Albicidin Biosynthesis Research Articles

In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-l-isoasparagine or a β-methoxy-l-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound l-asparagine. Furthermore, CysQ O-methylates hydroxyl-l-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

Full Genome Sequence Analysis of Two Isolates Reveals a Novel Xanthomonas Species Close to the Sugarcane Pathogen Xanthomonas albilineans.

Xanthomonas albilineans is the bacterium responsible for leaf scald, a lethal disease of sugarcane. Within the Xanthomonas genus, X. albilineans exhibits distinctive genomic characteristics including the presence of significant genome erosion, a non-ribosomal peptide synthesis (NRPS) locus involved in albicidin biosynthesis, and a type 3 secretion system (T3SS) of the Salmonella pathogenicity island-1 (SPI-1) family. We sequenced two X. albilineans-like strains isolated from unusual environments, i.e., from dew droplets on sugarcane leaves and from the wild grass Paspalum dilatatum, and compared these genomes sequences with those of two strains of X. albilineans and three of Xanthomonas sacchari. Average nucleotide identity (ANI) and multi-locus sequence analysis (MLSA) showed that both X. albilineans-like strains belong to a new species close to X. albilineans that we have named “Xanthomonas pseudalbilineans”. X. albilineans and “X. pseudalbilineans” share many genomic features including (i) the lack of genes encoding a hypersensitive response and pathogenicity type 3 secretion system (Hrp-T3SS), and (ii) genome erosion that probably occurred in a common progenitor of both species. Our comparative analyses also revealed specific genomic features that may help X. albilineans interact with sugarcane, e.g., a PglA endoglucanase, three TonB-dependent transporters and a glycogen metabolism gene cluster. Other specific genomic features found in the “X. pseudalbilineans” genome may contribute to its fitness and specific ecological niche.

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.

Abstracts Submitted for Presentation at the 2008 APS Centennial Meeting

Xanthomonas albilineans produces albicidin, a unique and specific toxin that causes foliar symptoms of sugarcane leaf scald disease. In the related species Xanthomonas campestris pv. campestris, a cluster of genes called rpf (for regulation of pathogenicity factors) is involved in control of various cellular processes. Marker exchange mutants of the rpfF gene in strain XaFL07-1 of X. albilineans from Florida were generated, and the mutants were verified by PCR analysis and by use of an X. campestris diffusible signal factor (DSF) reporter strain. Both albicidin and protease production by these mutants appeared significantly reduced compared to XaFL07-1, suggesting that DSF is involved in regulation of albicidin biosynthesis by X. albilineans. However, sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, exhibited pencil line symptoms indicative of albicidin production on emerging leaves after inoculation of stalks by the decapitation method. Preliminary experiments indicated that at least some rpfF mutants colonized sugarcane stalks less efficiently, both spatially and in intensity, than wild type X. albilineans. Additional inoculation experiments are in progress to assess disease severity caused by rpfF mutants, and to study the role of DSF production in sugarcane stalk colonization by X. albilineans. (Resume d'auteur)

DNA Gyrase from the Albicidin Producer Xanthomonas albilineans Has Multiple-Antibiotic-Resistance and Unusual Enzymatic Properties

The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase. The X. albilineans gyrA and gyrB genes are not clustered with previously described albicidin biosynthesis and self-protection genes. The GyrA and GyrB products differ from Escherichia coli homologues through several insertions and through changes in several amino acid residues implicated in quinolone and coumarin resistance. Reconstituted X. albilineans DNA gyrase showed 20- to 25-fold-higher resistance than E. coli DNA gyrase to albicidin and ciprofloxacin and 8-fold-higher resistance to novobiocin in the supercoiling assay. The X. albilineans DNA gyrase is unusual in showing a high degree of distributive supercoiling and little DNA relaxation activity. X. albilineans GyrA (XaA) forms a functional gyrase heterotetramer with E. coli GyrB (EcB) and can account for albicidin and quinolone resistance and low levels of relaxation activity. XaB probably contributes to both coumarin resistance and the distributive supercoiling pattern. Although XaB shows fewer apparent changes relative to EcB, the EcA.XaB hybrid relaxed DNA in the presence or absence of ATP and was unable to supercoil. A fuller understanding of structural differences between albicidin-sensitive and -resistant gyrases may provide new clues into features of the enzyme amenable to interference by novel antibiotics.

The phytotoxin albicidin is a novel inhibitor of DNA gyrase.

