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 ale 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.

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 xab 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 rhp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis. (C) 2000 Elsevier Science B.V. All rights reserved.

Expression, purification, and characterization of biol: A carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene biol. We have subcloned bioI and overexpressed the encoded protein, BioI. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography, Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coil acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production. (C) 2000 Academic Press.

Sex pheromone biosynthesis in the female olive fruit-fly. Double labelling from [O-18(2)]-dioxygen into 1,7-dioxaspiro[5.5]undecane

The demonstration that both oxygen atoms of 1,7-dioxaspiro[5.5] undecane (1), the sex-pheromone of the female olive fly, originate from dioxygen, strongly implicates monooxygenase mediated processes in assembly of (1), and reveals unexpected complexity in the formation of its nine-carbon precursor.

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.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Advanced precursors in marine biosynthetic study. Part 2: The biosynthesis of isocyanides and isothiocyanates in the tropical marine sponge Axinyssa n.sp.

The biosynthetic origins of the isocyanide and isothiocyanate groups in 9-isocyanop upukeanane (2) and 9-isothiocyanato-pupukeanane (3) are investigated by incorporation of [C-14]-labelled advanced precursors into the sponge Axinyssa n.sp. (C) 2001 Elsevier Science Ltd. All rights reserved.

Carbon hydroxylation of alkyltetrahydropyranols: A paradigm for spiroacetal biosynthesis in Bactrocera sp.

[GRAPHICS] In a number of Bactrocera species the penultimate step in the biosynthesis of spiroacetals is shown to be the hydroxylation of an alkyltetrahydropyranol followed by cyclization, The monooxygenases that catalyze this side chain hydroxylation show a strong preference for oxidation four carbons from the hemiketal center, to produce the spiroacetal, The hydroxy spiroacetals observed in Bactrocera appear to derive from direct oxidation of the parent spiroacetals and not from alternate precursors.

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cycloticles or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata Ell as well as three other clones for related cycloticles from the African plant Olden-landia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cycloticles have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

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 Mr 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)–ß-ketoacyl synthase (KS)–ß-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.

[18O]-Oxygen incorporation reveals novel pathways in spiroacetal biosynthesis by Bactrocera cacuminata and B. cucumis

The origins of the oxygen atoms in 1,7-dioxaspiro[5.5]undecane (1) and hydroxyspiroacetal (2) from Bactrocera cacuminata, and in 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (3) and hydroxyspiroacetal (4) from B. cucumis, have been investigated by incorporation studies from both [18O2]-dioxygen and [18O]-water. Combined GC-MS examination and high-field NMR analysis have demonstrated that all oxygen atoms in 1 and 2 from B. cacuminata are dioxygen derived, but in contrast, the spiroacetals 3 and 4 from B. cucumis incorporate one ring oxygen from water and one ring oxygen (and the hydroxyl oxygen in 4) from [18O2]-dioxygen. These results reveal not only the generality of monoxygenase mediation of spiroacetal formation in Bactrocera sp., but also an unexpected complexity in their biosynthesis. A general paradigm accommodating these and other observations is presented.

Monooxygenase stereoselectivity in the biosynthesis of stereoisomeric spiroacetals in the cucumber fly, Bactrocera cucumis

The stereoselectivity of hydroxylation of alkyltetrahydropyran-2-ols (or their biological equivalents) in the formation of stereoisomers of 2,8-dimethyl-1,7-dioxaspiro[5.5]undecanes in male Bactrocera cucumis has been investigated. Racemic, (6R)-, and (6S)-6-methyl-2-[5-H-2(1)]-n-pentyltetrahydropyran-2-ol was administered under an [O-18(2)]-enriched atmosphere. The stereochemistry and isotopic composition of generated spiroacetals were monitored by combined enantioselective GC-MS. The monooxygenase(s) strongly prefers the (6S)-substrate and furnishes predominantly the (S)-alcohol and then the (2S,6R,8S)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The (2S,6S,8R) and (2R,6S,8S) (E,Z)-isomers appear to be derived in vivo predominantly from (R)-hydroxylation of the (6S)-tetrahydropyranol.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or IgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.