia fiber explosion, supercritical CO2 treatment, alkali or acid pretreatment, ozone pre-treatment, physicochemical pretreatment, dilute-acid pretreatment and biological pre-treatment. Biomass pretreatment with hot water (HW) is the most investigated physicochemical method use the differences in the thermal stabilities of the major components of lignocellulosic materials. Acid pretreatment of lignocellulosic biomass aims at increasing the sugar substrate digestibility, defined as the concentration of reducing sugars after the hydrolysis, by microorganisms. Acid hydrolysis is an attractive pretreatment method as the hemicellulose degradation runs with the efficiency of approximately 20-90%, depending on the process conditions. Dilute acid (DA) processes with continued research and development, no significant breakthroughs have been made to raise the glucose yields much higher than 65-70%. Acid pretreatment is much more effective than water and alkaline pretreatment in terms of cellulose accessibility increase compared with DA and HW pretreatment.</p><p> </p><p><strong>Keywords:</strong> ethanol, cellulosic, pre-treatment</p>

Cloning and Functional Assessments of Floral-Expressed SWEET Transporter Genes from Jasminum sambac.

Sugar transporters of the SWEET family mediate cross membrane movement of mono- and disaccharides and play vital roles in diverse physiological and pathophysiological processes, including sink–source relationship, pathogen responses, reproductive growth, and development. However, it remains to be determined how these transporters function in non-module plants of agricultural significance, given the evolutionarily diverse traits. In this study, we combined transcriptome analysis, rapid amplification of cDNA ends-cloning (RACE-cloning), expression profiling, and heterologous functional assay to identify SWEET genes that may have potential roles during flower opening and sexual reproduction in Jasminum sambac . During the anthesis, the floral organs of J. sambac express seven SWEET homologous genes from all four clades of the family. JsSWEET9 and 2 are significantly upregulated when flowers are fully opened, up to 6- and 3-fold compared to unopened buds, respectively. The other transporters, JsSWEET1, 5, 10, and 17 are also accumulated slightly at stage associated with fragrance release, whereas only the vacuole transporter JsSWEET16 showed small decrease in transcript level after anthesis. The JsSWEET5, a clade II member, is capable to complement yeast cell uptake on most tested sugar substrates with a preference for hexoses, while the clade I transporter JsSWEET1 mediates merely galactose import when expressed in yeast. Our results provide first evidence for further investigation on sugar transport and allocation during flowering and reproductive processes in J. sambac.

Factors determining the reaction temperature of the solvent-free enzymatic synthesis of trehalose esters

Solvent-free synthesis encourages the design of processes and products that reduce the use and generation of hazardous chemicals. Given the importance of developing greener methodologies, we sought to determine the factors influencing the reaction temperature required for solvent-free, enzymatic synthesis of sugar esters such as trehalose (TRE) esters, using Novozyme 435 as the enzyme catalyst. The use of lauric acid (La) and ethyl laurate (LaEt) as acyl donors did not affect the activation temperature for the generation of trehalose diesters (TDEs), despite the differences in corresponding by-products (water and ethanol). However, when glucose (GLU) and La were employed as reaction substrates as a comparison, glucose monoester (GME) generation readily occurred at much lower temperatures than with the TRE esters, even without a water collection device. Moreover, when the glass transition temperature (Tg) of the sugar substrates increased, a higher reaction temperature was required. These results suggest that while the activation temperature of the reaction did not correlate with the boiling point of the by-product, it did correlate with the glass transition temperature (Tg) of the trehalose substrates. Thus, our work demonstrates the importance of the physical state of amorphous matrices in determining the optimal reaction temperature of a solvent-free sugar synthesis.

Exploring two contrasting surface-active exopolysaccharides from a single strain of Ochrobactrum utilizing different hydrocarbon substrates.