Xanthomonas albilineans produces a family of polyketide-peptide compounds called albicidins which are highly potent antibiotics and phytotoxins as a result of their inhibition of prokaryotic DNA replication. Here we show that albicidin is a potent inhibitor of the supercoiling activity of bacterial and plant DNA gyrases, with 50% inhibitory concentrations (40 to 50 nM) less than those of most coumarins and quinolones. Albicidin blocks the religation of the cleaved DNA intermediate during the gyrase catalytic sequence and also inhibits the relaxation of supercoiled DNA by gyrase and topoisomerase IV. Unlike the coumarins, albicidin does not inhibit the ATPase activity of gyrase. In contrast to the quinolones, the albicidin concentration required to stabilize the gyrase cleavage complex increases 100-fold in the absence of ATP. The slow peptide poisons microcin B17 and CcdB also access ATP-dependent conformations of gyrase to block religation, but in contrast to albicidin, they do not inhibit supercoiling under routine assay conditions. Some mutations in gyrA, known to confer high-level resistance to quinolones or CcdB, confer low-level resistance or hypersensitivity to albicidin in Escherichia coli. Within the albicidin biosynthesis region in X. albilineans is a gene encoding a pentapeptide repeat protein designated AlbG that binds to E. coli DNA gyrase and that confers a sixfold increase in the level of resistance to albicidin in vitro and in vivo. These results demonstrate that DNA gyrase is the molecular target of albicidin and that X. albilineans encodes a gyrase-interacting protein for self-protection. The novel features of the gyrase-albicidin interaction indicate the potential for the development of new antibacterial drugs.

High Variation in Pathogenicity of Genetically Closely Related Strains of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen, in Guadeloupe

ABSTRACT Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.

Variation in Albicidin Biosynthesis Genes and in Pathogenicity of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

ABSTRACT Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.

Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox − mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.

Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS-NRPS gene cluster for toxin production in the genus Xanthomonas.

A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans.

Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted M:(r) 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS-NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus-acyl-CoA ligase (AL)-acyl carrier protein (ACP)-beta-ketoacyl synthase (KS)-beta-ketoacyl reductase (KR)-ACP-ACP-KS-peptidyl carrier protein (PCP)-condensation (C)-adenylation-PCP-C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS-NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.

Albicidin antibiotic and phytotoxin biosynthesis in Xanthomonas albilineans requires a phosphopantetheinyl transferase gene

Xanthomonas albilineans produces a family of highly potent antibiotics and phytotoxins called albicidins, which are key pathogenesis factors in the systemic development of leaf scald disease of sugarcane. A gene ( xabA) required for albicidin biosynthesis encodes a peptide of 278 amino acids, including the signature sequence motifs for phosphopantetheinyl transferases (PPTases) that activate polyketide and non-ribosomal peptide synthetases. The Escherichia coli entD gene, which encodes a PPTase involved in biosynthesis of enterobactin (a siderophore), restored biosynthesis of albicidin (a DNA replication inhibitor) in X. albilineans Tox − LS156 ( xabA::Tn5). We conclude that XabA is a PPTase required for post-translational activation of synthetases in the albicidin biosynthetic pathway. This is the first antibiotic or toxin biosynthesis gene characterized from the genus Xanthomonas, and the first demonstration that antibiotic synthetases in the Pseudomonadaceae, like those in the Enterobacteriaceae and in Gram-positive bacteria, can require activation by a PPTase. Coupled with the recent demonstration of a separate albicidin biosynthetic gene cluster, the results indicate the possibility for overproduction of albicidins,which allows better understanding and application of these potent inhibitors of prokaryote DNA replication.

Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans

Transposon mutagenesis and complementation studies previously identified a gene ( xabB) for a large (526 kDa) polyketide–peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343 aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xabB promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239 aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of ‘in trans’ complementation of thp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis.

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR.

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.

Factors affecting biosynthesis by Xanthomonas albilineans of albicidin antibiotics and phytotoxins.

Albicidins are important factors in systemic pathogenesis by Xanthomonas albilineans, which causes the devastating leaf scald disease of sugar cane. They are also of substantial interest as antibiotics that selectively block prokaryote DNA replication. Albicidin biosynthesis is highly sensitive to medium composition. An optimized, chemically defined medium (SMG3) yielded 30-fold more albicidin from half the accumulated biomass, relative to sucrose peptone (SP) medium. Phosphate starvation stimulated albicidin production in SMG3 and SP media. Addition of other amino acids, ammonium ions or peptones to the defined medium increased the growth rate of X. albilineans XA3, but differentially inhibited albicidin biosynthesis. Knowledge of these factors indicates new approaches to understanding mechanisms of pathogenesis and resistance to sugar cane leaf scald disease, and to strain improvement for production of albicidin antibiotics.

Genes for albicidin biosynthesis and resistance span at least 69 kb in the genome of Xanthomonas albilineans.

All Tn5 insertion mutants of Xanthomonas albilineans, the cause of leaf scald disease of sugar cane, which failed to produce albicidin antibiotics failed to cause chlorosis in inoculated sugar cane but remained resistant to albicidin. Southern analysis revealed that mutants deficient in albicidin production carried the transposon on different chromosomal restriction fragments spanning at least 50 kb in the X. albilineans genome, which is larger than any reported cluster of genes involved in the production of a bacterial phytotoxin. Albicidin-resistant cosmid clones from a Tox- Tn5 insertion mutant did not carry the transposon, and the subcloned albicidin resistance gene did not hybridize to any of the restriction fragments carrying Tn5 in the Tox- mutants, indicating that the albicidin biosynthesis and resistance genes are not closely linked in X. albilineans.