Sugar Precursors Research Articles

Polyamine Synthesis Effects Capsule Expression by Reduction of Precursors in Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus, Spn) colonizes the human nasopharynx asymptomatically but can cause infections such as otitis media, and invasive pneumococcal disease such as community-acquired pneumonia, meningitis, and sepsis. Although the success of Spn as a pathogen can be attributed to its ability to synthesize and regulate capsular polysaccharide (CPS) for survival in the host, the mechanisms of CPS regulation are not well-described. Recent studies from our lab demonstrate that deletion of a putative polyamine biosynthesis gene (ΔcadA) in Spn TIGR4 results in the loss of the capsule. In this study, we characterized the transcriptome and metabolome of ΔcadA and identified specific mechanisms that could explain the regulatory role of polyamines in pneumococcal CPS biosynthesis. Our data indicate that impaired polyamine synthesis impacts galactose to glucose interconversion via the Leloir pathway which limits the availability of UDP-galactose, a precursor of serotype 4 CPS, and UDP-N-acetylglucosamine (UDP-GlcNAc), a nucleotide sugar precursor that is at the intersection of CPS and peptidoglycan repeat unit biosynthesis. Reduced carbon flux through glycolysis, coupled with altered fate of glycolytic intermediates further supports impaired synthesis of UDP-GlcNAc. A significant increase in the expression of transketolases indicates a potential shift in carbon flow toward the pentose phosphate pathway (PPP). Higher PPP activity could constitute oxidative stress responses in ΔcadA which warrants further investigation. The results from this study clearly demonstrate the potential of polyamine synthesis, targeted for cancer therapy in human medicine, for the development of novel prophylactic and therapeutic strategies for treating bacterial infections.

Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis.

Glycosylation is a common modification reaction in natural products biosynthesis and has been known to be a post assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation, and in vitro pathway reconstitution we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-[3-aminophenyl]3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free β-ketoacid that subsequently undergoes decarboxylation.

Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.

4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.

Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs

Sialic acids cap the glycans of cell surface glycoproteins and glycolipids. They are involved in a multitude of biological processes and aberrant sialic acid expression is associated with several pathologies. Sialic acids modulate the characteristics and functions of glycoproteins and regulate cell-cell as well as cell-extracellular matrix interactions. Pathogens such as influenza virus use sialic acids to infect host cells and cancer cells exploit sialic acids to escape from the host's immune system. The introduction of unnatural sialic acids with different functionalities into surface glycans enables the study of the broad biological functions of these sugars and presents a therapeutic option to intervene with pathological processes involving sialic acids. Multiple chemically modified sialic acid analogs can be directly utilized by cells for sialoglycan synthesis. Alternatively, analogs of the natural sialic acid precursor sugar N-Acetylmannosamine (ManNAc) can be introduced into the sialic acid biosynthesis pathway resulting in the intracellular conversion into the corresponding sialic acid analog. Both, ManNAc and sialic acid analogs, have been employed successfully for a large variety of glycoengineering applications such as glycan imaging, targeting toxins to tumor cells, inhibiting pathogen binding, or altering immune cell activity. However, there are significant differences between ManNAc and sialic acid analogs with respect to their chemical modification potential and cellular metabolism that should be considered in sialic acid glycoengineering experiments.

Synthesis of 6′‐Methyl‐2′‐O,4′‐C‐methylene‐α‐L‐ ribofuranosyl‐pyrimidine Nucleosides

