The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Misannotations of the genes encoding sugar N‐formyltransferases

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N‐formyltransferases have been annotated as l ‐methionyl‐tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l ‐methionyl‐tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose to dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose utilizing N¹⁰‐formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X‐ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis–Menten kinetics and adopt the characteristic three‐dimensional structural fold previously observed for other sugar N‐formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.     

Three‐dimensional structure of a sugar N‐formyltransferase from Francisella tularensis

N‐formylated sugars have been observed on the O‐antigens of such pathogenic Gram‐negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N‐formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N‐formyltransferase that functions on dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N¹⁰‐formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three‐dimensional structure of the enzyme was solved in the presence of dTDP‐sugar ligands to a nominal resolution of 2.1 Å. Each subunit of the dimeric enzyme is dominated by a “core” domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two α‐helices and a β‐hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C‐4′ amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C‐3′ amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Increasing l‐isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two‐component export system BrnFE

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.     

Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

Structure of the external aldimine form of PglE,an aminotransferase required for N,N'‐diacetylbacillosamine biosynthesis

N,N'‐diacetylbacillosamine is a novel sugar that plays a key role in bacterial glycosylation. Three enzymes are required for its biosynthesis in Campylobacter jejuni starting from UDP‐GlcNAc. The focus of this investigation, PglE, catalyzes the second step in the pathway. It is a PLP‐dependent aminotransferase that converts UDP‐2‐acetamido‐4‐keto‐2,4,6‐trideoxy‐d ‐glucose to UDP‐2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d ‐glucose. For this investigation, the structure of PglE in complex with an external aldimine was determined to a nominal resolution of 2.0 Å. A comparison of its structure with those of other sugar aminotransferases reveals a remarkable difference in the manner by which PglE accommodates its nucleotide‐linked sugar substrate.     

Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type‐2 and Their Major Compounds

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non‐cytotoxic and anti‐dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP‐SPE method allowed isolating the antiviral flavanone (2S)‐isosakuranetin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 1 ) from both species and yielded less active sub‐fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3′,5′‐trihydroxyflavanone‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 2a / 2b ) from F. hyacinthina; the known narigenin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 3 ) from both species; rutin ( 4 ) and quercetin‐4′‐β‐d ‐O‐glucopyranosyl‐3‐O‐rutinoside ( 5 ) from F. hyacinthina, and kaempferol‐3‐O‐rutinoside ( 6 ), erythroxyloside A ( 7 ) and asperuloside ( 8 ) from F. truncata have been isolated from these sub‐fractions. Compounds 4  –  8 are reported for the first time in Faramea spp.     

Molecular mechanism underlying pseudopeloria in Habenaria radiata (Orchidaceae)

Habenaria radiata (Orchidaceae) has two whorls of perianth, comprising three greenish sepals, two white petals and one lip (labellum). By contrast, the pseudopeloric (with a decreased degree of zygomorphy) mutant cultivar of H. radiata , ‘Hishou’, has changes in the identities of the dorsal sepal to a petaloid organ and the two ventral sepals to lip‐like organs. Here, we isolated four DEFICIENS ‐ like and two AGL 6 ‐like genes from H. radiata , and characterized their expression. Most of these genes revealed similar expression patterns in the wild type and in the ‘Hishou’ cultivar, except Hr DEF ‐C3. The Hr DEF ‐C3 gene was expressed in petals and lip in the wild type but was ectopically expressed in sepal, petals, lip, leaf, root and bulb in ‘Hishou’. Sequence analysis of the Hr DEF ‐C3 loci revealed that the ‘Hishou’ genome harbored two types of Hr DEF ‐C3 genes: one identical to wild‐type Hr DEF ‐C3 and the other carrying a retrotransposon insertion in its promoter. Genetic linkage analysis of the progeny derived from an intraspecific cross between ‘Hishou’ and the wild type demonstrated that the mutant pseudopeloric trait was dominantly inherited and was linked to the Hr DEF ‐C3 gene carrying the retrotransposon. These results indicate that the pseudopeloric phenotype is caused by retrotransposon insertion in the Hr DEF ‐C3 promoter, resulting in the ectopic expression of Hr DEF ‐C3 . As the expression of Hr AGL 6‐C2 was limited to lateral sepals and lip, the overlapping expression of Hr DEF ‐C3 and Hr AGL 6‐C2 is likely to be responsible for the sepal to lip‐like identity in the lateral sepals of the ‘Hishou’ cultivar.     

Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum

Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms and red tides in estuarine ecosystems. While known to be mixotrophic, previous attempts to document feeding on algal prey have yielded low grazing rates. In this study, growth and ingestion rates of P. minimum were measured as a function of nitrogen (‐N) and phosphorous (‐P) starvation. A P. minimum isolate from Chesapeake Bay was found to ingest cryptophyte prey when in stationary phase and when starved of N or P. Prorocentrum minimum ingested two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte species. In all cases ‐P treatments resulted in the highest grazing. Ingestion rates of ‐P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by ‐N cells saturated at 1 prey per predator per day. In the presence of prey, ‐P treated cells reached a maximum mixotrophic growth rate (μ_max) of 0.5 d^?1, while ‐N cells had a μ_max of 0.18 d^?1. Calculations of ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotrophy can be an important source of all three elements. While P. minimum is a proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Comparison of transmission efficiency of various isolates of Potato virus Y among three aphid vectors

Potato virus Y (PVY) strains are transmitted by different aphid species in a non‐persistent, non‐circulative manner. Green peach aphid (GPA), Myzus persicae Sulzer, is the most efficient vector in laboratory studies, but potato aphid (PA), Macrosiphum euphorbiae Thomas (both Hemiptera: Aphididae, Macrosiphini), and bird cherry‐oat aphid (BCOA), Rhopalosiphum padi L. (Hemiptera: Aphididae, Aphidini), also contribute to PVY transmission. Studies were conducted with GPA, PA, and BCOA to assess PVY transmission efficiency for various isolates of the same strain. Treatments included three PVY strains (PVY^O, PVY^N:O, PVY^NTN) and two isolates of each strain (Oz and NY090031 for PVY^O; Alt and NY090004 for PVY^N:O; N4 and NY090029 for PVY^NTN), using each of three aphid species as well as a sham inoculation. Virus‐free tissue‐cultured plantlets of potato cv. Russet Burbank were used as virus source and recipient plants. Five weeks post inoculation, recipient plants were tested with quantitative DAS‐ELISA to assess infection percentage and virus titer. ELISA‐positive recipient plants were assayed with RT‐PCR to confirm presence of the expected strains. Transmission efficiency (percentage infection of plants) was highest for GPA, intermediate for BCOA, and lowest for PA. For all aphid species, transmission efficiency did not differ significantly between isolates within each strain. No correlations were found among source plant titer, infection percentage, and recipient plant titer. For both GPA and BCOA, isolates of PVY^NTN were transmitted with greatest efficiency followed by isolates of PVY^O and PVY^N:O, which might help explain the increasing prevalence of necrotic strains in potato‐growing regions. Bird cherry‐oat aphid transmitted PVY with higher efficiency than previously reported, suggesting that this species is more important to PVY epidemiology than has been considered.     

Regulation of polyamine metabolism in Pyropia cinnamomea (W.A. Nelson), an important mechanism for reducing UV‐B‐induced oxidative damage

It is generally accepted that ultraviolet (UV) radiation can have adverse affects on phototrophic organisms, independent of ozone depletion. The red intertidal seaweed Pyropia cinnamomea W.A. Nelson (previously Porphyra cinnamomea Sutherland et al. 2011), similar to many other intertidal macrophytes, is exposed to high levels of UV radiation on a daily basis due to emersion in the upper littoral zone. It has been shown that seaweeds, like higher plants, respond to an increased activity of antioxidative enzymes when exposed to stress. However, earlier investigations have shown that P. cinnamomea also compensates for stress due to UV radiation by increasing polyamine (PA) levels, especially bound‐soluble and bound‐insoluble PAs. The PA precursor putrescine (PUT) can be synthesized via two enzymatic pathways: arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Both of these enzymes showed increased activity in P. cinnamomea under UV stress. In higher plants, ADC is the enzyme responsible for increased PA levels during stress exposure, while ODC is correlated with cell division and reproduction. However, there are contrary findings in the literature. Using two irreversible inhibitors, we identified the enzyme most likely responsible for increased PUT synthesis and therefore increased stress tolerance in P. cinnamomea. Our results show that changes in the PA synthesis pathway in P. cinnamomea under UV stress are based on an increased activity of ADC. When either inhibitor was added, lipid hydroperoxide levels increased even under photosynthetically active radiation, suggesting that PAs are involved in protection mechanisms under normal light conditions as well. We also show that under optimum or low‐stress conditions, ODC activity is correlated with PUT synthesis.     

Effect of Aglycon Structure on Saccharide Elongation by Cells

Alkyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐C12), tridecan‐7‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C6), and pentacosan‐13‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C12) primers were synthesized by glycosylation of dodecan‐1‐ol, tridecan‐7‐ol, and pentacosan‐13‐ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc‐C12 was elongated to give NeuAc‐Gal‐GlcNAc‐C12. GlcNAc‐2C6 was also elongated to afford Gal‐GlcNAc‐2C6 and NeuAc‐Gal‐GlcNAc‐2C6. On the other hand, GlcNAc‐2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5‐times by modifying the aglycon structure of GlcNAc from C₁₂ to 2 C₆ despite having almost the same number of C‐units.     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.