Shi-En Lu

Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing

ABSTRACT The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. “Candidatus Liberibacter asiaticus” was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of “Candidatus Liberibacter asiaticus” in symptomatic leaves. These data implicate “Candidatus Liberibacter asiaticus” as the pathogen responsible for HLB disease.

Characterization of the salA, syrF, and syrG Regulatory Genes Located at the Right Border of the Syringomycin Gene Cluster of Pseudomonas syringae pv. syringae

Sequence analysis of the right border of the syr gene cluster of Pseudomonas syringae pv. syringae strain B301D revealed the presence of the salA gene 8,113 bp downstream of syrE. The predicted SalA protein of strain B301D differs by one amino acid from that of strain B728a. Two homologs of salA, designated syrF and syrG, were identified between syrE and salA. All three proteins contain helix-turn-helix DNA-binding motifs at their C termini and exhibit homology to regulatory proteins of the LuxR family. A salA mutant failed to produce syringomycin, whereas syrF and syrG mutants produced 12 and 50%, respectively, of syringomycin relative to the wild-type strain. The salA, syrF, and syrG mutants were significantly reduced in virulence, forming small, nonspreading lesions in immature cherry fruits. Translational fusions to the uidA gene were constructed to evaluate expression of syrB1 in regulatory mutant backgrounds and to determine the relationship among the three regulatory loci. Expression of a syrB1::uidA fusion required functional salA and syrF genes and, in series, the expression of a syrF::uidA fusion required a functional salA gene. These results demonstrate that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production and virulence in P. syringae pv. syringae.

Occidiofungin, a unique antifungal glycopeptide produced by a strain of burkholderia contaminans

Bacterial strain Burkholderia contaminans MS14 was isolated from soil that suppressed brown patch disease of lawn grass. An antifungal compound was purified from the liquid culture of this bacterium. In this study, complete covalent structures of two purified closely related antifungal compounds were determined by the experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR, and ESI-MS and GC. The analysis of monoisotopic masses of the purified preparation indicated the presence of two related compounds with masses determined to be 1199.543 and 1215.518 Da; the difference corresponds to the mass of an oxygen atom. GC analysis identified a xylose sugar attached to the antifungal compound. NMR experiments revealed that the compound is cyclic and composed of eight amino acids, two of which are beta-hydroxy derivatives of Tyr and Asn, and one being a novel amino acid. The novel amino acid serves as the scaffold for the attachment of the xylose and a short acyl chain. The spectrum and concentration of antifungal activity were determined using a microtiter plate assay. The antifungal compound demonstrated potent antifungal activities against a broad panel of fungal plant and animal pathogens, as well as two Pythium spp. Microscopic observations showed that the antifungal compound disrupts normal membrane morphology. The cells fill with large inclusion bodies and the membrane becomes irregularly shaped and swollen following the exposure to subinhibitory concentrations of the antifungal compound. Our data support the identification of a novel fungicide and the compound has been named occidiofungin, meaning fungal killer.

Oligonucleotide Microarray Analysis of the SalA Regulon Controlling Phytotoxin Production by Pseudomonas syringae pv. syringae

The salA gene is a key regulatory element for syringomycin production by Pseudomonas syringae pv. syringae and encodes a member of the LuxR regulatory protein family. Previous studies revealed that salA, a member of the GacS/GacA signal transduction system, was required for bacterial virulence, syringomycin production, and expression of the syrB1 synthetase gene. To define the SalA regulon, the spotted oligonucleotide microarray was constructed using gene-specific 70-mer oligonucleotides of all open reading frames (ORFs) predicted in the syringomycin (syr) and syringopeptin (syp) gene clusters along with representative genes important to bacterial virulence, growth, and survival. The microarray containing 95 oligos was used to analyze transcriptional changes in a salA mutant (B301DSL07) and its wild-type strain, B301D. Expression of 16 genes was significantly higher (> twofold) in B301D than in the salA mutant; the maximum change in expression was 15-fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion, and regulation of syringomycin and syringopeptin. The positive regulatory effect of SalA on transcription of sypA, syrB1, syrC, and sylD was verified by reporter fusions or real-time polymerase chain reaction analysis. None of the genes or ORFs was significantly down-regulated by the salA gene. These results demonstrated that a subgenomic oligonucleotide microarray is a powerful tool for defining the SalA regulon and its relationship to other genes important to plant pathogenesis.

