The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases

Background: The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes.Results: The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598 kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297 kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723 kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant.Conclusions: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.     

Kalihinols from two Acanthella cavernosa sponges: inhibitors of bacterial folate biosynthesis

Acanthella spp. sponges have been prolific sources of highly functionalized diterpene antibiotics. Two Acanthella cavernosa sponges were investigated based on the activity of their extracts in a screen designed to detect bacterial folate biosynthesis inhibitors. Bacillus subtilis PY79 strain harboring a lacZ reporter gene fusion to a trimethoprim-responsive promoter (PpanB) was used for the screen. The ability of kalihinols to inhibit bacterial folate biosynthesis was investigated resulting in preliminary structure activity relationships. Eight kalihinol type diterpenes were isolated from two Philippine Acanthella cavernosa specimens including two new 10- and 15-formamido-kalihinol F analogs.     

The first total synthesis of lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.     

Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil

Cupriavidus necator H16C_Ac, previously constructed for production of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil, was further engineered aiming to increase 3HHx composition in the copolyester. PHA synthase gene derived from Aeromonas caviae on the H16C_Ac chromosome was replaced by a gene encoding the N149S/D171G mutant and this recombination enhanced PHA productivity as well as slightly increased 3HHx composition. Manipulation of phaA_Cn locus partially reduced the amount of 3HB unit concomitantly with relative increase of 3HHx composition, whereas deletion of phaB1_Cn resulted in drastic decline of 3HB unit in P(3HB-co-3HHx). Insertion of phaJ_Ac encoding (R)-specific enoyl-CoA hydratase from A. caviae into pha operon significantly enlarged 3HHx fraction without negative effects on the cell growth and polyester accumulation. Consequently, efficient production of P(3HB-co-3HHx) with 3HHx composition of 5.7-9.9 mol% was successfully achieved from soybean oil by the engineered strains.     

Cloning and characterization of an environmental DNA-derived gene cluster that encodes the biosynthesis of the antitumor substance BE-54017

Soil is predicted to contain thousands of unique bacterial species per gram. Soil DNA libraries represent large reservoirs of biosynthetic diversity from which diverse secondary metabolite gene clusters can be recovered and studied. The screening of an archived soil DNA library using primers designed to target oxytryptophan dimerization genes allowed us to identify and functionally characterize the first indolotryptoline biosynthetic gene cluster. The recovery and heterologous expression of an environmental DNA-derived gene cluster encoding the biosynthesis of the antitumor substance BE-54017 is reported here. Transposon mutagenesis identified two monooxygenases, AbeX1 and AbeX2, as being responsible for the transformation of an indolocarbazole precursor into the indolotryptoline core of BE-54017.     

Direct interaction of a vancomycin derivative with bacterial enzymes involved in cell wall biosynthesis.

BACKGROUND: The glycopeptide antibiotic vancomycin complexes DAla-DAla termini of bacterial cell walls and peptidoglycan precursors and interferes with enzymes involved in murein biosynthesis. Semisynthetic vancomycins incorporating hydrophobic sugar substituents exhibit efficacy against DAla-DLac-containing vancomycin-resistant enterococci, albeit by an undetermined mechanism. Contrasting models that invoke either cooperative dimerization and membrane anchoring or direct inhibition of bacterial transglycosylases have been proposed to explain the bioactivity of these glycopeptides. RESULTS: Affinity chromatography has revealed direct interactions between a semisynthetic hydrophobic vancomycin (DCB-PV), and select Escherichia coli membrane proteins, including at least six enzymes involved in peptidoglycan assembly. The N(4)-vancosamine substituent is critical for protein binding. DCB-PV inhibits transglycosylation in permeabilized E. coli, consistent with the observed binding of the PBP-1B transglycosylase-transpeptidase. CONCLUSIONS: Hydrophobic vancomycins interact directly with a select subset of bacterial membrane proteins, suggesting the existence of discrete protein targets. Transglycosylase inhibition may play a role in the enhanced bioactivity of semisynthetic glycopeptides.     

Insights into the biosynthesis of hormaomycin, an exceptionally complex bacterial signaling metabolite

Hormaomycin produced by Streptomyces griseoflavus is a structurally highly modified depsipeptide that contains several unique building blocks with cyclopropyl, nitro, and chlorine moieties. Within the genus Streptomyces, it acts as a bacterial hormone that induces morphological differentiation and the production of bioactive secondary metabolites. In addition, hormaomycin is an extremely potent narrow-spectrum antibiotic. In this study, we shed light on hormaomycin biosynthesis by a combination of feeding studies, isolation of the biosynthetic nonribosomal peptide synthetase (NRPS) gene cluster, and in vivo and in vitro functional analysis of enzymes. In addition, several nonnatural hormaomycin congeners were generated by feeding-induced metabolic rerouting. The NRPS contains numerous highly repetitive regions that suggest an evolutionary scenario for this unusual bacterial hormone, providing new opportunities for evolution-inspired metabolic engineering of novel nonribosomal peptides.     

