Loss of mitochondrial Ca2+ uptake protein 3 impairs skeletal muscle calcium handling and exercise capacity.

Mitochondrial calcium concentration ([Ca2+ ]m ) plays an essential role in bioenergetics, and loss of [Ca2+ ]m homeostasis can trigger diseases and cell death in numerous cell types. Ca2+ uptake into mitochondria occurs via the mitochondrial Ca2+ uniporter (MCU), which is regulated by three mitochondrial Ca2+ uptake (MICU) proteins localized in the intermembrane space, MICU1, 2, and 3. We generated a mouse model of systemic MICU3 ablation and examined its physiological role in skeletal muscle. We found that loss of MICU3 led to impaired exercise capacity. When the muscles were directly stimulated there was a decrease in time to fatigue. MICU3 ablation significantly increased the maximal force of the KO muscle and altered fibre type composition with an increase in the ratio of type IIb (low oxidative capacity) to type IIa (high oxidative capacity) fibres. Furthermore, MICU3-KO mitochondria have reduced uptake of Ca2+ and increased phosphorylation of pyruvate dehydrogenase, indicating that KO animals contain less Ca2+ in their mitochondria. Skeletal muscle from MICU3-KO mice exhibited lower net oxidation of NADH during electrically stimulated muscle contraction compared with wild-type. These data demonstrate that MICU3 plays a role in skeletal muscle physiology by setting the proper threshold for mitochondrial Ca2+ uptake, which is important for matching energy demand and supply in muscle. KEY POINTS: Mitochondrial calcium uptake is an important regulator of bioenergetics and cell death and is regulated by the mitochondrial calcium uniporter (MCU) and three calcium sensitive regulatory proteins (MICU1, 2 and 3). Loss of MICU3 leads to impaired exercise capacity and decreased time to skeletal muscle fatigue. Skeletal muscle from MICU3-KO mice exhibits a net oxidation of NADH during electrically stimulated muscle contractions, suggesting that MICU3 plays a role in skeletal muscle physiology by matching energy demand and supply.

Role of Different Low-Density Lipoprotein-Lowering Medications on Secondary Prevention of Atherosclerotic Cardiovascular Disease in Patients With Diabetes Mellitus.

Purpose The objective of this study was to explore the optimal cholesterol-lowering therapy for diabetic patients categorized as having a very high risk for future atherosclerotic cardiovascular disease (ASCVD) events. The primary medications under investigation were statins, ezetimibe, and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors (PCSK9-Is). The efficacy of different medication regimens helped to draw conclusions regarding the evolution of cholesterol management recommended under the American College of Cardiology's (ACC) 2013 and 2018 guidelines. Methods A retrospective chart review was conducted on a cohort of patients from a large, community-based cardiology practice. Inclusion criteria specified patients aged 30-82 with a past medical history of two or more ASCVD events or one ASCVD event and at least two high-risk comorbidities. Acquired data included demographics, all lipid panels, medications used, and ASCVD events between December 1, 2013, and December 31, 2019. The data were stored and encrypted on a REDCap account. Sub-group analysis was conducted on only diabetic patients, who were then categorized by medication regimen. The statistical analysis was completed using Fisher's exact test. A p-value <0.05 was considered significant. Results A total of 102 diabetic patients met the inclusion criteria. Our primary analysis determined the percentage of patients who achieved their goals on each medication regimen. The goal was defined as a low-density lipoprotein cholesterol (LDL-C) level of less than 70 mg/dL or at least a 50% reduction from baseline levels. The results are as follows: none (0%), statin (33.9%), ezetimibe (21.1%), statin + ezetimibe (73.5%), PCSK9-Is ± statin (83.3%), and PCSK9-Is and ezetimibe ± statin (100%). There proved to be a significant difference favoring all combination regimens over statins alone; however, there was no significant difference between these advanced regimens. A follow-up analysis determined if these patients were able to maintain their goals in the subsequent lipid panel after achieving their goals. The results are as follows: none (0%), statin (61.5%), ezetimibe (50%), statin + ezetimibe (77.8%), PCSK9-Is ± statin (100%), and PCSK9-Is and ezetimibe ± statin (66.6%). The only significant difference found was between PCSK9-Is ± statins and statins alone. Conclusions Our study revealed that regimens using PCSK9 inhibitors and ezetimibe, in addition to maximally tolerated statin therapy, were more effective than statin therapy alone in achieving the goal. On extended analysis, only PCSK9 inhibitors showed superior ability in terms of maintaining the goals for diabetic patients at very high risk for future ASCVD events. This implies that statins alone may be inadequate to properly treat this specific patient population. In the context of clinical practice, physicians could have heightened consideration for dual therapy consisting of maximally tolerated statins and a secondary agent in accordance with the 2018 ACC guidelines.

