Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose.

BACKGROUND: The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. RESULTS: In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant-) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. CONCLUSIONS: Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases.

ArnT is a glycosyltransferase that catalyzes the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyze lipid A modification with l-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

NagR Differentially Regulates the Expression of the glmS and nagAB Genes Required for Amino Sugar Metabolism by Streptococcus mutans.

UNLABELLED: The ability of bacteria to metabolize glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) is considered important for persistent colonization of the oral cavity. In the dental caries pathogen Streptococcus mutans, the NagR protein regulates the expression of glmS, which encodes a GlcN-6-P synthetase, and nagA (GlcNAc-6-P deacetylase) and nagB (GlcN-6-P deaminase), which are required for the catabolism of GlcNAc and GlcN. Two NagR-binding sites (dre) were identified in each of the promoter regions for nagB and glmS. Using promoter-reporter gene fusions, the role of each dre site was examined in the regulation of glmS and nagB promoter activities in cells grown with glucose, GlcNAc, or GlcN. A synergistic relationship between the two dre sites in the glmS promoter that required proper spacing was observed, but that was not the case for nagB. Binding of purified NagR to DNA fragments from both promoter regions, as well as to dre sites alone, was strongly influenced by particular sugar phosphates. Using a random mutagenesis approach that targeted the effector-binding domain of NagR, mutants that displayed aberrant regulation of both the glmS and nagAB genes were identified. Collectively, these findings provide evidence that NagR is essential for regulation of genes for both the synthesis and catabolism of GlcN and GlcNAc in S. mutans, and that NagR engages differently with the target promoter regions in response to specific metabolites interacting with the effector-binding domain of NagR. IMPORTANCE: Glucosamine and N-acetylglucosamine are among the most abundant naturally occurring sugars on the planet, and they are catabolized by many bacterial species as sources of carbon and nitrogen. Representing a group called lactic acid bacteria (LAB), the human dental caries pathogen Streptococcus mutans is shown to differ from known paradigm organisms in that it possesses a GntR/HutC-type regulator, NagR, that is required for the regulation of both catabolism of GlcN and biosynthesis. Results reported here reveal a simple and elegant mechanism whereby NagR differentially regulates two opposing biological processes by surveying metabolic intermediates. This study provides insights that may contribute to the development of novel therapeutic tools to combat dental caries and other infectious diseases.

Regulation of the Utilization of Amino Sugars by Escherichia coli and Bacillus subtilis: Same Genes, Different Control.

Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan (PG) and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell. The enzymes for both the synthesis and degradation of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are highly conserved but during evolution have become subject to different regulatory regimes. Escherichia coli grows more rapidly using GlcNAc as a carbon source than with GlcN. On the other hand, Bacillus subtilis, but not other Bacilli tested, grows more efficiently on GlcN than GlcNAc. The more rapid growth on this sugar is associated with the presence of a second, GlcN-specific operon, which is unique to this species. A single locus is associated with the genes for catabolism of GlcNAc and GlcN in E. coli, although they enter the cell via different transporters. In E. coli the amino sugar transport and catabolic genes have also been requisitioned as part of the PG recycling process. Although PG recycling likely occurs in B. subtilis, it appears to have different characteristics.

[The role of human 6-sulfo LacNAc dendritic cells (slanDCs) in autoimmunity and tumor diseases]. / Die Bedeutung von humanen 6-sulfo LacNAc dendritischen Zellen (slanDCs) bei Autoimmunität und Tumorerkrankungen.

Dendritic cells play a central role in the regulation of immunological reactivity. The existence of functionally specialized populations of skin dendritic cells is a consequence of qualitatively different attacks on our organism. slanDCs are human inflammatory dendritic cells that are characterized by the specific expression of the carbohydrate 6-sulfo LacNAc (slan). After phenotypic maturation slanDCs are capable of producing very high amounts of proinflammatory mediators like IL-12, TNF-α, IL-1ß â and IL-23. Recent data describe a potential role of slanDCs in a number of different diseases like psoriasis, lupus erythematosus but also tumor diseases and therefore open up new areas of research on their respective pathogenesis. Furthermore, as a basis of a directed therapeutic manipulation,a slanDC-specific targeting system has been developed. Future challenges of slanDC research include the elaboration of a deeper understanding of the significance of slanDCs for the regulation of adaptive and innate immune responses as well as a translation of this knowledge into therapeutic options.

