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1.  Discovery of Plant Phenolic Compounds That Act as Type III Secretion System Inhibitors or Inducers of the Fire Blight Pathogen, Erwinia amylovora 
Applied and Environmental Microbiology  2013;79(18):5424-5436.
Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.
PMCID: PMC3754148  PMID: 23770912
2.  Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon ‘Ca. Liberibacter asiaticus’ Infection 
PLoS ONE  2013;8(6):e67442.
Citrus huanglongbing (HLB) is a highly destructive disease of citrus presumably caused by ‘Candidatus Liberibacter asiaticus’ (Las), a gram-negative, insect-transmitted, phloem-limited α-proteobacterium. Although almost all citrus plants are susceptible to HLB, reports have shown reduced susceptibility to Las infection in lemon (Citruslimon) plants. The aim of this study is to identify intra-species specific molecular mechanisms associated with Las-induced responses in lemon plants. To achieve this, comparative 2-DE and mass spectrometry, in addition to Inductively Coupled Plasma Spectroscopy (ICPS) analyses, were applied to investigate differences in protein accumulation and the concentrations of cationic elements in leaves of healthy and Las-infected lemon plants. Results showed a differential accumulation of 27 proteins, including an increase in accumulation of starch synthase but decrease in the production of photosynthesis-related proteins in Las-infected lemon plants compared to healthy plants. Furthermore, there was a 6% increase (P > 0.05) in K concentration in leaves of lemon plants upon Las infection, which support results from previous studies and might represent a common response pattern of citrus plants to Las infection. Interestingly, contrary to reports from prior studies, this study showed a general reduction in the production of defense-related pathogen-response proteins but a 128% increase in Zn concentration in lemon plants in response to Las infection. Taken together, this study sheds light on general and intra-species specific responses associated with the response of citrus plants to Las.
PMCID: PMC3686881  PMID: 23922636
3.  Effects of 14-Alpha-Lipoyl Andrographolide on Quorum Sensing in Pseudomonas aeruginosa 
Antimicrobial Agents and Chemotherapy  2012;56(12):6088-6094.
In Pseudomonas aeruginosa, the quorum-sensing (QS) system is closely related to biofilm formation. We previously demonstrated that 14-alpha-lipoyl andrographolide (AL-1) has synergistic effects on antibiofilm and antivirulence factors (pyocyanin and exopolysaccharide) of P. aeruginosa when combined with conventional antibiotics, while it has little inhibitory effect on its growth. However, its molecular mechanism remains elusive. Here we investigated the effect of AL-1 on QS systems, especially the Las and Rhl systems. This investigation showed that AL-1 can inhibit LasR–3-oxo-C12-homoserine lactone (HSL) interactions and repress the transcriptional level of QS-regulated genes. Reverse transcription (RT)-PCR data showed that AL-1 significantly reduced the expression levels of lasR, lasI, rhlR, and rhlI in a dose-dependent manner. AL-1 not only decreased the expression level of Psl, which is positively regulated by the Las system, but also increased the level of secretion of ExoS, which is negatively regulated by the Rhl system, indicating that AL-1 has multiple effects on both the Las and Rhl systems. It is no wonder that AL-1 showed synergistic effects with other antimicrobial agents in the treatment of P. aeruginosa infections.
PMCID: PMC3497202  PMID: 22802260
4.  SlyA Regulates Type III Secretion System (T3SS) Genes in Parallel with the T3SS Master Regulator HrpL in Dickeya dadantii 3937 
The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.
PMCID: PMC3318817  PMID: 22267675
5.  Presence of Hydrogen Peroxide, a Source of Hydroxyl Radicals, in Acid Electrolyzed Water 
PLoS ONE  2012;7(9):e46392.
Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.
Methodology/Principal Findings
The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.
It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.
PMCID: PMC3460889  PMID: 23029505
6.  Erwinia amylovora CRISPR Elements Provide New Tools for Evaluating Strain Diversity and for Microbial Source Tracking 
PLoS ONE  2012;7(7):e41706.
Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.
PMCID: PMC3409226  PMID: 22860008
7.  Defence Signalling Triggered by Flg22 and Harpin Is Integrated into a Different Stilbene Output in Vitis Cells 
PLoS ONE  2012;7(7):e40446.
