Characterization of the Serralysin-Like Gene of 'Candidatus Liberibacter solanacearum' Associated with Potato Zebra Chip Disease.

The nonculturable bacterium 'Candidatus Liberibacter solanacearum' is the causative agent of zebra chip disease in potato. Computational analysis of the 'Ca. L. solanacearum' genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The 'Ca. L. solanacearum' serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional 'Ca. L. solanacearum' serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the 'Ca. L. solanacearum' serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the 'Ca. L. solanacearum' serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-'Ca. L. solanacearum' serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the 'Ca. L. solanacearum' protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the 'Ca. L. solanacearum' serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites.

Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. The transcript profiles of Pseudomonas syringae pv. syringae B728a support a model in which leaf surface, or epiphytic, sites specifically favor flagellar motility, swarming motility based on 3-(3-hydroxyalkanoyloxy) alkanoic acid surfactant production, chemosensing, and chemotaxis,indicating active relocation primarily on the leaf surface. Epiphytic sites also promote high transcript levels for phenylalanine degradation, which may help counteract phenylpropanoid-based defenses before leaf entry. In contrast, intercellular, or apoplastic,sites favor the high-level expression of genes for GABA metabolism (degradation of these genes would attenuate GABA repression of virulence) and the synthesis of phytotoxins, two additional secondary metabolites, and syringolin A. These findings support roles for these compounds in virulence, including a role for syringolin A in suppressing defense responses beyond stomatal closure. A comparison of the transcriptomes from in planta cells and from cells exposed to osmotic stress, oxidative stress, and iron and nitrogen limitation indicated that water availability, in particular,was limited in both leaf habitats but was more severely limited in the apoplast than on the leaf surface under the conditions tested. These findings contribute to a coherent model of the adaptations of this widespread bacterial phytopathogen to distinct habitats within its host.

Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of 'candidatus Liberibacter solanacearum' in potatoes and Psyllids.

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of 'Candidatus Liberibacter solanacearum', the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of 'Ca. Liberibacter solanacearum' was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected 'Ca. Liberibacter solanacearum' and the closely related species 'Ca. Liberibacter asiaticus', the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting 'Ca. Liberibacter' pathogens in psyllids and field-grown potato plants and tubers.

Development of Primers for Improved PCR Detection of the Potato Zebra Chip Pathogen, 'Candidatus Liberibacter solanacearum'.

Zebra chip disease poses a major economic threat to potato production. The causative agent is a phloem-limited bacterium identified as 'Candidatus Liberibacter solanacearum' that is transmitted by the potato/ tomato psyllid. Currently, there are no effective controls and existing control strategies depend largely on the early detection of the pathogen via polymerase chain reaction (PCR) assays. Most primer sets used for PCR detection target a region of the bacterial 16S rDNA gene, and detection of the pathogen in symptomatic potato tissue with existing primers has been variable depending on the specific primer sets used. This study describes the development of two new primer sets that target a conserved intergenic region between the 16S and 23S rDNA genes and a conserved bacterial housekeeping gene, adenylate kinase (adk). Results demonstrate that the new primer sets are more reliable in detecting 'Ca. L. solanacearum' in field and glasshouse samples than the currently used LsoF/OI2 primers. The newly developed primers differentiated between 'Ca. L. solanacearum' and a closely related 'Ca. Liberibacter' spp. and were more sensitive than the LsoF/OI2 primers. The low detection limit for the new primers was four times lower (0.65 ng) than the limit (2.5 ng) for the LsoF/OI2 primers.

Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors.

Pseudomonas syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This study demonstrates that a 29.9-kb cluster of genes in the B728a genome shares homology to the novel type VI secretion system (T6SS) locus recently described for other gram-negative bacteria. Western blot analyses showed that B728a secretes Hcp, a T6SS protein, in culture and that this secretion is dependent on clpV, a gene that likely encodes an AAA(+) ATPase. In addition, we have identified two B728a sensor kinases that have homology to the P. aeruginosa proteins RetS and LadS. We demonstrate that B728a RetS and LadS reciprocally regulate the T6SS and collectively modulate several virulence-related activities. Quantitative PCR analyses indicated that RetS and LadS regulate genes associated with the type III secretion system and that LadS controls the expression of genes involved in the production of the exopolysaccharides alginate and levan. These analyses also revealed that LadS and the hybrid sensor kinase GacS positively regulate the expression of a putative novel exopolysaccharide called Psl. Plate assays demonstrated that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affected B728a population levels on the surfaces of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed.

Drippy Pod of White Lupine: A New Bacterial Disease Caused by a Pathovar of Brenneria quercina.

