Towards identification of pathogenicity genes specific to Xanthomonas albilineans strains closely associated with sugarcane leaf disease outbreaks

Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, one of the major diseases of sugarcane. Interestingly, this pathogen lacks both the xanthan gum genes cluster and a type III secretion system (T3SS) of the Hrp1 and Hrp2 injectisome families (1). X. albilineans produces a unique and specific toxin, albicidin, which is a powerful DNA gyrase inhibitor. Consequently, albicidin blocks chloroplasts differentiation, resulting in leaf scald symptoms. High genetic and pathogenic variability exists among strains of X. albilineans, and 10 genetic groups were identified by pulsed field gel electrophoresis (PFGE). All strains involved in sugarcane leaf scald disease outbreaks since the late 1980s belong to the same genetic group called PFGE-B, whereas the strains isolated previously belong to other groups, especially to group PFGE-A. These two groups were also revealed by multilocus sequence analysis (MLSA) using seven housekeeping genes (groEL, dnaK, gyrB, atpD, efp, recA and glnA). The complete genome sequence of strain GPE PC73 belonging to PFGE-B is now available (1). To better understand the genetic differences between the PFGE-A and PFGE-B strains, Suppression Subtractive Hybridization (SSH) analysis was performed to compare the genomes of XaFL07-1 (PFGE-B) and Xa23R1 (PFGE-A), both isolated in Florida. SSH is a method used to identify DNA fragments that are uniquely found in one strain when compared with another, closely related bacterial strain (2,3). We enriched a library of unique DNA sequences from strain XaFL07-1 (tester strain), using Xa23R1 DNA as the driver strain. A total of 188 XaFL07-1-specific clones were generated and sequenced. Sequences were all compared against the GPE PC73 genome and against the GenBank non redundant database (NCBI). Initial screening focused on 12 genes with potential pathogenicity function and for which SSH data were confirmed by PCR and Southern blot hybridization. These included a DNA methyltransferase, a chemotaxis protein, a permease, a CRISPR protein, and an Rhs protein. However, after further distribution studies of these genes among strains of X. albilineans representing the genetic diversity observed in this pathogen, only one gene encoding a DNA (cytosine-5)-methyltransferase was found to be specific to PFGE-B strains. In GPE PC73, this gene is localized in a 53 kb chromosome region of phage origin that includes also two other DNA methyltransferases. SSH often results in identification of phage DNA (3); DNA methyltransferases are known to play a role in genetic regulation by modifying the binding of DNA polymerases on promoters. Additional PCR and Southern blot screening of all SSH clones, including genes with unknown functions, could also lead to the identification of other pathogenicity genes specific to PFGE-B strains of X. albilineans. Knockout mutagenesis of the DNA methyltransferases and of other SSH candidate genes will be performed to investigate the role of these genes in pathogenicity of Xanthomonas albilineans strains closely associated with sugarcane leaf scald disease outbreaks. (Texte intégral

Bioactivity-guided mass spectral net working reveals a new set of allbicidin derivatives from Xanthomonas albilineans

