A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli.

AIMS: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. METHODS AND RESULTS: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. CONCLUSIONS: AlbF is the first apparent single-component antibiotic-specific efflux pump from a gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis.

AIMS: Isolation, identification and characterization of a highly efficient isomaltulose producer. METHODS AND RESULTS: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. SIGNIFICANCE AND IMPACT OF STUDY: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

Xanthomonas albilineans and the antipathogenesis approach to disease control.

UNLABELLED: Summary Molecular studies into sugarcane leaf scald disease, caused by X. albilineans, revealed an unusual pathogenesis strategy, a new family of antibiotics, an extraordinary biosynthetic apparatus, and a new approach to disease control in plants and animals. TAXONOMY: Bacteria; Proteobacteria; gamma subdivision; Xanthomonadales; Xanthomonas group; X. albilineans (Ashby 1929) Dowson 1943. Microbiological properties: Gram-negative, slender rod-shaped, nonsporing, aerobic, motile by a single polar flagellum; producing slow-growing, pale yellow, nonmucoid colonies in culture; ecologically obligate plant parasite. HOST RANGE: Monocotyledonous plants in the Poaceae family, including Saccharum spp. and other grasses. Causal agent of sugarcane leaf scald. Disease symptoms: Characteristic white leaf stripes with necrotic zones at leaf margins, extensive chlorosis of emerging leaves, vascular reddening and cavity formation in invaded stems, production of side shoots, rapid wilting and death of plants. Prolonged latent infection can occur, necessitating detection by isolation or sensitive molecular assays. PATHOGENESIS: Xylem-invading pathogen, transmitted in cuttings, mechanically, and by wind-blown rain. Produces albicidin toxins that block prokaryotic DNA replication and plastid development, causing chlorosis in emerging leaves. Albicidins interfere with host resistance mechanisms, allowing systemic invasion. Strains vary in virulence. Agronomic importance and control: Sugarcane leaf scald is a widespread and devastating disease. Eradication is impractical because of alternative hosts. Measures to reduce inoculum sources and transmission can reduce losses. Long-term control requires sugarcane varieties with introgressed resistance, thus limiting gains from breeding. Antipathogenesis approach: By understanding key pathogenicity factors (such as albicidins), it may be possible to develop new control strategies, including novel resistance genes to rescue susceptible varieties. Useful web site:http://cygnus.tamu.edu/Texlab/Sugarcrops/Sugarcane/sugarc.html.

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.

Characterization of the acyl carrier protein gene and the fab gene locus in Xanthomonas albilineans.

A genomic region containing the fatty acid biosynthetic (fab) genes was isolated from the sugarcane leaf-scald pathogen Xanthomonas albilineans. The order and predicted products of fabG (beta-ketoacyl reductase), acpP (acyl carrier protein), fabF (ketoacyl synthase II) and downstream genes in X. albilineans are very similar to those in Escherichia coli, with one exception. Sequence analysis, confirmed by insertional knockout and specific substrate feeding experiments, shows that the position occupied by pabC (encoding aminodeoxychorismate lyase) in other bacteria is occupied instead by pabB (encoding aminodeoxychorismate synthase component I) in X. albilineans. Downstream of pabB, X. albilineans resumes the arrangement common to characterized Gram-negative bacteria, with three transcriptionally coupled genes, encoding an ORF340 protein of undefined function, thymidylate kinase and delta' subunit of DNA polymerase III holoenzyme (HolB). Different species may obtain a common advantage from coordinated regulation of the same biosynthetic pathways using different genes in this region.

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 (526kDa) 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 343aa 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 239aa 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.

Expression of the Shpx2 peroxidase gene of Stylosanthes humilis in transgenic tobacco leads to enhanced resistance to Phytophthora parasitica pv. nicotianae and Cercospora nicotianae.