AbstractHerein, we report the efficient synthesis of (6′R)‐ and (6′S)‐6′‐methyl‐2′‐O,4′‐C‐methylene‐α‐L‐ribofuranosyl‐thymine, and (6′R)‐ and (6′S)‐6′‐methyl‐2′‐O,4′‐C‐methylene‐α‐L‐ribofuranosyl‐uracil starting from diacetone glucofuranose in overall yields of 6.3, 4.7, 5.4 and 4.0%, respectively. The key step in the synthesis of stereochemically defined 6′‐Me‐bicyclic‐nucleosides is the nucleophilic addition of methyl group at methylene carbon of 4‐C–CH2OH moiety of the 4‐C‐tert‐butyldiphenylsilyloxymethylated sugar precursor. Thus, the methyl group was added on the aldehyde obtained from Dess‐Martin periodinane oxidation of the precursor alcohol employing AlMe3 in hexane. Both (6′R)‐ and (6′S)‐stereoisomers of bicyclic nucleosides T and U were successfully synthesized following Vorbrüggen nucleobase coupling of T and U with triacetylated glycosyl donor obtained from acetolysis of (5R)‐ and (5 S)‐4‐C‐(tert‐butyldiphenylsilyloxymethyl)‐5‐C‐methyl‐1,2‐O‐isopropylidene‐3‐O‐(2‐naphthylmethyl)‐α‐D‐xylofuranoses and further cyclization and deprotection of the resulted nucleoside. One of the nucleosides, (6′R)‐6′‐methyl‐2′‐O,4′‐C‐methylene‐α‐L‐ribofuranosyl‐uracil has been reported earlier in 1.8% yield, while the present methodology yielded the nucleoside in 5.4% yield. All the synthesized 6′‐Me‐bicyclic‐nucleosides showed no significant anti‐viral activity against H1 N1 strain of influenza A virus (A/Puerto Rico/8/1934).

Glycosylation in health and disease.

The glycome describes the complete repertoire of glycoconjugates composed of carbohydrate chains, or glycans, that are covalently linked to lipid or protein molecules. Glycoconjugates are formed through a process called glycosylation and can differ in their glycan sequences, the connections between them and their length. Glycoconjugate synthesis is a dynamic process that depends on the local milieu of enzymes, sugar precursors and organelle structures as well as the cell types involved and cellular signals. Studies of rare genetic disorders that affect glycosylation first highlighted the biological importance of the glycome, and technological advances have improved our understanding of its heterogeneity and complexity. Researchers can now routinely assess how the secreted and cell-surface glycomes reflect overall cellular status in health and disease. In fact, changes in glycosylation can modulate inflammatory responses, enable viral immune escape, promote cancer cell metastasis or regulate apoptosis; the composition of the glycome also affects kidney function in health and disease. New insights into the structure and function of the glycome can now be applied to therapy development and could improve our ability to fine-tune immunological responses and inflammation, optimize the performance of therapeutic antibodies and boost immune responses to cancer. These examples illustrate the potential of the emerging field of'glycomedicine'.

Remodeling of O Antigen in Mucoid Pseudomonas aeruginosa via Transcriptional Repression of wzz2.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people with cystic fibrosis (CF). Chronic P. aeruginosa isolates generally do not express O antigen and often have a mucoid phenotype, which is characterized by the overproduction of the exopolysaccharide alginate. Therefore, O antigen expression and the mucoid phenotype may be coordinately regulated upon chronic adaption to the CF lung. Here we demonstrate that PDO300, a mucoid strain derived from the nonmucoid laboratory isolate PAO1, does not produce very long O antigen due to decreased expression of Wzz2, the very long O antigen chain length control protein, and that mucoid clinical isolates express reduced levels of Wzz2 compared to nonmucoid isolates. Further, we show that forcing the expression of very long O antigen by PDO300, by providing wzz2 in trans, does not alter alginate production, suggesting that sugar precursors are not limited between the two biosynthesis pathways. Moreover, we confirm that AmrZ, a transcription factor highly expressed in mucoid strains, is a negative regulator of wzz2 promoter activity and very long O antigen expression. These experiments identify the first transcriptional regulator of O antigen chain length in P. aeruginosa and support a model where transition to a chronic mucoid phenotype is correlated with downregulation of very long O antigen through decreased Wzz2 production.IMPORTANCE Detection of mucoid Pseudomonas aeruginosa, characterized by the overproduction of alginate, is correlated with the establishment of a chronic pulmonary infection and disease progression in people with cystic fibrosis (CF). In addition to the overproduction of alginate, loss of O antigen lipopolysaccharide production is also selected for in chronic infection isolates. In this study, we have identified the regulatory network that inversely regulates O antigen and alginate production. Understanding the regulation of these chronic phenotypes will elucidate mechanisms that are important for the establishment of a long-term P. aeruginosa lung infection and ultimately provide an opportunity for intervention. Preventing P. aeruginosa from chronically adapting to the CF lung environment could provide a better outcome for people who are infected.

Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.