Plants can activate defence to pathogen attack by two layers of innate immunity: basal immunity triggered by pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) linked with programmed cell death. Flg22 and Harpin are evolutionary distinct bacterial PAMPs. We have previously shown that Harpin triggers hypersensitive cell death mimicking ETI in Vitis rupestris, but not in the Vitis vinifera cultivar ‘Pinot Noir’. In contrast, the bacterial PAMP flg22 activating PTI does not trigger cell death. To get insight into the defence signalling triggered by flg22 and Harpin, we compared cellular responses upon flg22 and Harpin treatment in the two Vitis cell lines. We found that extracellular alkalinisation was blocked by inhibition of calcium influx, and modulated by pharmacological manipulation of the cytoskeleton and mitogen-activated protein kinase activity with quantitative differences between cell lines and type of PAMPs. In addition, an oxidative burst was detected that was much stronger and faster in response to Harpin as compared to flg22. In V. rupestris, both flg22 and Harpin induced transcripts of defence-related genes including stilbene synthase, microtubule disintegration and actin bundling in a similar way, whereas they differed in V. vinifera cv. ‘Pinot Noir’. In contrast to Harpin, flg22 failed to trigger significant levels of the stilbene trans-resveratrol, and did not induce hypersensitive cell death even in the highly responsive V. rupestris. We discuss these data in a model, where flg22- and Harpin-triggered defence shares a part of early signal components, but differs in perception, oxidative burst, and integration into a qualitatively different stilbene output, such that for flg22 a basal PTI is elicited in both cell lines, while Harpin induces cell death mimicking an ETI-like pattern of defence.
PMCID: PMC3391249  PMID: 22792328
8.  Derivatives of Plant Phenolic Compound Affect the Type III Secretion System of Pseudomonas aeruginosa via a GacS-GacA Two-Component Signal Transduction System 
Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.
PMCID: PMC3256035  PMID: 21968370
9.  Fructose-Bisphophate Aldolase Exhibits Functional Roles between Carbon Metabolism and the hrp System in Rice Pathogen Xanthomonas oryzae pv. oryzicola 
PLoS ONE  2012;7(2):e31855.
Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.
PMCID: PMC3285194  PMID: 22384086
10.  Non-Host Defense Response in a Novel Arabidopsis-Xanthomonas citri subsp. citri Pathosystem 
PLoS ONE  2012;7(1):e31130.
Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gene transfer technology.
PMCID: PMC3267768  PMID: 22299054
11.  Hpa2 Required by HrpF To Translocate Xanthomonas oryzae Transcriptional Activator-Like Effectors into Rice for Pathogenicity▿† 
Applied and Environmental Microbiology  2011;77(11):3809-3818.
Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2 and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX. In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.
PMCID: PMC3127602  PMID: 21478322
12.  Genome Sequence of the Plant-Pathogenic Bacterium Dickeya dadantii 3937▿ 
Journal of Bacteriology  2011;193(8):2076-2077.
Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.
PMCID: PMC3133054  PMID: 21217001
13.  Persistence of Escherichia coli O157:H7 and Its Mutants in Soils 
PLoS ONE  2011;6(8):e23191.
The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx1, stx2, stx1–2, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx1, stx2, stx1–2, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated Td (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that Td was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx1−stx2−) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples.
PMCID: PMC3149627  PMID: 21826238
14.  Commensal Effect of Pectate Lyases Secreted from Dickeya dadantii on Proliferation of Escherichia coli O157:H7 EDL933 on Lettuce Leaves ▿  
The outbreaks caused by enterohemorrhagic Escherichia coli O157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms of Dickeya dadantii 3937, a broad-host-range phytopathogen, on the proliferation of the human pathogen E. coli O157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 with D. dadantii 3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-type D. dadantii 3937. Increased expression of pelD (encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS) in planta. These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases in D. dadantii 3937.
PMCID: PMC3019694  PMID: 21075884
15.  Ralstonia solanacearum Extracellular Polysaccharide Is a Specific Elicitor of Defense Responses in Wilt-Resistant Tomato Plants 
PLoS ONE  2011;6(1):e15853.
Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps− mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps− mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps− mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.