Deoxysugar pathway interchange for erythromycin analogues heterologously produced through Escherichia coli.

The overall erythromycin biosynthetic pathway can be sub-divided into macrocyclic polyketide formation and polyketide tailoring to produce the final bioactive molecule. In this study, the native deoxysugar tailoring reactions were exchanged for the purpose of demonstrating the production of alternative final erythromycin compounds. Both the d-desosamine and l-mycarose deoxysugar pathways were replaced with the alternative d-mycaminose and d-olivose pathways to produce new erythromycin analogues through the Escherichia coli heterologous system. Both analogues exhibited bioactivity against multiple antibiotic-resistant Bacillus subtilis strains. Besides demonstrating an intrinsic flexibility for the biosynthetic system to accommodate alternative tailoring pathways, the results offer an initial attempt to leverage the E. coli platform for erythromycin analogue production.

Metabolism of vertebrate amino sugars with N-glycolyl groups: intracellular ß-O-linked N-glycolylglucosamine (GlcNGc), UDP-GlcNGc, and the biochemical and structural rationale for the substrate tolerance of ß-O-linked ß-N-acetylglucosaminidase.

The O-GlcNAc modification involves the attachment of single ß-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.

Metabolism of vertebrate amino sugars with N-glycolyl groups: elucidating the intracellular fate of the non-human sialic acid N-glycolylneuraminic acid.

The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.

Dorsal radial glial cells have the potential to generate cortical interneurons in human but not in mouse brain.

Radial glial (RG) cells, in the neocortical ventricular/subventricular zone (VZ/SVZ), generate cortical projection neurons both in rodents and humans, but whether they can also generate cortical interneurons is not clear. We demonstrated both on cryosections and in cell cultures that in the human VZ/SVZ, cells can be double labeled with RG markers and calretinin (CalR) and GABA, markers that suggest interneuronal lineage. We examined in more detail the cell fate of human RG cells isolated from the VZ/SVZ at midterm. After 24 h, no CalR(+) or GABA(+) cells were seen in cultures, whereas 5-10% cells expressed Nkx2.1 and Dlx, two ventral transcription factors. CalR(+) and GABA(+) cells were apparent for the first time after 3 d in vitro, and their number increased in subsequent days, consistent with the gradual transition of RG cells into CalR(+) or GABA(+) cells. Indeed, the progeny of genetically labeled RG cells could be immunolabeled with antibodies to CalR and GABA or ventral transcription factors (Nkx2.1(+), Dlx(+)). In contrast to humans, in the embryonic mouse, similar experiments showed that only RG cells isolated from the subpallium (ganglionic eminence) generate CalR(+) or GABA(+) cells, whereas this was not the case with RG cells isolated from the pallium. These findings support the idea that human, but not mouse, dorsal RG cells have the potential to generate various subtypes of neocortical interneurons. Multiple progenitors and sites of cortical interneuron origin in human might be an evolutionary adaptation underlying brain expansion and the increased complexity of cortical circuitry in humans.

Characterization of the enzymes encoded by the anthrose biosynthetic operon of Bacillus anthracis.

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-ß-L-rhamnose α-1,3-L-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit ß-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.

The effects of organic solvents on the efficiency and regioselectivity of N-acetyl-lactosamine synthesis, using the ß-galactosidase from Bacillus circulans in hydro-organic media.