The ice composition in the disk around V883 Ori revealed by its stellar outburst

Complex organic molecules (COMs), which are the seeds of prebiotic material and precursors of amino acids and sugars, form in the icy mantles of circumstellar dust grains but cannot be detected remotely unless they are heated and released to the gas phase. Around solar-mass stars, water and COMs only sublimate in the inner few au of the disk, making them extremely difficult to spatially resolve and study. Sudden increases in the luminosity of the central star will quickly expand the sublimation front (so-called snow line) to larger radii, as seen previously in the FU Ori outburst of the young star V883 Ori. In this paper, we take advantage of the rapid increase in disk temperature of V883 Ori to detect and analyze five different COMs, methanol, acetone, acetonitrile, acetaldehyde, and methyl formate, in spatially-resolved submillimeter observations. The COMs abundances in V883 Ori is in reasonable agreement with cometary values. This result suggests that outbursting young stars can provide a special opportunity to study the ice composition of material directly related to planet formation.

Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification.

SummaryBiosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC50 of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.

Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity.

Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity.

Radiolytic Synthesis of Cyanogen Chloride, Cyanamide and Simple Sugar Precursors

AbstractChemical evolution, from simple molecules to complex systems, is fundamental to modern origins of life research, and complex reaction networks are often supposed to have operated on the early Earth. Herein, a variety of compounds useful particularly for RNA synthesis − namely, cyanogen chloride, cyanamide, and nitrile precursors to simple sugars − are produced in short order by a chemical reaction network starting from hydrogen cyanide and driven by gamma radiolysis. The radiolytic yield of cyanamide was found to be proportional to the concentration of chloride, an often overlooked spectator anion. Aqueous irradiation of hydrogen cyanide in the presence of sodium chloride also affords cyanogen chloride, a possible intermediate in the radiolytic synthesis of cyanamide. Meanwhile, the synthesis of simple sugar precursors is proposed to proceed through a Kiliani‐Fischer homologation mechanism made possible by the reducing power of the solvated electron and hydrogen atom. Such a reaction network has the potential to serve as a model for better understanding and engineering chemical evolution of complex mixtures in the laboratory that could have happened on the early Earth.

Stability of aqueous formaldehyde under γ irradiation: prebiotic relevance

AbstractFormaldehyde is a precursor of sugars, which are compounds essential in all forms of life and a necessary molecule for prebiotic processes. This work focuses on evaluating the stability of formaldehyde exposed to a high radiation field simulating prebiotic conditions on primitive Earth, such as the ocean or shallow waters. Formaldehyde may have been formed from reactions in the atmosphere and from rainout processes reached water bodies. In our experiments, we employed γ radiation and found that formaldehyde was labile towards radiation and decomposed even at low irradiation doses due to the fact that aldehyde/hydrate groups present in formaldehyde structure are very reactive under irradiation. However, after exposing this molecule to several doses of irradiation, we detected the formation of formic acid and glycolaldehyde – both of which are of prebiotic interest. We also observed formaldehyde regeneration by one of its radiolytic products: formic acid.

Enzymatic synthesis of xylulose from formaldehyde

Xylulose as a metabolic intermediate is the precursor of rare sugars, and its unique pattern of biological activity plays an important role in the fields of food, health, medicine and so on. The aim of this study was to design a new pathway for xylulose synthesis from formaldehyde, which is one of the most simple and basic organic substrate. The pathway was comprised of 3 steps: (1) formaldehyde was converted to glycolaldehyde by benzoylformate decarboxylase mutant BFD-M3 (from Pseudomonas putida); (2) formaldehyde and glycolaldehyde were converted to dihydroxyacetone by BFD-M3 as well; (3) glycolaldehyde and dihydroxyacetone were converted to xylulose by transaldolase mutant TalB-F178Y (from Escherichia coli). By adding formaldehyde (5 g/L), BFD-M3 and TalB-F178Y in one pot, xylulose was produced at a conversion rate of 0.4%. Through optimizing the concentration of formaldehyde, the conversion rate of xylulose was increased to 4.6% (20 g/L formaldehyde), which is 11.5 folds higher than the initial value. In order to further improve the xylulose conversion rate, we employed Scaffold Self-Assembly technique to co-immobilize BFD-M3 and TalB-F178Y. Finally, the xylulose conversion rate reached 14.02%. This study provides a new scheme for the biosynthesis of rare sugars.

Comparative transcription profiling of two fermentation cultures of Xanthomonas campestris pv. campestris B100 sampled in the growth and in the stationary phase.