Drippy pod is a unique bacterial disease of Mediterranean white lupine (Lupinus albus) that first appeared in commercial fields in Eastern Washington State in the mid-1980s. The disease is most noticeable in the field as water-soaked lesions on lupine pods that produce an abundance of whitish-colored ooze with a sticky and foamy consistency. As the disease progresses, yellowing of lupine plants occurs with ooze characteristically dripping down the infected pods and stems and solidifying. A gram-negative rod-shaped bacterium with facultative anaerobic growth was repeatedly isolated from infected lupine tissues, and subsequently confirmed by Koch's postulates to infect lupines. Physiological and biochemical tests, including the API 20E and 50CHE strip assays, showed a highly uniform phenotype for the lupine strains that was distinctive for the genus Brenneria and most closely resembled the oak pathogen Brenneria quercina. Furthermore, sequence analyses of the 16S rDNA gene and the 16S-23S intergenic region of lupine strains revealed the highest similarity (>97%) to the corresponding regions of B. quercina and less similarity to the next closest species, B. salicis. Fatty acid profiling demonstrated that lupine strains were qualitatively similar in composition to Brenneria spp., and supported placement of the drippy pod bacterium in the species B. quercina. Oak strains of B. quercina, however, did not incite drippy pod disease on lupine. Consequently, the lupine strains that cause bacterial drippy pod disease were classified as B. quercina pv. lupinicola pv. nov.

Mutational analysis and homology modelling of SyrC, the aminoacyltransferase in the biosynthesis of syringomycin.

SyrC, a component of the multienzyme system of syringomycin biosynthesis, has been shown to shuttle Thr/4-Cl-Thr between the thiolation domains SyrB1-T1 and SyrE-T8,9 by transiently linking it to Cys224 in the enzyme active site. We present data on the structure-function relationship in vivo of this protein and an in silico model of its three-dimensional structure. The biosynthetic activity of SyrC was not influenced when either Asp348 or His376 that together with Cys224 form a putative catalytic triad, were replaced with Ala, but it was abolished by the exchange Cys224 with Ser. The presence of the FLAG peptide on either the N- or C-terminus of the protein did not affect activity, whereas the deletion of the first 16 amino acids at the N-terminus or the insertion of Maltose Binding Protein abolished the production of syringomycin. We present the model of the three-dimensional structure of SyrC suggesting a homodimeric structure for the protein and biochemical data that are supportive of this model.

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.

Characterization of salA, syrF, and syrG Genes and Attendant Regulatory Networks Involved in Plant Pathogenesis by Pseudomonas syringae pv. syringae B728a.

Pseudomonas syringae pv. syringae B728a, causal agent of brown spot on bean, is an economically important plant pathogen that utilizes extracellular signaling to initiate a lifestyle change from an epiphyte to a pathogen. LuxR regulatory proteins play an important role in the transcriptional regulation of a variety of biological processes involving two-component signaling, quorum sensing, and secondary metabolism. Analysis of the B728a genome identified 24 LuxR-like proteins, three of which are encoded by salA, syrF, and syrG located adjacent to the syringomycin gene cluster. The LuxR-like proteins encoded by these three genes exhibit a domain architecture that places them in a subfamily of LuxR-like proteins associated with regulation of secondary metabolism in B728a. Deletion mutants of salA, syrF, and syrG failed to produce syringomycin and displayed reduction of virulence on bean. The transcriptional start sites of salA, syrG, and syrF were located 63, 235, and 498 bp upstream of the start codons, respectively, using primer extension analysis. The predicted -10/-35 promoter regions of syrF and syrG were confirmed using site-directed mutagenesis and GFP reporters that showed conserved promoter sequences around the -35 promoter region. Overexpression analysis and GFP reporters identified SyrG as an upstream transcriptional activator of syrF, where both SyrG and SyrF activate promoters of syringomycin biosynthesis genes. This study shows that syrG and syrF encode important transcriptional regulators of syringomycin biosynthesis genes.

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.

Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen "Candidatus Liberibacter solanacearum".

An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers.

Comparative genomics of Pseudomonas syringae pv. syringae strains B301D and HS191 and insights into intrapathovar traits associated with plant pathogenesis.

Pseudomonas syringae pv. syringae is a common plant-associated bacterium that causes diseases of both monocot and dicot plants worldwide. To help delineate traits critical to adaptation and survival in the plant environment, we generated complete genome sequences of P. syringae pv. syringae strains B301D and HS191, which represent dicot and monocot strains with distinct host specificities. Intrapathovar comparisons of the B301D (6.09 Mb) and HS191 (5.95 Mb plus a 52 kb pCG131 plasmid) genomes to the previously sequenced B728a genome demonstrated that the shared genes encompass about 83% of each genome, and include genes for siderophore biosynthesis, osmotolerance, and extracellular polysaccharide production. Between 7% and 12% of the genes are unique among the genomes, and most of the unique gene regions carry transposons, phage elements, or IS elements associated with horizontal gene transfer. Differences are observed in the type III effector composition for the three strains that likely influences host range. The HS191 genome had the largest number at 25 of effector genes, and seven effector genes are specific to this monocot strain. Toxin production is another major trait associated with virulence of P. syringae pv. syringae, and HS191 is distinguished by genes for production of syringopeptin SP25 and mangotoxin.