During production and characterization of exopolysaccharides (EPS) of Ochrobactrum pseudintermedium C1, it was observed that an experimental change in the basic hydrocarbon type of substrate for bacterial utilization led to elicitation of different surface-active properties in the EPS produced. In the sugar substrate, it elicited surfactant property, while in oil substrates it elicited emulsifying property, which indicated that the EPS might be different. Consequently, attention was focused on a detailed analysis of this substrate-specific EPS. Utilizing waste sugar, edible, and mineral oil substrates, EPS produced in each situation was characterized. Besides estimating surface activity and thermostability, each substrate-specific EPS was analyzed by Fourier-transform infrared spectroscopy, gas chromatography-mass spectroscopy, 1 H-nuclear magnetic resonance, and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy to find any structural difference. The results were significantly contrasting although the similarity in molecular mass suggested a basic similarity in polysaccharide structure. Morphological differences were also evident both macroscopically and microscopically with scanning electron microscopy. As the surface-active property of EPS was dependent on the substrate utilized, their structural differences might account for it. These diverse surface activities of EPS produced by a single bacterial strain simply by changing the nature of substrate would also augment their bioapplications. Moreover, utilization of waste and easily available substrates should make such applications convenient, ecofriendly, and cost-worthy.

Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars

BackgroundIn the context of sustainable development, yeast are one class of microorganisms foreseen for the production of oil from diverse renewable feedstocks, in particular those that do not compete with the food supply. However, their use in bulk production, such as for the production of biodiesel, is still not cost effective, partly due to the possible poor use of desired substrates or poor robustness in the practical bioconversion process. We investigated the natural capacity of Blastobotrys adeninivorans, a yeast already used in biotechnology, to store lipids under different conditions.ResultsThe genotyping of seven strains showed the species to actually be composed of two different groups, one that (including the well-known strain LS3) could be reassigned to Blastobotrys raffinosifermentans. We showed that, under nitrogen limitation, strains of both species can synthesize lipids to over 20% of their dry-cell weight during shake-flask cultivation in glucose or xylose medium for 96 h. In addition, organic acids were excreted into the medium. LS3, our best lipid-producing strain, could also accumulate lipids from exogenous oleic acid, up to 38.1 ± 1.6% of its dry-cell weight, and synthesize lipids from various sugar substrates, up to 36.6 ± 0.5% when growing in cellobiose. Both species, represented by LS3 and CBS 8244T, could grow with little filamentation in the lipogenic medium from 28 to 45 °C and reached lipid titers ranging from 1.76 ± 0.28 to 3.08 ± 0.49 g/L in flasks. Under these conditions, the maximum bioconversion yield (YFA/S = 0.093 ± 0.017) was obtained with LS3 at 37 °C. The presence of genes for predicted subunits of an ATP citrate lyase in the genome of LS3 reinforces its oleaginous character.ConclusionsBlastobotrys adeninivorans and B. raffinosifermentans, which are known to be xerotolerant and genetically-tractable, are promising biotechnological yeasts of the Saccharomycotina that could be further developed through genetic engineering for the production of microbial oil. To our knowledge, this is the first report of efficient lipid storage in yeast when cultivated at a temperature above 40 °C. This paves the way to help reducing costs through consolidated bioprocessing.

Itaconic acid fermentation using activated charcoal-treated corn stover hydrolysate and process evaluation based on Aspen plus model

Itaconic acid production using lignocellulose materials is a promising approach to replace sugar substrates such as glucose that are expensive. However, the complicated detoxification of the hydrolysate is pre-requisite to remove the lignocellulose-derived inhibitors to enable high organic acid fermentation. In this study, the hydrolysate prepared using dry acid pretreated and biodetoxified corn stover was tested for itaconic acid production by the fungal strain Aspergillus terreus M69. Corn stover hydrolysate containing 0.85 g/L acetic acid severely inhibited the organic acid production, but a treatment on the hydrolysate with activated charcoal to remove partial acetic acid helped to produce 33.6 g/L itaconic acid at a yield of 0.56 g/g. Most acetic acid released during enzymatic hydrolysis other than pretreatment was responsible for the inhibition of itaconic acid production. The technol-economic analysis showed that the minimum itaconic acid selling price was $1.647 per kg, which was lower than its market price. This study demonstrates the great potential of itaconic acid production using lignocellulose.