A Physical Map of the Syringomycin and Syringopeptin Gene Clusters Localized to an Approximately 145-kb DNA Region of Pseudomonas syringae pv. syringae Strain B301D

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.

Biosynthesis of an antifungal oligopeptide in Burkholderia contaminans strain MS14

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. When compared with the wild type strain, antifungal activities of mutants MS14MT13 and MS14MT18 against Geotrichum candidum were eliminated, and the activity of mutant MS14MT15 was reduced by approximately 80%. Sequence analysis of a 22.7-kb DNA segment revealed the presence of six open-reading frames (ORFs), including the genes disrupted in the mutants. The wild type level of antifungal activity was observed for mutant MS14MT15 (ambR2::Tn5) complemented in trans with the intact ambR2 gene, which encodes a LuxR type regulator. AmbR2 positively regulates the transcription of the other five ORFs identified, which including two nonribosomal peptide synthetase genes required for biosynthesis of antifungal compound. The compound consists of amino acid residues having a molecular weight of 1200.6 Da. These results show that strain MS14 possesses a novel set of genes dedicated to the biosynthesis of an antifungal oligopeptide.

Characterization of the Transcriptional Activators SalA and SyrF, Which Are Required for Syringomycin and Syringopeptin Production by Pseudomonas syringae pv. syringae

Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of beta-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes.

Genetic and biochemical map for the biosynthesis of occidiofungin, an antifungal produced by Burkholderia contaminans strain MS14.

ABSTRACT A striking feature of Burkholderia contaminans strain MS14 is the production of a glycolipopeptide named occidiofungin. Occidiofungin has a broad range of antifungal activities against plant and animal pathogens. In this study, a complete covalent structure characterization and identification of the whole genomic DNA region for the occidiofungin gene (ocf) cluster are described. Discovery of the presence of 2,4-diaminobutyric acid and 3-chloro-β-hydroxytyrosine and elucidation of the structure of a novel C18 fatty amino acid residue have been achieved. In addition, seven additional putative open reading frames (the genes from ocfI to ocfN [ocfI-N] and ORF16) were identified. Transcription of all the putative genes ocfI-N identified in the region except ORF16 was regulated by both ambR1 and ambR2. Elucidation of the structure and the ocf gene cluster provides insight into the biosynthesis of occidiofungin and promotes future aims at understanding the biosynthetic machinery. This work provides new avenues for optimizing the production and synthesis of structural analogs of occidiofungin.

The expression of genes encoding lipodepsipeptide phytotoxins by Pseudomonas syringae pv. syringae is coordinated in response to plant signal molecules.

Specific plant signal molecules are known to induce syringomycin production and expression of syrB1, a syringomycin synthetase gene, in Pseudomonas syringae pv. syringae. This report demonstrates that syringopeptin production likewise is activated by plant signal molecules and that the GacS, SalA, and SyrF regulatory pathway mediates transmission of plant signal molecules to the syr-syp biosynthesis apparatus. Syringopeptin production by BR132 was increased two-fold by addition of arbutin (100 microM) and D-fructose (0.1%) to syringomycin minimal medium (SRM). Among 10 plant phenolic compounds tested, only the phenolic glucosides arbutin, salicin, and phenyl-beta-D-glucopyranoside induced substantially the beta-glucuronidase (GUS) activity of a sypA::uidA reporter from 242 U per 10(8) CFU without plant signal molecules up to 419 U per 10(8) CFU with plant signal molecules. Syringopeptin production was found to be controlled by the SalA/SyrF regulon because no toxin was detected from cultures of B301DSL7 (i.e., salA mutant) and B301DSL1 (i.e., syrF mutant), and the expression of sypA::uidA was decreased approximately 99 and 94% in salA (B301DSL30) and syrF (B301DNW31) mutant backgrounds, respectively. Subgenomic analysis of transcriptional expression with a 70-mer oligonucleotide microarray demonstrated that the syr-syp genes are induced 2.5- to 10.5-fold by addition of arbutin and D-fructose to SRM. This study establishes that plant signal molecules are transmitted through the GacS, SalA/SyrF pathway to activate the coordinated transcriptional expression of the syr-syp genes.

Occidiofungin's Chemical Stability and In vitro Potency Against Candida species

ABSTRACT Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between 0.5 and 2.0 μg/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal concentration (MLC) of 2 and 4 μg/ml, respectively. Time-kill and postantifungal effect (PAFE) experiments of occidiofungin against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also found to be a very stable molecule. It is resistant to extreme temperatures and pH and maintains its activity following exposure to gastric proteases.