Albicidin is produced by the sugarcane pathogenic bacterium Xanthomonas albilineans and is a potent antibiotic specifically targeting the bacterial DNA gyrase with an IC50 value in the nanomolar range. The structure of albicidin, which has remained unsolved for more than three decades since its first description, has been solved by a combination of extensive NMR and HR-MS/MS experiments. An inherent problem for the structure elucidation was the low amount of the albicidin production in cell culture. The low production forced us to cultivate several hundred liters in order to purify a few milligrams required for NMR experiments. In comparison, LC-HR-MS/MS has the great advantage of several orders of magnitude and high sensitivity. Thus, this approach requires much less material. But more importantly, purification of compounds is not necessary for structural analysis. Due to the application of targeted LC-MS/MS experiments, we were able to propose the structure of N-terminal carba- S158 moylated and b-methoxylated albicidin derivatives. Both of them are currently chemically synthesized and studied in a medicinal chemistry campaign. In order to search for more pharmaceutically relevant albicidin derivatives, we applied non-targeted LC-HR-MS/MS approaches combined with mass spectral networking, a recently introduced approach for non-targeted MS/MS data-analysis. However, as for the structural elucidation of albicidin, the even lower concentrated derivatives resulted in difficulties to obtain useful MS/ MS spectra. Since we typically operate the mass spectrometer in data-dependent acquisition mode (DDA), it inherently selects high abundant compounds of cell extracts for MS/ MS scans and possibly ignore low abundant compounds. The same goes for the network analysis which is based on similarity of MS/MS spectra and may not take potential ion species into account. To overcome this problem, we implemented a bioactivity-guided fractionation by solid phase extraction and semi-preparative HPLC. Fractions with antibacterial activity were subsequently submitted to several LC-MS/MS runs in DDA mode with shifted survey scan windows (stitched DDA). Besides the increased amount of MS/MS events per mass range, narrowing down the m/z range of the survey scan also increased the dynamic range of the orbitrap analyzer, which enabled us to significantly increase the method's sensitivity. Finally, we were able to detect a whole series of new albicidin derivatives and to propose their structures based on exact mass determination and MS/MS fragmentation patterns. These new compounds provide great opportunities in lead optimization of albicidin as a new anti-infective drug. Apart from the pharmaceutical relevance, some of the identified by-products give us important insights into the formation of albicidin and its intriguing biosynthetic machinery. (Résumé d'auteur

CRISPR-associated sequence diversity within Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane

Co-ordinated efforts to collect and maintain cotton genetic resources have increased over the last 100 years to insure the worldwide economic value of cotton fibre and cotton by-products. The classified genetic resources of cotton are extensive and include five tetraploid species in the primary gene pool, 20 diploid species in the secondary gene pool, and 25 diploid species in the tertiary gene pool. There are at least eight major cotton collections worldwide and their status and contents are discussed. An overview of the collections suggest that there is a substantial coverage of the Gossypium genome but some recently identified species are not yet maintained and several species are underrepresented and threatened by loss of their natural habitat. Meeting the high demand for cotton genetic resources and increasing the coverage of the genus with decreasing budgets are a few of the challenges facing individual collections. These types of challenges and the opportunities for international collaboration that they create are discussed. One desirable outcome of co-ordinated efforts among collections would be finding gaps in the collections and sharing of the workload to conserve the genus. Multinational communication and collaboration are critical for the evaluation of rare and unique cotton germplasm and protection of the global cotton germplasm resources. (Résumé d'auteur

Engineered transfer of the PKS/NRPS biosynthesis pathway of albicidin: A promising approach to overproduce this potent antibiotic

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin and antibiotic called albicidin that was shown to inhibit DNA replication in both sugarcane proplastids and Escherichia coli. Low yields of albicidin production in slow growing X. albilineans have slowed studies of its chemical structure and potential therapeutic applications. Albicidin is synthesized by a unique hybrid PKS/NRPS (polyketide synthase/nonribosomal peptide synthase) pathway that does not resemble any other described to date. We report here the transfer of the entire 49 kb albicidin biosynthetic gene cluster from X. albilineans into X. axonopodis pv. vesicatoria and the subsequent production of an antibiotic active against E. coli that shows cross-resistance with albicidin. The yield of this antibiotic in X. axonopodis pv. vesicatoria is 6 times higher than in X. albilineans. This study demonstrates the feasibility to transfer the albicidin pathway into an heterologous host and offers a promising strategy to overproduce, characterize and explore potential therapeutic applications of this potent antibiotic. (Texte intégral

CRISPR-associated sequence diversity within Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane.

Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, a lethal disease of sugarcane. Unlike other xanthomonads, X. albilineans exhibits a large intra-species variability which was previously observed with different genetic markers (PFGE for Pulsed Field Gel Electrophoresis and MLSA for Multi Locus Sequence Analysis). The CRISPR systems (Clustered Regularly Interspaced Short Palindromic Repeats) are repetitive structures in bacteria and Archaea composed of exact 24- to 48-bp repeated sequences (or "repeats") separated by unique sequences of similar length (or "spacers"). Over 40 gene families, which are found nowhere except near these repeats, have been designated collectively as CRISPR-associated (cas) genes. CRISPR/cas systems participate in an antiviral response, probably by an RNA interference-like mechanism. Analysis of the variability of CRISPR spacers is currently used to perform diversity or epidemiological studies in bacteria. The genome sequence of X. albilineans revealed the occurrence of two different CRISPR/cas systems in this pathogen. The first system, called CRISPR-1, is associated with seven cas genes and contains repeats of 31 base pairs. It is similar to the CRISPR system found in several sequenced species of Xanthomonas. The second system, called CRISPR-2, is associated with six cas genes and contains repeats of 28 base pairs. There is only one Xanthomonas pathovar that is known to contain a similar CRISPR-2 system, namely X. campestris pv. raphani. In this study, we analyzed the polymorphism of the two different CRISPR/cas systems among 21 strains spanning the genetic diversity of X. albilineans. We have either sequenced PCR products resulting from amplification of spacers or cas genes, or used sequences from draft genome sequences. Whereas CRISPR-2 is ubiquitous within the 21 strains, CRISPR-1 is absent in three strains. The loss of CRISPR-1 by a common ancestor of these three strains is in accordance with the MLSA phylogeny. As described in other bacteria, we observed a variability of the CRISPR spacers, not only between phylogenetically distant strains, but also between closely related strains (acquisition of new spacers at the 5' leader-proximal end of CRISPR and deletion or replacement of some spacers in the central region). This polymorphism within X. albilineans, which is congruent with previous MLSA and PFGE results, provides a better resolution of the phylogeny of X. albilineans strains. (Résumé d'auteur

Deciphering small molecules as new virulence factors in the bacterial sugarcane pathogen Xanthomonas albilineans

Xanthomonas albilineans is a xylem-invading plant pathogen that causes leaf scald disease of sugarcane. Unlike most plant pathogenic bacteria, X. albilineans does not possess a Hrp-Type Three Secretion System. Pathogenicity of this bacterial species must therefore rely on other virulence factors. X. albilineans produces albicidin, a toxin and potent inhibitor of DNA gyrase which inhibits proplastic DNA replication. Consequently, chloroplast differentiation is blocked and disease symptoms develop. Albicidin is also bactericidal at nanomolar concentrations against a range of Gram-positive and Gram-negative bacteria. This potent and novel antibiotic is especially of interest because of its activity against Escherichia coli, a species causing nosocomial diseases. Sequencing and annotation of the entire genome of X. albilineans recently revealed that X. albilineans possesses 12 large genes encoding nonribosomal peptide synthetases (NRPSs) which are located in four gene clusters covering 4% of the genome (1). One of these NRPS clusters corresponds to the previously identified albicidin biosynthesis gene cluster. The mode of action of this antibiotic was extensively studied but its structure remains unknown. Characterization of albicidin is the main bottleneck which slows development of its therapeutic application. X. albilineans is a slow growing bacterium and production yields of albicidin are low, i.e. it turned out to be extremely tedious to obtain sufficient amounts for structure elucidation. To overcome this problem, we successfully considered heterologous overproduction by transferring all albicidin biosynthesis genes into the fast growing bacterium Xanthomonas axonopodis pv. vesicatoria (2). Production of albicidin in this heterologous system already allowed us to obtain several milligrams of pure compound and promising preliminary results regarding the structural characterization of albicidin. As an example, 1H-NMR analyses showed the presence of a number of para-substituted aromates, putatively tyrosine or 4-hydroxyphenylglycines. However, the number of amide protons found in 1H-NMR spectra is lower than the number expected from the biosynthesis assembly lines, suggesting the involvement of tailoring steps during or post-NRPS biosynthesis. In silico analysis of the three other NRPS gene clusters resulted in partial prediction of the sequences of the precursor peptides synthesized by these clusters which do not resemble any peptide described to date. One of these NRPS gene clusters encodes a complete machinery predicted to be required for secretion and tailoring of small molecules: ABC transporter, MbtH like protein, isomerase, aminotransferase, acyltransferase, enoyl-CoA hydratase. The two other NRPS gene clusters encode only NRPSs which are thought to trans-act with the first one. Interestingly, the NRPSs encoded by X. albilineans share characteristics with NRPSs encoded by the root and stem-nodulating Bradyrhizobium sp. BTAi1, suggesting structural similarities between small molecules produced by these two plant interacting species. In X. albilineans, functional analyses of the phosphopantheteinyl transferase gene (which is required for activation of NRPSs) showed that these non-albicidin NRPS gene clusters are most likely involved in the biosynthesis of at least one new virulence factor. Future work will focus on characterization of the full structure of albicidin as well as the isolation and characterization of other small molecules assembled by NRPS. These compounds will be chromatographically isolated and characterized by high-resolving FTICR mass spectrometry, 2D-NMR spectroscopy and X-ray crystallography. Isolation and structural characterization of these new bioactive molecules will facilitate the annotation of biosynthesis gene clusters and the study of their role during interactions between sugarcane and X. albilineans. On a biochemical level, deciphering their role in pathogenicity should result in the identifi