The potential of indigenous bacteria from oil sludge for biosurfactant production using hydrolysate of agricultural waste

Abstract. Ni’matuzahroh, Sari SK, Ningrum IP, Pusfita AD, Marjayandari L, Trikurniadewi N, Ibrahim SNMM, Fatimah, Nurhariyati T, Surtiningsih T, Yuliani H. 2019. The potential of indigenous bacteria from oil sludge for biosurfactant production using hydrolysate of agricultural waste. Biodiversitas 20: 1374-1379. Biosurfactants are amphipathic compounds which are useful in various fields of health, industry, and remediation. Biosurfactants are produced by bacteria that grow in hydrocarbon or sugar substrates. Hydrolysis product of agricultural waste can be used as a biosurfactant production medium. This research aims to obtain biosurfactant producing bacteria from Balongan oil sludge, Indonesia. The ability to grow and produce biosurfactant by indigenous bacteria was tested using a medium of Synthetic Mineral Water (SMW) added by 209.3 ppm of rice straw hydrolysis product (RSHP). The growth of bacteria was evaluated through Total Plate Count (TPC) and biosurfactant production was evaluated through measurement of emulsification activity and surface tension. Six indigenous bacteria were capable to produce biosurfactants in the RSHP. Emulsification activity was not detected, but surface tension reduction was founded. The best biosurfactant was indicated by surface tension value of 53.56 mN/m with TPC value of 20.07 CFU/mL at the 5th day of incubation by BP (1) 5. The indigenous bacteria were identified as Propionibacterium BP (1) 1, Propionibacterium BP (1) 3, Bacillus BP (1) 4, Corynebacterium BP (1) 5, Corynebacterium BP (1) 8, and Rothia BP (1) 6. Utilization of sugar as hydrolysis product of agricultural waste is an innovation of raw materials for biosurfactant production.

A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. melilotiIMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.

Understanding the multifaceted roles of the phosphoenolpyruvate: Phosphotransferase system in regulation of Salmonella virulence using a mutant defective in ptsI and crr expression

The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) catalyzes the translocation of sugar substrates with their concomitant phosphorylation in bacteria. In addition to its intrinsic role in sugar transport and metabolism, numerous recent studies report the versatility of the PTS to interconnect energy and signal transduction in response to sugar availability. In this study, the role of PTS in Salmonella virulence regulation was explored. To decipher the regulatory network coordinated by the PTS during Salmonella infection, a transcriptomic approach was applied to a transposon insertion mutant with defective expression of ptsI and crr, which encode enzyme I and enzyme IIAGlc of the PTS, respectively. There were 114 differentially expressed genes (DEGs) exhibiting two-fold or higher expression changes in the transposon mutant strain, with 13 up-regulated genes versus 101 down-regulated genes. One-third of the DEGs were associated with energy production and carbohydrate/amino acid metabolism pathways, implicating the prominent role of the PTS in carbohydrate transport. With regard to regulation of virulence, the tested mutant decreased the expression of genes associated with quorum sensing, Salmonella pathogenicity islands, flagella, and the PhoPQ regulon. We investigated the possibility of PTS-mediated regulation of virulence determinants identified in the transcriptomic analysis and proposed a regulatory circuit orchestrated by the PTS in Salmonella infection of host cells. These results suggest that Salmonella divergently controls virulence attributes in accordance with the availability of carbohydrates in the environment.

16S rRNA gene profiling and genome reconstruction reveal community metabolic interactions and prebiotic potential of medicinal herbs used in neurodegenerative disease and as nootropics.

