NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 µM cf. 1.5 nM). A comparison of the GccFα-methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production.

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino-acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting the growth of the most susceptible bacterial strains at picomolar concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here, we report the development of pRV610gcc, an easily modifiable 11.2-kbp plasmid that enables the production of GccF in L. plantarum NC8. gcc gene expression relies on native promoters in the cloned cluster, and NC8(pRV610gcc) produces mature GccF at levels similar to KW30. Key findings are that the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence but glycosylation of the loop serine is both sequence and spatially specific, that glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation, that neither of the putative thioredoxin proteins is essential for peptide maturation, and that removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity.IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen.

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d-glucose, d-fructose, d-galactose, cellobiose, and sucrose supplementation. d-Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.

Monoglobus pectinilyticus gen. nov., sp. nov., a pectinolytic bacterium isolated from human faeces.

A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae, but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7 % sequence similarity). Strain 14T shared ~99 % sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6 µm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae. The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium†M1 binds a broad range of hydrogen-producing microorganisms.

Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein.

Synthesis of the antimicrobial S-linked glycopeptide, glycocin†F.

The first total synthesis of glycocin F, a uniquely diglycosylated antimicrobial peptide bearing a rare S-linked N-acetylglucosamine (GlcNAc) moiety in addition to an O-linked GlcNAc, has been accomplished using a native chemical ligation strategy. The synthetic and naturally occurring peptides were compared by HPLC, mass spectrometry, NMR and CD spectroscopy, and their stability towards chymotrypsin digestion and antimicrobial activity were measured. This is the first comprehensive structural and functional comparison of a naturally occurring glycocin with an active synthetic analogue.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Tyrosine latching of a regulatory gate affords allosteric control of aromatic amino acid biosynthesis.

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (ß/α)(8) barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å. In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å. Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.

Genomic insights into the physiology of Quinella, an iconic uncultured rumen bacterium.

Quinella is a genus of iconic rumen bacteria first reported in 1913. There are no cultures of these bacteria, and information on their physiology is scarce and contradictory. Increased abundance of Quinella was previously found in the rumens of some sheep that emit low amounts of methane (CH4) relative to their feed intake, but whether Quinella contributes to low CH4 emissions is not known. Here, we concentrate Quinella cells from sheep rumen contents, extract and sequence DNA, and reconstruct Quinella genomes that are >90% complete with as little as 0.20% contamination. Bioinformatic analyses of the encoded proteins indicate that lactate and propionate formation are major fermentation pathways. The presence of a gene encoding a potential uptake hydrogenase suggests that Quinella might be able to use free hydrogen (H2). None of the inferred metabolic pathways is predicted to produce H2, a major precursor of CH4, which is consistent with the lower CH4 emissions from those sheep with high abundances of this bacterium.

Structural, dynamic, and chemical characterization of a novel S-glycosylated bacteriocin.

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two ß-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.

Bacteriocin ASM1 is an O/S-diglycosylated, plasmid-encoded homologue of glycocin F.

Here, we report on the biochemical characterization of a new glycosylated bacteriocin (glycocin), ASM1, produced by Lactobacillus plantarum A-1 and analysis of the A-1 bacteriocinogenic genes. ASM1 is 43 amino acids in length with Ser18-O- and Cys43-S-linked N-acetylglucosamine moieties that are essential for its inhibitory activity. Its only close homologue, glycocin F (GccF), has five amino acid substitutions all residing in the flexible C-terminal 'tail' and a lower IC50 (0.9 nm) compared to that of ASM1 (1.5 nm). Asm/gcc genes share the same organization (asmH← â†’asmABCDE→F), and the asm genes reside on an 11 905-bp plasmid dedicated to ASM1 production. The A-1 genome also harbors a gene encoding a 'rare' bactofencin-type bacteriocin. As more examples of prokaryote S-GlcNAcylation are discovered, the functions of this modification may be understood.

Genomic insights from Monoglobus pectinilyticus: a pectin-degrading specialist bacterium in the human colon.

Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.