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) by the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), catalyzed by the ß-galactosidase from Bacillus circulans (BcßGal), was studied in hydro-organic media, starting from o-nitrophenyl-ß-D-galactopyranoside (oNPG) as a galactosyl donor. Thermal stability and synthesis activity of BcßGal were shown to depend on the organic solvent polarity, characterized by its Log P value. BcßGal was thus most stable in 10% (v/v) t-BuOH, an organic solvent found to have a stabilizing and/or weakly denaturing property, which was confirmed for high t-BuOH concentrations. In the same manner, the optimal synthesis yield increased as the Log P value of the organic solvent increased. The best results were obtained for reactions carried out in 10% (v/v) pyridine or 2-methyl-2-butanol, which gave 47% GlcNAc transgalactosylation yield based on starting oNPG, of which 23% (11 mM; 4.3 g/L) consisted in LacNAc synthesis. Furthermore, it was also established that both the GlcNAc transgalactosylation yield and the enzyme regioselectivity depended on the percentage of organic solvent used, the optimal percentage varying from 10 to 40% (v/v), depending on the solvent. This phenomenon was found to correlate mainly with the thermodynamic activity of water (a(w)) in the aqueous organic solvent mixture, which was found to be optimal when close to 0.96, whatever the organic solvent used. Finally, this study highlighted the fact that the regioselectivity of BcßGal for 1-4 linkage formation could be advantageously managed by controlling the a(w) parameter.

An undecaprenyl phosphate-aminoarabinose flippase required for polymyxin resistance in Escherichia coli.

Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA(c) parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. Lipid A was no longer modified with L-Ara4N, even though the levels of the lipid-linked donor of the L-Ara4N moiety, undecaprenyl phosphate-alpha-L-Ara4N, were not reduced in the mutants. However, the undecaprenyl phosphate-alpha-L-Ara4N present in the mutants was less concentrated on the periplasmic surface of the inner membrane, as judged by 4-5-fold reduced labeling with the inner membrane-impermeable amine reagent N-hydroxysulfosuccin-imidobiotin. In an arnT mutant of the same pmrA(c) parent, which lacks the enzyme that transfers the L-Ara4N unit to lipid A but retains the same high levels of undecaprenyl phosphate-alpha-L-Ara4N as the parent, N-hydroxysulfosuccinimidobiotin labeling was not reduced. These results implicate pmrL and pmrM, but not arnT, in transporting undecaprenyl phosphate-alpha-L-Ara4N across the inner membrane. PmrM and PmrL, now renamed ArnE and ArnF because of their involvement in L-Ara4N modification of lipid A, may be subunits of an undecaprenyl phosphate-alpha-L-Ara4N flippase.

Transcriptional regulation of the 4-amino-4-deoxy-L-arabinose biosynthetic genes in Yersinia pestis.

Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe(3+), which is sensed by PmrA cognate sensor PmrB, and by low Mg(2+), in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg(2+)-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins.

Biosynthesis of deoxyaminosugars in antibiotic-producing bacteria.

Deoxyaminosugars comprise an important class of deoxysugars synthesized by a variety of different microorganisms; they can be structural components of lipopolysaccharides, extracellular polysaccharides, and secondary metabolites such as antibiotics. Genes involved in the biosynthesis of the deoxyaminosugars are often clustered and are located in the vicinity of other genes required for the synthesis of the final compound. Most of the gene clusters for aminosugar biosynthesis have common features, as they contain genes encoding dehydratases, isomerases, aminotransferases, methyltransferases, and glycosyltransferases. In the present mini-review, the proposed biosynthetic pathways for deoxyaminosugar components of both macrolide and non-macrolide antibiotics are highlighted. The possibilities for genetic manipulations of the deoxyaminosugar biosynthetic pathways aimed at production of novel secondary metabolites are discussed.

Beta-glucosylation as a part of self-resistance mechanism in methymycin/pikromycin producing strain Streptomyces venezuelae.

In our study of the biosynthesis of D-desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to beta-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemical characterization of the purified protein confirmed the catalytic function of this enzyme as a beta-glycosidase capable of catalyzing the hydrolysis of glucosylated methymycin/neomethymycin produced by S. venezuelae. These results provide strong evidence substantiating glycosylation/deglycosylation as a likely self-resistance mechanism of S. venezuelae. However, further experiments have suggested that such a glycosylation/deglycosylation is only a secondary self-defense mechanism in S. venezuelae, whereas modification of 23S rRNA, which is the target site for methymycin and its derivatives, by PikR1 and PikR2 is a primary self-resistance mechanism. Considering that postsynthetic glycosylation is an effective means to control the biological activity of macrolide antibiotics, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be a valuable part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach.

Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose.

In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.

Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae.

The antibacterial properties of macrolide antibiotics (such as erythromycin, tylosin, and narbomycin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as narbomycin) have only single sugar substituents. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the narbomycin producer Streptomyces narbonensis. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.