Genomic instability of TnSMU2 contributes to Streptococcus mutans biofilm development and competence in a cidB mutant.

Streptococcus mutans is a key pathogenic bacterium in the oral cavity and a primary contributor to dental caries. The S. mutans Cid/Lrg system likely contributes to tolerating stresses encountered in this environment as cid and/or lrg mutants exhibit altered oxidative stress sensitivity, genetic competence, and biofilm phenotypes. It was recently noted that the cidB mutant had two stable colony morphologies: a "rough" phenotype (similar to wild type) and a "smooth" phenotype. In our previously published work, the cidB rough mutant exhibited increased sensitivity to oxidative stress, and RNAseq identified widespread transcriptomic changes in central carbon metabolism and oxidative stress response genes. In this current report, we conducted Illumina-based genome resequencing of wild type, cidB rough, and cidB smooth mutants and compared their resistance to oxidative and acid stress, biofilm formation, and competence phenotypes. Both cidB mutants exhibited comparable aerobic growth inhibition on agar plates, during planktonic growth, and in the presence of 1 mM hydrogen peroxide. The cidB smooth mutant displayed a significant competence defect in BHI, which was rescuable by synthetic CSP. Both cidB mutants also displayed reduced XIP-mediated competence, although this reduction was more pronounced in the cidB smooth mutant. Anaerobic biofilms of the cidB smooth mutant displayed increased propidium iodide staining, but corresponding biofilm CFU data suggest this phenotype is due to cell damage and not increased cell death. The cidB rough anaerobic biofilms showed altered structure relative to wild type (reduced biomass and average thickness) which correlated with decreased CFU counts. Sequencing data revealed that the cidB smooth mutant has a unique "loss of read coverage" of ~78 kb of DNA, corresponding to the genomic island TnSMU2 and genes flanking its 3' end. It is therefore likely that the unique biofilm and competence phenotypes of the cidB smooth mutant are related to its genomic changes in this region.

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.

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.

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.

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.

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.

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.

Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.

This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids.

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.

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.

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.

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.

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.

Translocation of 'Candidatus Liberibacter solanacearum', the Zebra Chip pathogen, in potato and tomato.

Zebra Chip disease is a serious threat to potato production. The pathogen, the phloem-limited bacterium 'Candidatus Liberibacter solanacearum,' is vectored by the potato and tomato psyllid Bactericerca cockerelli to potato and tomato. Patterns of pathogen translocation through phloem in potato and tomato plants were examined to determine whether rate or direction of translocation vary by host species or potato cultivars. Two insects were given a 7-day inoculation access period on a single leaf. Weekly, leaves from upper-, middle-, and lower-tier branches were tested for the presence of 'Ca. L. solanacearum' by polymerase chain reaction (PCR). In tomato and potato, 'Ca. L. solanacearum' was detected 2 to 3 weeks after infestation, most frequently in upper- and middle-tier leaves. In potato, the pathogen was detected in leaves on a second, noninfested stem when the stems remained joined via the tuber. Although rates of pathogen movement were similar among potato cultivars, symptoms developed earlier in more susceptible cultivars. Quantitative PCR indicated that bacterial titers were frequently low in tomato and potato samples (<20 genome units per nanogram of DNA). Results establish that, for improved detection, samples should include newly developing leaves and consider that, under low insect pressure, the pathogen may be undetectable by PCR until 3 weeks after infestation.

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.