Synthesis of a carbohydrate-derived hydroxamic acid inhibitor of the bacterial enzyme (LpxC) involved in lipid A biosynthesis

[reaction: see text] The enzyme LpxC (UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc deacetylase) catalyzes the second step of lipid A biosynthesis and is essential for bacterial growth. A GlcNAc-derived hydroxamic acid inhibitor 8 of this enzyme was synthesized using two different routes. Compound 8 exhibits activity toward LpxC enzymes from a wider spectrum of bacterial species than any of the previously reported hydroxamic acid inhibitors.     

Biosynthesis of the fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid and mechanism of self-resistance

Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure–activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in the HA cluster was characterized to be less sensitive to HA, and therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which was confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions, knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors.

The structural basis and self-resistance mechanism of fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid are uncovered.     

Vancomycin resistance in enterococci: reprogramming of the D-ala-D-Ala ligases in bacterial peptidoglycan biosynthesis

Vancomycin binds to bacterial cell-wall intermediates to achieve its antibiotic effect. Infections of vancomycin-resistant enterococci are, however, becoming an increasing problem; the bacteria are resistant because they synthesize different cell-wall intermediates. The enzymes involved in cell-wall biosynthesis, therefore, are potential targets for combating this resistance. Recent biochemical and crystallographic results are providing mechanistic and structural details about some of these targets.     

Characterization and biochemical investigation of the potential inositol monophosphate phosphatase involved in bacterial mycothiol biosynthesis

Abstract

Mycothiol (MSH) is the predominant form of small molecule thiols produced by actinomycetes and plays a pivotal role in the bacterial detoxication process. The only enzyme involved in MSH biosynthesis that has not been characterized yet is a phosphatase MshA2, which was proposed to catalyze the hydrolysis of 3-phospho-1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins-3-P) to GlcNAc-Ins. In this study, a new inositol monophosphate phosphatase from Corynebacterium glutamicum, designated as Cg0911, was discovered, expressed and characterized. Detailed biochemical studies on Cg0911 revealed that GlcNAc-Ins-3-P was its preferred substrate for efficient conversion into GlcNAc-Ins.     

Synthesis of analogs of D-Ala-D-Ala as potential inhibitors of bacterial cell wall biosynthesis

The syntheses of the L,L- and D,D-stereoisomers of N- phenoxyacetyl -X-alanine in which X = Ser, Ala( beta Cl ) or Arg, are described. The antibacterial activity of these peptides and some of their synthetic intermediates has been examined. Four of the intermediates in which X = Ala( beta Cl ) and Arg(NO2), which possess C-terminal benzyl ester groups, were active against viridans streptococci and Streptococcus agalactiae. The D,D-enantiomers were more active than the corresponding L,L-isomers. None of the compounds were active against beta-lactamase producing bacteria or acted as beta-lactamase inhibitors.     

Biological solubilization of untreated north dakota lignite by a mixed bacterial and a mixed bacterial/fungal culture

Four microbial cultures, two pure and two mixed, were examined for their abilities to solubilize chemically treated (oxidized) lignites, thermally treated (wet-carbonized) lignite, and untreated lignites. Extensive solubilization of oxidized lignites and limited solubilization of untreated North Dakota lignite was observed by three of the four cultures tested. Solubilization of wet-carbonized (a technique to reduce equilibrium moisture and oxygen contents) lignite was not demonstrated. The increase in solubilization correlated with the increase in the oxygen content of lignite and the pH of culture broths. These results also suggest that microbial solubilization of coal may involve nonlignin degrading organisms capable of producing alkaline conditions in the presence of coal.

     

The biosynthesis of pentalenolactone

Feeding of [UL-¹³C₆]-glucose, an $\text{in}$$\text{vivo}$ precursor of [1,2-¹³C₂]-acetyl-CoA, gave pentalenolactone in which the pattern of ¹³C-enrichments and couplings supported a mevalonoid biosynthetic pathway.     

The biosynthesis of cyclitols

Information has recently been obtained, mainly with the aid of radioactive compounds, about the biosynthesis of various cyclitols. It was found that the most widely occurring cyclitol, meso-inositol, is biosynthesized by a pathway probably common to all organisms, in which the cyclohexane ring is formed by ring closure between the two terminal carbon atoms of D -glucose. Cell-free extracts or enzyme systems that catalyse the conversion of D -glucose into meso-inositol can be prepared from various biological materials. The biosynthesis of the other hexahydroxycyclohexanes (inositols) involves meso-inositol as an intermediate. A few steps in the conversion of meso-inositol into other inositols have been studied.