The prebiotic potential of nervine herbal medicines has been scarcely studied. We therefore used anaerobic human fecal cultivation to investigate whether medicinal herbs commonly used as treatment in neurological health and disease in Ayurveda and other traditional systems of medicine modulate gut microbiota. Profiling of fecal cultures supplemented with either Kapikacchu, Gotu Kola, Bacopa/Brahmi, Shankhapushpi, Boswellia/Frankincense, Jatamansi, Bhringaraj, Guduchi, Ashwagandha or Shatavari by 16S rRNA sequencing revealed profound changes in diverse taxa. Principal coordinate analysis highlights that each herb drives the formation of unique microbial communities predicted to display unique metabolic potential. The relative abundance of approximately one-third of the 243 enumerated species was altered by all herbs. Additional species were impacted in an herb-specific manner. In this study, we combine genome reconstruction of sugar utilization and short chain fatty acid (SCFA) pathways encoded in the genomes of 216 profiled taxa with monosaccharide composition analysis of each medicinal herb by quantitative mass spectrometry to enhance the interpretation of resulting microbial communities and discern potential drivers of microbiota restructuring. Collectively, our results indicate that gut microbiota engage in both protein and glycan catabolism, providing amino acid and sugar substrates that are consumed by fermentative species. We identified taxa that are efficient amino acid fermenters and those capable of both amino acid and sugar fermentation. Herb-induced microbial communities are predicted to alter the relative abundance of taxa encoding SCFA (butyrate and propionate) pathways. Co-occurrence network analyses identified a large number of taxa pairs in medicinal herb cultures. Some of these pairs displayed related culture growth relationships in replicate cultures highlighting potential functional interactions among medicinal herb-induced taxa.

Isolation, characterization and optimization of EPSs produced by a cold-adaptedMarinobacterisolate from Antarctic seawater

AbstractMarinobactersp. W1-16 from Antarctic surface seawater was analysed for the production of extracellular polymeric substances (EPSs). Enhancement of the EPS biosynthesis was carried out by evaluating the influences of the carbon source (type and concentration), temperature, pH and salinity. EPS yields varied strongly depending on sugar substrate and temperature, while pH and salinity did not strongly affect levels of EPS production.Marinobactersp. W1-16 produced the highest quantity of EPSs when growing at 15°C and pH 8, in the presence of 2% glucose and 3% NaCl. The EPS chemical characterization revealed a molecular weight of about 260 kDa. Colorimetric assays determined a higher quantity of carbohydrate than of proteins and uronic acids, as well as the presence of sulphate, in the extracted EPSs. The monosaccharidic composition resulted in Glc:Man:Gal:GalN:GalA:GlcA in relative molar proportions of 1:0.9:0.2:0.1:0.1:0.01. Some biotechnological potentialities (i.e. emulsifying and cryoprotective actions, and heavy metal binding properties) of the EPSs were proved, suggesting possible industrial and bioremediation applications.

Monitoring glycosidase activity for clustered sugar substrates, a study on β-glucuronidase.

Determination of glycosidase hydrolysis kinetics for a monovalent sugar substrate is relatively straightforward and classically achieved by monitoring the fluorescence signal released from the sugar-conjugated probe after enzymatic hydrolysis. Naturally occuring sugar epitopes are, however, often clustered on biopolymers or at biological surfaces, and previous reports have shown that glycosidase hydrolytic rates can differ greatly with multivalent presentation of the sugar epitopes. New probes are needed to make it easier to interpret the importance of substrate clustering towards a specific enzyme activity. In this work, we developed multivalent glucuronide substrates attached to fluorescent amino-coumarines through self-immolative linkers to enable real time-monitoring of the hydrolysing activity of E.coli β-glucuronidases (GUS) towards clustered substrates. GUS are exoglycosidases of considerable therapeutic interest cleaving β-d-glucuronides and are found in the lysosomes, in the tumoral microenvironment, and are expressed by gut microbiota. GUS showed a much lower catalytic efficiency in hydrolysing clustered glucuronides due to a significantly lower enzymatic velocity and affinity for the substrates. GUS was 52-fold less efficient in hydrolysing GlcA substrates presented on an octameric silsequioxane (COSS) compared with a monovalent GlcA of similar chemical structure. Thus, kinetic and thermodynamic data of GUS hydrolysis towards multivalent glucuronides were easily obtained with these new types of enzymatically-triggered probes. More generally, adapting the substrate nature and valency of these new probes, should improve understanding of the impact of multivalency for a specific enzyme.