Heterologous production and characterization of albicidin, a potent DNA gyrase inhibitor

Albicidin is a toxin and an antibiotic produced by the slow-growing bacterium Xanthomonas albilineans, the causal agent of sugarcane leaf scald. Albicidin is involved in the pathogenicity of X. albilineans and inhibits the replication of chloroplastic DNA. It also inhibits DNA replication in Escherichia coli at nanomolar concentrations, whereas mammalian cells are unaffected at 8 ?g/ml. Albicidin targets DNA gyrase with features of inhibition that differ from those of other known antibiotics. It is synthesized by a unique hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway that does not resemble any other pathway described to date. The antibiotic activity of albicidin against a wide range of gram-positive and gram-negative pathogenic bacteria (Enterobacter aerogenes, E. coli, Haemophilus influenzae, Klebsiella pneumoniae, Shigella sonnei, and Staphylococcus aureus) is of interest for the development of new antibacterial drugs. Low yields of albicidin production in slow-growing X. albilineans have slowed studies of its chemical structure and potential therapeutic applications. Therefore, we have developed a heterologous system for albicidin overproduction using a Xanthomonas axonopodis pv. vesicatoria strain transformed with two plasmids harbouring the complete albicidin biosynthetic gene set. A sixty-fold increase in albicidin production was obtained when compared to albicidin production by the native host, X. albilineans. Heterologous production of albicidin was confirmed by FTICR-MS (high resolving Fournier Transform Ion Cyclotron Resonance Mass Spectrometry). (Texte intégral

Overproduction and isolation from Xanthomonas albilineans of a new non-ribosomally synthesized peptide putatively involved in plant-bacteria interactions : [Poster 538]

Background: To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in the genus Xanthomonas is the phytotoxin albicidin produced by Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Recent analyses of available genomic sequences indicated that X. albilineans, as well as Xanthomonas oryzae and Xanthomonas translucens, possess a novel NRPS gene cluster called META-B whose architecture is strainspecific (each sequenced strain is predicted to synthesize a different peptide) and doesn´t resemble to any NRPS gene cluster described to date. Because the META-B NRPS gene cluster is specific to three phylogenetically distant species of Xanthomonas associated with monocotyledonous plants, we assume a putative involvement of the new family of small molecules produced by this gene cluster in plant-bacteria interactions. Objectives: We aimed at isolating and characterizing the small molecule encoded by the META-B NRPS gene cluster of X. albilineans in order to study its putative involvement in plant-bacteria interactions. Methods: We developed a META-B overproducing X. albilineans strain by over-expressing the AraC transcriptional regulator which is present within the META-B NRPS gene cluster. HPLC profiles obtained from cultures of wild type vs the AraC over-expressing strain were compared. Peaks with significantly increased amplitude in the HPLC profile from the AraC over-expressing strain were collected and their content analyzed by mass spectrometry. Conclusions: We identified a peptide with a nominal molecular mass > 2 kDa and with an amino acid sequence (MS/MS experiments) which excellently matches the one predicted by A-domain specificities of META-B NRPS genes. (Texte intégral