The sypA, sypS, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D.

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.

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.

Construction of pMEKm12, an expression vector for protein production in Pseudomonas syringae.

Characterization of the biological roles of proteins is essential for functional genomics of pseudomonads. Heterologous proteins overproduced in Escherichia coli frequently fail to exhibit biological function. To circumvent this problem, vector pMEKm12 was constructed and used to overexpress proteins in Pseudomonas. The vector contains the pRO1600 replication origin, the maltose-binding protein (MBP) fusion system, and an inducible tac promoter. The pMEKm12 was successfully used to overexpress the syringomycin synthetase SyrB1 protein fused to MBP in Pseudomonas syringae pv. syringae. Furthermore, expression of the MBP-SyrB1 protein in the syrB1 mutant BR132A1 resulted in the restoration of syringomycin production. This vector will facilitate confirmation of the biochemical roles of nonribosomal peptide synthetase genes in Pseudomonas syringae, and studies of gene function from a wide spectrum of pseudomonads.

Transcriptional analysis of the global regulatory networks active in Pseudomonas syringae during leaf colonization.

UNLABELLED: The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean (Phaseolus vulgaris). To understand the contribution of distinct regulators to B728a fitness and pathogenicity, we performed a transcriptome analysis of strain B728a and nine regulatory mutants recovered from the surfaces and interior of leaves and exposed to environmental stresses in culture. The quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator, SalA, formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals and a plant signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. SalA functioned as a central regulator of iron status based on its reciprocal regulation of pyoverdine and achromobactin genes and also sulfur uptake, suggesting a role in the iron-sulfur balance. RetS functioned almost exclusively to repress secondary metabolite genes when the cells were not on leaves. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN, and AlgU in global regulation in B728a in planta and a high level of plasticity in these regulators' responses to distinct environmental signals. IMPORTANCE: Leaves harbor abundant microorganisms, all of which must withstand challenges such as active plant defenses and a highly dynamic environment. Some of these microbes can influence plant health. Despite knowledge of individual regulators that affect the fitness or pathogenicity of foliar pathogens, our understanding of the relative importance of various global regulators to leaf colonization is limited. Pseudomonas syringae strain B728a is a plant pathogen and a good colonist of both the surfaces and interior of leaves. This study used global transcript profiles of strain B728a to investigate the complex regulatory network of putative quorum-sensing regulators, two-component regulators, and sigma factors in cells colonizing the leaf surface and leaf interior under stressful in vitro conditions. The results highlighted the value of evaluating these networks in planta due to the impact of leaf-specific environmental signals and suggested signal differences that may enable cells to differentiate surface versus interior leaf habitats.

Characterization of five ECF sigma factors in the genome of Pseudomonas syringae pv. syringae B728a.

Pseudomonas syringae pv. syringae B728a, a bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome revealed 10 ECF sigma factors. This study analyzed deletion mutants of five previously uncharacterized ECF sigma factor genes in B728a, including three FecI-type ECF sigma factors (ECF5, ECF6, and ECF7) and two ECF sigma factors placed in groups ECF11 and ECF18. Transcriptional profiling by qRT-PCR analysis of ECF sigma factor mutants was used to measure expression of their associated anti-sigma and outer membrane receptor proteins, and expression of genes associated with production of extracellular polysaccharides, fimbriae, glycine betaine and syringomycin. Notably, the B728aΔecf7 mutant displayed reduced swarming and had decreased expression of CupC fimbrial genes. Growth and pathogenicity assays, using a susceptible bean host, revealed that none of the tested sigma factor genes are required for in planta growth and lesion formation.

Development of a real-time microchip PCR system for portable plant disease diagnosis.

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.

RNA-seq analysis reveals that an ECF σ factor, AcsS, regulates achromobactin biosynthesis in Pseudomonas syringae pv. syringae B728a.

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a.

Insight into the structure-function relationship of the nonheme iron halogenases involved in the biosynthesis of 4-chlorothreonine --Thr3 from Streptomyces sp. OH-5093 and SyrB2 from Pseudomonas syringae pv. syringae B301DR.

Molecular cloning of the biosynthetic gene cluster involved in the production of free 4-chlorothreonine in Streptomyces sp. OH-5093 showed the presence of six ORFs: thr1, thr2, thr3, orf1, orf2 and thr4. According to bioinformatic analysis, thr1, thr2, thr3 and thr4 encode a free-standing adenylation domain, a carrier protein, an Fe(II) nonheme α-ketoglutarate-dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4-chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from Pseudomonas syringae pv. syringae B301DR. Orf1 and orf2 show sequence similarity with alanyl/threonyl-tRNA synthetases editing domains and drug metabolite transporters, respectively. We show that thr3 can replace the halogenase gene syrB2 in the biosynthesis of syringomycin, by functional complementation of the mutant P. s. pv. syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structure-function relationship of halogenases Thr3 and SyrB2 using homology modelling and site-directed mutagenesis.