PMCID: PMC3017055  PMID: 21253019
16.  Quantification of Persistence of Escherichia coli O157:H7 in Contrasting Soils 
Persistence of Escherichia coli (E. coli) O157:H7 in the environment is a major concern to vegetable and fruit growers where farms and livestock production are in close proximity. The objectives were to determine the effects of preplant fumigation treatment on the survival of E. coli O157:H7 in two soils and the effects of indigenous bacterial populations on the survival of this pathogen. Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in two contrasting soils after fumigation with methyl bromide (MeBr) and methyl iodide (MeI). Ten days after fumigation, E. coli O157:H7 counts were significantly lower (P = .0001) in fumigated soils than in the non-fumigated. Direct comparison between MeBr and MeI within each soil indicated that these two fumigants showed similar impacts on E. coli O157:H7 survival. Microbial species diversity as determined by DGGE was significantly higher in clay soil than sandy soil and this resulted in higher initial decline in population in clay soil than in sandy soil. This study shows that if soil is contaminated with E. coli O157:H7, fumigation alone may not eliminate the pathogen, but may cause decrease in microbial diversity which may enhance the survival of the pathogen.
PMCID: PMC2943103  PMID: 20871863
17.  Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk 
PLoS ONE  2010;5(6):e11047.
Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.
Methods and Findings
Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5–0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.
We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.
PMCID: PMC2883556  PMID: 20548779
18.  Light-Dependent Electrogenic Activity of Cyanobacteria 
PLoS ONE  2010;5(5):e10821.
Cyanobacteria account for 20–30% of Earth's primary photosynthetic productivity and convert solar energy into biomass-stored chemical energy at the rate of ∼450 TW [1]. These single-cell microorganisms are resilient predecessors of all higher oxygenic phototrophs and can be found in self-sustaining, nitrogen-fixing communities the world over, from Antarctic glaciers to the Sahara desert [2].
Methodology/Principal Findings
Here we show that diverse genera of cyanobacteria including biofilm-forming and pelagic strains have a conserved light-dependent electrogenic activity, i.e. the ability to transfer electrons to their surroundings in response to illumination. Naturally-growing biofilm-forming photosynthetic consortia also displayed light-dependent electrogenic activity, demonstrating that this phenomenon is not limited to individual cultures. Treatment with site-specific inhibitors revealed the electrons originate at the photosynthetic electron transfer chain (P-ETC). Moreover, electrogenic activity was observed upon illumination only with blue or red but not green light confirming that P-ETC is the source of electrons. The yield of electrons harvested by extracellular electron acceptor to photons available for photosynthesis ranged from 0.05% to 0.3%, although the efficiency of electron harvesting likely varies depending on terminal electron acceptor.
The current study illustrates that cyanobacterial electrogenic activity is an important microbiological conduit of solar energy into the biosphere. The mechanism responsible for electrogenic activity in cyanobacteria appears to be fundamentally different from the one exploited in previously discovered electrogenic bacteria, such as Geobacter, where electrons are derived from oxidation of organic compounds and transported via a respiratory electron transfer chain (R-ETC) [3], [4]. The electrogenic pathway of cyanobacteria might be exploited to develop light-sensitive devices or future technologies that convert solar energy into limited amounts of electricity in a self-sustainable, CO2-free manner.
PMCID: PMC2876029  PMID: 20520829
19.  Characterization of the Interaction and Cross-Regulation of Three Mycobacterium tuberculosis RelBE Modules 
PLoS ONE  2010;5(5):e10672.
RelBE represents a typical bacterial toxin-antitoxin (TA) system. Mycobacterium tuberculosis H37Rv, the pathogen responsible for human tuberculosis, contains three RelBE-like modules, RelBE, RelFG, and RelJK, which are at least partly expressed in human macrophages during infection. RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated. In the present study, we characterized the interaction and cross-regulation of these Rel toxin-antitoxin modules from this unique pathogen. The physical interactions between the three pairs of RelBE proteins were confirmed and the DNA-binding domain recognized by three RelBE-like pairs and domain structure characteristics were described. The three RelE-like proteins physically interacted with the same RelB-like protein, and could conditionally regulate its binding with promoter DNA. The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth. The relB antitoxin gene could replace relF in cross-neutralizing the relG toxin gene. Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK. This is the first report of interactions between different pairs of RelBE modules of M. tuberculosis.
PMCID: PMC2871789  PMID: 20498855
20.  A Boolean Model of the Pseudomonas syringae hrp Regulon Predicts a Tightly Regulated System 
PLoS ONE  2010;5(2):e9101.