Arabinose 5-phosphate analogues as mechanistic probes for Neisseria meningitidis 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the condensation reaction between phosphoenolpyruvate and D-arabinose 5-phosphate (D-A5P) in a key step in lipopolysaccharide biosynthesis in Gram-negative bacteria. The KDO8P synthase from Neisseria meningitidis was cloned into Escherichia coli, overexpressed and purified. A variety of D-A5P stereoisomers were tested as substrates, of these only D-A5P and l-X5P were substrates. The Asn59Ala mutant of N. meningitidis KDO8P synthase was constructed and this mutant retained less than 1% of the wild-type activity. These results are consistent with a catalytic mechanism for this enzyme in which the C2 and C3 hydroxyl groups of D-A5P and Asn59 are critical.

Expression of Lactobacillus plantarum KW30 gcc genes correlates with the production of glycocin F in late log phase.

Antibacterial compounds known as bacteriocins are microbial inventions designed to reduce the competition for limited resources by inhibiting the growth of closely related bacteria. Glycocin F (GccF) is an unusually di-glycosylated bacteriocin produced in a lactic acid bacterium, Lactobacillus plantarum KW30 that has been shown to be resistant to extreme conditions. It is bacteriostatic rather than bactericidal, and all its post-translational modifications (a pair of nested disulfide bonds, and O-linked and S-linked N-acetylglucosamines) are required for full activity. Here, we examine a cluster of genes predicted to be responsible for GccF expression and maturation. The expression of eight genes, previously reported to make up the gcc operon, was profiled for their expression during cell culture. We found that all but one of the genes of the gcc cluster followed a pattern of expression that correlated with the stage of growth observed for the producer organism along with the increase in GccF secretion. We also found that most of the gcc genes are transcribed as a single unit. These data provide evidence that the gcc cluster genes gccABCDEF constitute a true operon for regulated GccF production, and explain the observed increase in GccF concentration that accompanies an increase in cell numbers.

Total chemical synthesis of glycocin F and analogues: S-glycosylation confers improved antimicrobial activity.

Glycocin F (GccF) is a unique diglycosylated bacteriocin peptide that possesses potent and reversible bacteriostatic activity against a range of Gram-positive bacteria. GccF is a rare example of a 'glycoactive' bacteriocin, with both the O-linked N-acetylglucosamine (GlcNAc) and the unusual S-linked GlcNAc moiety important for antibacterial activity. In this report, glycocin F was successfully prepared using a native chemical ligation strategy and folded into its native structure. The chemically synthesised glycocin appeared to be slightly more active than the recombinant material produced from Lactobacillus plantarum. A second-generation synthetic strategy was used to prepare 2 site selective 'glyco-mutants' containing either two S-linked or two O-linked GlcNAc moieties; these mutants were used to probe the contribution of each type of glycosidic linkage to bacteriostatic activity. Replacing the S-linked GlcNAc at residue 43 with an O-linked GlcNAc decreased the antibacterial activity, while replacing O-linked GlcNAc at position 18 with an S-linked GlcNAc increased the bioactivity suggesting that the S-glycosidic linkage may offer a biologically-inspired route towards more active bacteriocins.

Using Chemical Synthesis to Probe Structure-Activity Relationships of the Glycoactive Bacteriocin Glycocin F.

Glycocin F, a bacteriocin produced by Lactobacillus plantarum KW30, is glycosylated with two N-acetyl-d-glucosamine sugars, and has been shown to exhibit a rapid and reversible bacteriostasis on susceptible cells. The roles of certain structural features of glycocin F have not been studied to date. We report here the synthesis of various glycocin F analogues through solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), allowing us to probe the roles of different structural features of this peptide. Our results indicate that the bacteriostatic activity of glycocin F is controlled by the glycosylated interhelical loop, while the glycosylated flexible tail appears to be involved in localizing the peptide to its cellular target.

Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.

DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

The glycocins: in a class of their own.

First reported in 2011, glycocins (glycosylated bacteriocins) are bacterial toxins that constitute a subset of ribosomally synthesised and post-translationally modified peptide (RiPP) natural products. Three NMR structures (glycocin F, ASM1 and sublancin 168), two with helix-loop-helix Cs α/α folds, are deposited in the PDB. Each structure contains a monosaccharide ß-S-linked to a cysteine side chain. Three more glycocins (thurandacin, and enterocins F4-9 and 96) have been biochemically characterised, and others predicted on the basis of bioinformatic analyses. Only glycocin F, ASM1 and enterocin F4-9 are unequivocally glycoactive. This review probes the structure-function relationships of four types of nested disulfide-bonded glycocins.

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus.

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD(+)/H or NADP(+)/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD(+)/H than with NADP(+)/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.