Abstract Previous research indicated that the constitutive expression of a pathogen-inducible peroxidase gene (Shpx6a) from Stylosanthes humilis in transgenic plants resulted in enhanced resistance to fungal pathogens ( Kazan, K., Goulter, K.C., Way, H.M. and Manners, J.M. (1998) Expression of a pathogenesis-related peroxidase of Stylosanthes humilis in transgenic tobacco and canola and its effect on disease development. Plant Sci. 136, 207-217). We have now investigated another pathogen-inducible peroxidase gene of S. humilis, termed Shpx2, which is highly divergent from Shpx6a. Constitutive expression of the Shpx2 cDNA was obtained in tobacco using the 35S CaMV promoter, and up to a 12-fold increase in total peroxidase activity was observed in the leaves of transgenic plants compared to nontransgenic controls. Disease development was evaluated after inoculations in T(1) and T(2) transgenic lines expressing Shpx2. Lesion expansion was significantly (P < 0.05) reduced by up to 25% and 50% on leaves and stems, respectively, of transgenic plants expressing high levels of peroxidase compared to nontransgenic controls, following inoculation with Phytophthora parasitica pv. nicotianae, the cause of black shank disease. In addition, plant survival and recovery were greatly enhanced in transgenic plants following stem inoculations of plants with this plant pathogen. A significant (55%, P < 0.05) reduction in lesion number was observed in transgenic plants with high levels of peroxidase activity following inoculation with the fungus Cercospora nicotianae, the cause of frog-eye disease. No significant differences in disease development were observed between the lines expressing Shpx2 and controls following inoculation with the bacterium Pseudomonas syringae pv. tabaci, the cause of wildfire disease. These results provide evidence for a role for this peroxidase gene in plant defence, and suggest that diverse peroxidase genes may be employed as components of strategies aimed at the engineering of disease resistance.

Engineered detoxification confers resistance against a pathogenic bacterium.

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

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.

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane.

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35 degrees C. Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

PLANT TRANSFORMATION: Problems and Strategies for Practical Application.

Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

Mechanisms of biocontrol by Pantoea dispersa of sugar cane leaf scald disease caused by Xanthomonas albilineans.

Albicidins, a family of phytotoxins and antibiotics produced by Xanthomonas albilineans, are important in sugar cane leaf scald disease development. The albicidin detoxifying bacterium Pantoea dispersa (syn. Erwinia herbicola) SB1403 provides very effective biocontrol against leaf scald disease in highly susceptible sugar cane cultivars. The P. dispersa gene (albD) for enzymatic detoxification of albicidin has recently been cloned and sequenced. To test the role of albicidin detoxification in biocontrol of leaf scald disease, albD was inactivated in P. dispersa by site-directed mutagenesis. The mutants, which were unable to detoxify albicidin, were less resistant to the toxin and less effective in biocontrol of leaf scald disease than their parent strain. This indicates that albicidin detoxification contributes to the biocontrol capacity of P. dispersa against X. albilineans. Rapid growth and ability to acidify media are other characteristics likely to contribute to the competitiveness of P. dispersa against X. albilineans at wound sites used to invade sugar cane.

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.

A gene from Alcaligenes denitrificans that confers albicidin resistance by reversible antibiotic binding.

Albicidin antibiotics specifically block prokaryote DNA replication. The albicidin resistance gene (albB) cloned from a soil isolate of Alcaligenes denitrificans encodes a 23 kDa protein capable of detoxifying albicidin by reversible binding. This mechanism operates intracellularly to protect DNA replication in albicidin-sensitive Escherichia coli expressing the cloned resistance gene, which can be induced fivefold in the presence of 1.5 micrograms albicidin ml-1 in the surrounding medium. The coding region of 621 bp has regions with partial DNA sequence homology to an albicidin resistance gene (albA) from Klebsiella oxytoca, but with rearrangements and frame-shifts resulting in loss of protein homology. There is a short region of N-terminal homology between the albicidin resistance (Albr) proteins from A. denitrificans and K. oxytoca, although the two genes use different codons for shared amino acids. The N-terminal homology suggested a common functional domain; this was confirmed by deletion analysis, translational fusions and albicidin binding by a synthetic oligopeptide. Antibiotic binding provides a high level of albicidin resistance in E. coli. The gene appears to be a useful candidate for transfer to plants to protect plastid DNA replication from inhibition by albicidin phytotoxins involved in sugarcane leaf scald disease.

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture.

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 107 kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.

Effects of promoter, intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts.

Various chimaeric promoter regions coupled to the uidA beta-glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5' transcribed region decreased expression strength.

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system.

Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E. coli host-cells. All stable spontaneous Albr mutants of E. coli simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E. coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.

Competence for gene transfer by electroporation in a sub-population of protoplasts from uniform carrot cell suspension cultures.

We have investigated the basis for increased transient reporter gene expression following electroporation of protoplasts from uniform carrot cell suspension cultures at increasing DNA concentrations. Use of a combination of histochemical and fluorometric GUS gene assays allowed differentiation between increases due to a higher proportion of expressing protoplasts and increases due to higher expression by each expressing protoplast. A plateau of 20-25% expressing protoplasts was reached by 50 µg ml(-1) DNA but total expression continued to increase in direct proportion to applied DNA concentration up to at least 100 µg ml(-1). This indicates the existence of a subpopulation of protoplasts competent for the uptake and expression of genes by electroporation.