The Type III secretion system (TTSS) is a protein secretion machinery used by certain gram-negative bacterial pathogens of plants and animals to deliver effector molecules to the host and is at the core of the ability to cause disease. Extensive molecular and biochemical study has revealed the components and their interactions within this system but reductive approaches do not consider the dynamical properties of the system as a whole. In order to gain a better understanding of these dynamical behaviours and to create a basis for the refinement of the experimentally derived knowledge we created a Boolean model of the regulatory interactions within the hrp regulon of Pseudomonas syringae pathovar tomato strain DC3000 Pseudomonas syringae. We compared simulations of the model with experimental data and found them to be largely in accordance, though the hrpV node shows some differences in state changes to that expected. Our simulations also revealed interesting dynamical properties not previously predicted. The model predicts that the hrp regulon is a biologically stable two-state system, with each of the stable states being strongly attractive, a feature indicative of selection for a tightly regulated and responsive system. The model predicts that the state of the GacS/GacA node confers control, a prediction that is consistent with experimental observations that the protein has a role as master regulator. Simulated gene “knock out” experiments with the model predict that HrpL is a central information processing point within the network.
PMCID: PMC2821412  PMID: 20169167
21.  A Feeding Induced Switch from a Variable to a Homogenous State of the Earthworm Gut Microbiota within a Host Population 
PLoS ONE  2009;4(10):e7528.
The distribution pattern of the earthworm gut microbiota at the host population level is of fundamental importance to understand host-microbiota interactions. Our current understanding of these interactions is very limited. Since feeding represents a main perturbation of the gut microbiota, we determined the effect of a single dose of feed on the microbiota associated with an earthworm population in a simulated microenvironment.
Earthworms were sampled 0, 1 and 7 days after feeding. We determined the overall composition of the earthworm-associated microbiota by 16S rRNA gene cloning and sequencing. Based on the 16S rRNA gene data we constructed quantitative PCR's (Q-PCR) for the seven most dominating bacterial groups.
Principal Findings
Q-PCR revealed low density and highly variable microbiota among the earthworms before feeding, while a high-density homologous microbiota resulted from feeding. We found that the microbiota 1 day after feeding was more equal to the microbiota after 7 days than before feeding. Furthermore, we found that the gut microbiota was very distinct from that of the bedding and the feed.
The homogenous population response represents fundamental new knowledge about earthworm gut associated bacteria.
PMCID: PMC2759579  PMID: 19841743
22.  Extracellular and Intracellular Polyphenol Oxidases Cause Opposite Effects on Sensitivity of Streptomyces to Phenolics: A Case of Double-Edged Sword 
PLoS ONE  2009;4(10):e7462.
Many but not all species of Streptomyces species harbour a bicistronic melC operon, in which melC2 encodes an extracellular tyrosinase (a polyphenol oxidase) and melC1 encodes a helper protein. On the other hand, a melC-homologous operon (melD) is present in all sequenced Streptomyces chromosomes and could be isolated by PCR from six other species tested. Bioinformatic analysis showed that melC and melD have divergently evolved toward different functions. MelD2, unlike tyrosinase (MelC2), is not secreted, and has a narrower substrate spectrum. Deletion of melD caused an increased sensitivity to several phenolics that are substrates of MelD2. Intracellularly, MelD2 presumably oxidizes the phenolics, thus bypassing spontaneous copper-dependent oxidation that generates DNA-damaging reactive oxygen species. Surprisingly, melC+ strains were more sensitive rather than less sensitive to phenolics than melC− strains. This appeared to be due to conversion of the phenolics by MelC2 to more hydrophobic and membrane-permeable quinones. We propose that the conserved melD operon is involved in defense against phenolics produced by plants, and the sporadically present melC operon probably plays an aggressive role in converting the phenolics to the more permeable quinones, thus fending off less tolerant competing microbes (lacking melD) in the phenolic-rich rhizosphere.