The ɣ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) is the producer of the biopolymer xanthan, a polysaccharide which is used as a thickener in numerous industrial applications. In this study, we present a global transcriptome profiling of two Xcc strain B100 cultures obtained from fermentation during the growth phase and the subsequent stationary phase associated with xanthan biosynthesis. During the xanthan production phase, highly abundant transcripts belonged to genes encoding for small RNAs, glycogen biosynthesis, and xanthan export. A total of 1850 (40%) genes were differentially transcribed during the stationary phase where 924 were transcriptionally up-regulated and 926 genes were down-regulated. An overview of differentially transcribed genes includes a significant down-regulation of genes involved in transcription, translation, and amino acid biosynthesis pathways. A group of up-regulated genes was involved in cellular response against oxidative stress, such as those coding for superoxide dismutase and catalase. Genes encoding enzymes involved in nucleotide sugar precursor synthesis of xanthan biosynthesis, such as xanA, galU, and ugd, exhibited a transcription pattern that did not change during the growth and stationary phase. Regarding the transcription pattern of the gum gene cluster that govern xanthan biosynthesis, a significant up-regulation of the genes gumB, gumC, and gumD was observed, while the transcript pools of the genes gumG, gumH, gumI, and gumJ were reduced and those of genes gumE, gumF, gumK, gumL, and gumM remained un-changed during the stationary phase compared to the growth phase. The obtained data represents the first analysis of gene expression patterns under xanthan production conditions and provides the bases for future studies aiming at enhancing xanthan yield.

Hyaluronan: A Master Switch Between Vascular Homeostasis and Inflammation.

Hyaluronan (HA) matrix responds quickly to changing conditions during atherogenesis and atheroprogression. Our evolving understanding of HA’s specific functions at the interface of inflammatory responses and phenotypic control of mesenchymal cells in the vessel wall points to its importance in inflammation and atherosclerosis. Additionally, the enzymes that mediate HA biosynthesis and degradation are potential target molecules for novel therapies, as are signal-transducing HA receptors: deciphering the HA matrix, thus, has significant translational possibilities. HA is glycosaminoglycan polymer without covalent binding to a core protein. A unique feature of HA is its de novo synthesis at the plasma membrane by HA synthases (HAS1–3) from UDP (uridine diphosphate)-sugar precursors. The chemical structure of HA is simple and comprises repeating disaccharides of β(1,4)- N -acetyl-d-glucosamine and β(1,3)-d-glucuronic acid that form an unbranched and unmodified polymer. This structure is conserved across all mammalian species. The biological functions of HA are complex and highly context-specific because as soon as HA is extruded to the extracellular space, it is subject to complex interactions with HA-binding proteins (hyaladherins). The function of the pericellular HA matrix depends on the particular hyaladherins present and their engagement with HA receptors, such as CD44, RHAMM (receptor of HA-mediated motility), and Lyve-1. The resulting interactions modulate the adhesion, migration, and proliferation of smooth muscle cells (SMC), fibroblasts, and other cell types present in vascular tissues. In parallel, HA complexes also influence important aspects of immune cell polarization, differentiation, and function. Beyond the immediate pericellular microenvironment, HA is also integrated into structures such as basement membranes, is present in circulation, and can function as a signaling molecule to activate HA receptors. The molecular weight of HA has critical functional importance in vivo. In the steady state, longer, high-molecular-weight HA polymers support homeostasis and immune surveillance. On injury, however, HA …

Fragmentation Pathways of Lithiated Hexose Monosaccharides.

We characterize the primary fragmentation reactions of three isomeric lithiated D-hexose sugars (glucose, galactose, and mannose) utilizing tandem mass spectrometry, regiospecific labeling, and theory. We provide evidence that these three isomers populate similar fragmentation pathways to produce the abundant cross-ring cleavage peaks (0,2A1 and 0,3A1). These pathways are highly consistent with the prior literature (Hofmeister et al. J. Am. Chem. Soc. 113, 5964-5970, 1991, Bythell et al. J. Am. Soc. Mass Spectrom. 28, 688-703, 2017, Rabus et al. Phys. Chem. Chem. Phys. 19, 25643-25652, 2017) and the present labeling data. However, the structure-specific energetics and rate-determining steps of these reactions differ as a function of precursor sugar and anomeric configuration. The lowest energy water loss pathways involve loss of the anomeric oxygen to furnish B1 ions. For glucose and galactose, the lithiated α-anomers generate ketone structures at C2 in a concerted reaction involving a 1,2-migration of the C2-H to the anomeric carbon (C1). In contrast, the β-anomers are predicted to form 1,3-anhydroglucose/galactose B1 ion structures. Initiation of the water loss reactions from each anomeric configuration requires distinct reactive conformers, resulting in different product ion structures. Inversion of the stereochemistry at C2 has marked consequences. Both lithiated mannose forms expel water to form 1,2-anhydromannose B1 ions with the newly formed epoxide group above the ring. Additionally, provided water loss is not instantaneous, the α-anomer can also isomerize to generate a ketone structure at C2 in a concerted reaction involving a 1,2-migration of the C2-H to C1. This product is indistinguishable to that from α-glucose. The energetics and interplay of these pathways are discussed. Graphical Abstract ᅟ.

Protein lysine acetylation is a regulatory mechanism for Bacillus subtilis multicellularity

Lysine acetylation is a post‐translational modification that alters the charge, conformation, and stability of proteins. Published lists of acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of function, including central metabolism, DNA transcription and binding, chemotaxis, and cell size. Bacillus subtilis is a non‐pathogenic soil bacterium, and model organism for sporulation, motility, biofilm formation, and general multicellular development. The genetic regulation of these processes has been extensively studied, thus, we aimed to investigate the role of lysine acetylation in multicellular development (or biofilm formation) in B. subtilis. To further investigate acetylation, we generated an acetylome under biofilm‐inducing conditions and identified acetylated proteins involved in multicellularity; specifically, swarming and biofilm formation. Upon deletion of known genes for acetylation, cells had significantly slower swarming kinetics and a significantly smaller final swarming radius compared to wildtype. This suggests that acetylation impacts bacterial swarming. Biofilm is a mixture of cells and secreted matrix consisting of exopolysaccharides, proteins, and nucleotides that assists bacterial communities in colonization and survival. Genetic analysis of acetylation in biofilm formation demonstrated that lack of acetylation impacts biofilm development as shown by a decrease in matrix production. Targeting the acetylated biofilm proteins, we performed site‐directed mutagenesis of known acetylated lysine residues and assayed for biofilm formation. Two biofilm proteins, YmcA, a biofilm regulatory protein, and GtaB, an enzyme for biosynthesis of a precursor sugar for exopolysaccharides, demonstrated severe biofilm defects upon mutation of lysine residues. This suggests the importance of YmcA Lys64 and GtaB Lys89 and Lys191 for function in biofilm formation. In summary, we believe that protein lysine acetylation is a novel regulatory mechanism linking cell metabolism to bacterial multicellular development.Support or Funding InformationNational Science Foundation and Northeastern UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new regulator of cellulase and xylanase in the thermophilic fungus Myceliophthora thermophila strain ATCC 42464.

Myceliophthora thermophila (ATCC 42464) is a thermophilic fungus that produces cellulolytic enzymes with high thermal stability. Unlike its mesophile counterparts, study on gene expression regulation of cellulolytic enzymes in M. thermophila is inadequate. This work identified the function of MHR1, a putative transcription regulator of cellulolytic enzymes in M. thermophila that was found through RNA-Seq based gene expression profile analysis. RNA interference was used to study the role of MHR1. A recombinant plasmid, pUC19-Ppdc-mhr1-Tpdc, which contained the RNAi sequence for mhr1 was constructed and transformed into M. thermophila. One of the transformants, MtR5, in which the RNA interference efficiency was the highest, was used for the following studies. In the mhr1-silenced strain MtR5, the filter paper hydrolyzing activity was 1.33-fold; β-1, 4-endoglucanase activity was 1.65-fold; and xylanase activity was 1.48-fold higher than those of the parental strain after induction, respectively, by wheat straw powder. qRT-PCR showed that gene expression of cbh1, cbh2, egl3 and xyr1 were 9.56-, 37.36-, 56.14- and 28.30-fold higher in MtR5 than in wild type, respectively. Our findings suggest that the transcription factor MHR1 of M. thermophila can repress cellulase and xylanase activities. Silenced mhr1 results in increased expression not only of the main cellulase genes, but also of the positive regulatory gene xyr1. This work is relevant to the development of M. thermophila as an industrial production host for cellulolytic and hemicellulolytic enzymes, which could be used to degrade a wide range of different biomass, converting lignocellulosic feedstock into sugar precursors for biofuels.