PMCID: PMC2758597  PMID: 19826489
23.  The Plant Phenolic Compound p-Coumaric Acid Represses Gene Expression in the Dickeya dadantii Type III Secretion System▿ †  
The type III secretion system (T3SS) is a major virulence factor in many gram-negative bacterial pathogens. This secretion system translocates effectors directly into the cytosol of eukaryotic host cells, where the effector proteins facilitate bacterial pathogenesis by interfering with host cell signal transduction and other cellular processes. Plants defend themselves against bacterial pathogens by recognizing either the type 3 effectors or their actions and initiating a cascade of defense responses that often results in programmed cell death of the plant cell being attacked. Here we show that a plant phenolic compound, p-coumaric acid (PCA), represses the expression of T3SS genes of the plant pathogen Dickeya dadantii, suggesting that plants can also defend against bacterial pathogens by manipulating the expression of the T3SS. PCA repressed the expression of T3SS regulatory genes through the HrpX/Y two-component system, a core regulator of the T3SS, rather than through the global regulator GacS/A, which indirectly regulates the T3SS. A further analysis of several PCA analogs suggests that the para positioning of the hydroxyl group in the phenyl ring and the double bond of PCA may be important for its biological activity.
PMCID: PMC2648153  PMID: 19114532
24.  Competition Triggers Plasmid-Mediated Enhancement of Substrate Utilisation in Pseudomonas putida 
PLoS ONE  2009;4(6):e6065.
Competition between species plays a central role in the activity and structure of communities. Stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. Outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. Survival and proliferation of a particular species is governed by its competitive fitness. Therefore, bacteria must be able to continuously sense their immediate environs for presence of competitors and prevailing conditions. Here we present results of our investigations on a novel competition sensing mechanism in the rhizosphere-inhabiting Pseudomonas putida KT2440, harbouring gfpmut3b-modified KanR TOL plasmid. We monitored benzyl alcohol (BA) degradation rate, along with GFP expression profiling in mono species and dual species cultures. Interestingly, enhanced plasmid expression (monitored using GFP expression) and consequent BA degradation were observed in dual species consortia, irrespective of whether the competitor was a BA degrader (Pseudomonas aeruginosa) or a non-degrader (E. coli). Attempts at elucidation of the mechanistic aspects of induction indicated the role of physical interaction, but not of any diffusible compounds emanating from the competitors. This contention is supported by the observation that greater induction took place in presence of increasing number of competitors. Inert microspheres mimicking competitor cell size and concentration did not elicit any significant induction, further suggesting the role of physical cell-cell interaction. Furthermore, it was also established that cell wall compromised competitor had minimal induction capability. We conclude that P. putida harbouring pWW0 experience a competitive stress when grown as dual-species consortium, irrespective of the counterpart being BA degrader or not. The immediate effect of this stress is a marked increase in expression of TOL, leading to rapid utilization of the available carbon source and massive increase in its population density. The plausible mechanisms behind the phenomenon are hypothesised and practical implications are indicated and discussed.
PMCID: PMC2698150  PMID: 19557171
25.  The Use of Flagella and Motility for Plant Colonization and Fitness by Different Strains of the Foodborne Pathogen Listeria monocytogenes 
PLoS ONE  2009;4(4):e5142.
The role of flagella and motility in the attachment of the foodborne pathogen Listeria monocytogenes to various surfaces is mixed with some systems requiring flagella for an interaction and others needing only motility for cells to get to the surface. In nature this bacterium is a saprophyte and contaminated produce is an avenue for infection. Previous studies have documented the ability of this organism to attach to and colonize plant tissue. Motility mutants were generated in three wild type strains of L. monocytogenes by deleting either flaA, the gene encoding flagellin, or motAB, genes encoding part of the flagellar motor, and tested for both the ability to colonize sprouts and for the fitness of that colonization. The motAB mutants were not affected in the colonization of alfalfa, radish, and broccoli sprouts; however, some of the flaA mutants showed reduced colonization ability. The best colonizing wild type strain was reduced in colonization on all three sprout types as a result of a flaA deletion. A mutant in another background was only affected on alfalfa. The third, a poor alfalfa colonizer was not affected in colonization ability by any of the deletions. Fitness of colonization was measured in experiments of competition between mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants of the three strain backgrounds were impaired in fitness of colonization of alfalfa and radish sprouts, and one strain background showed reduced fitness of both mutant types on broccoli sprouts. Together these data indicate a role for flagella for some strains to physically colonize some plants, while the fitness of that colonization is positively affected by motility in almost all cases.
PMCID: PMC2664462  PMID: 19357783

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