[show abstract][hide abstract] ABSTRACT: 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. Using a 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, T3SS inhibitors TMCA and BA, and one T3SS inducer, EHPES, were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the T3SS expression through HrpS-HrpL pathway. Additionally, TMCA altered the T3SS expression through rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) symptom 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 the virulence factor of bacterial pathogens.
Applied and environmental microbiology 06/2013; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Due to potential risks to the environment and human health arising from pathogens/chemical contaminants, novel devices are being developed for rapid and precise detection of those contaminants. Here, we demonstrate highly sensitive and selective field-effect transistor (FET) sensor devices for detection of Escherichia coli (E. coli) bacteria using thermally reduced monolayer graphene oxide (TRMGO) sheets as semiconducting channels. The graphene oxide (GO) sheets are assembled on the aminoethanethiol (AET)-functionalized gold (Au) electrodes through electrostatic interactions with ultrasonic assistance. Anti-Escherichia coli (anti-E. coli) antibodies are used as receptors for selective detection of E. coli cells and integrated on the FET device through covalent bonding with Au nanoparticles on the GO surface. The TRMGO FET device shows great electronic stability and high sensitivity to E. coli cells with a concentration as low as 10 colony-forming units (cfu) per mL. The biosensing platform reported here is promising for large-scale, sensitive, selective, low-cost, and real-time detection of E. coli bacteria.
[show abstract][hide abstract] ABSTRACT: Persistence of Escherichia coli O157:H7 in 32 (16 organically managed and 16 conventionally managed) soils from California (CA) and Arizona (AZ) was investigated. Results showed that the longest survival (ttd, time needed to reach detection limit, 100 CFU g(-1) dry soil) of E. coli O157:H7 was observed in the soils from Salinas Valley, CA and in organically managed soils from AZ. Detrended correspondence analysis revealed that the survival profiles in organically managed soils in Yuma, AZ were different from the ones in conventionally managed soils from the same site. Principal component analysis and stepwise regression analysis showed that E. coli O157:H7 survival in soils was negatively correlated with salinity (EC) (P < 0.001), while positively correlated with assimilable organic carbon (AOC) and total nitrogen (TN) (P < 0.01). Pearson correlation analysis revealed that a greater ttd was associated with a larger δ (time needed for first decimal reduction in E. coli population). EC was negatively correlated and TN was positively correlated (P < 0.05) with δ, suggesting that EC and TN likely have a direct impact on ttd. On the other hand, AOC showed a close correlation with p (the shape parameter) that was not directly related to ttd, indicating that AOC might have an indirect effect in the overall survival of E. coli O157:H7 in soils. Our data showed that AOC and EC significantly affected the survival of E. coli O157:H7 in leafy green producing soils and the development of good agricultural practices (manure/composting/irrigation water source management) in the preharvest environment must be followed to minimize foodborne bacterial contamination on fresh produce.
[show abstract][hide abstract] ABSTRACT: Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.
Annual Review of Phytopathology 06/2012; 50:425-49. · 10.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Assimilable organic carbon (AOC) is commonly used to measure the growth potential of microorganisms in water, but has not yet been investigated for measuring microbial growth potential in soils. In this study, a simple, rapid, and non-growth based assay to determine AOC in soil was developed using a naturally occurring luminous strain Vibrio harveyi BB721 to determine the fraction of low molecular weight organic carbon in soil water extract. Calibration of the assay was achieved by measuring the luminescence intensity of starved V. harveyi BB721 cells in the late exponential phase with a concentration range from 0 to 800 mg l 21 glucose (equivalent to 0–16.0 mg glucose C kg 21 soil) with the detection limit of 10 mg l 21 equivalent to 0.20 mg glucose C kg 21 soil. Results showed that bioluminescence was proportional to the concentration of glucose added to soil. The luminescence intensity of the cells was highly pH dependent and the optimal pH was about 7.0. The average AOC concentration in 32 soils tested was 2.962.2 mg glucose C kg 21 . Our data showed that AOC levels in soil water extracts were significantly correlated (P,0.05) with microbial biomass determined as microbial biomass carbon, indicating that the AOC concentrations determined by the method developed might be a good indicator of soil microbial biomass. Our findings provide a new approach that may be used to determine AOC in environmental samples using a non-growth bioluminescence based assay. Understanding the levels of AOC in soil water extract provides new insights into our ability to estimate the most available carbon pool to bacteria in soil that may be easily assimilated into cells for many metabolic processes and suggest possible the links between AOC, microbial regrowth potential, and microbial biomass in soils. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding:
[show abstract][hide abstract] ABSTRACT: Erwinia chrysanthemi 3937 is a soft-rotting plant pathogen in Enterobacteriaceae. It attacks a wide range of plant host species. Previously, we
identified dozens of E. chrysanthemi 3937 genes induced during plant infection by microarray differential display. Here, we have mutated plant-upregulated and
putatively plant-upregulated genes in E. chrysanthemi 3937 using a transposon insertion method. Of 57 mutants produced, 8 were significantly reduced in maceration in African violet
leaves. These 8 E. chrysanthemi genes are similar to Escherichia coli purU (formyltetrahydrofolate deformylase; ASAP20623) and wcaJ (undecaprenylphosphate glucosephosphotransferase; ASAP18556), Bacillus subtilis dltA (d-alanine-d-alanyl carrier protein ligase; ASAP19406), Pseudomonas syringae PSPTO2912 (ABC transporter, periplasmic glutamine-binding protein; ASAP15639), Pseudomonas aeruginosa pheC (cyclohexadienyl dehydratase; ASAP19773), P. syringae syrE (peptide synthase; ASAP19989), Vibrio vulnificus VV12303 (unknown protein; ASAP18555), and Yersinia pestis speD (S-adenosylmethionine decarboxylase; ASAP20536). In some of the genes, possible roles in virulence could be postulated based
on the functions of their homologues. This work demonstrated that a low proportion of pathogenicity-related genes were among
the plant-upregulated genes of E. chrysanthemi 3937. This study and further dissection of these putative virulence genes should lead to new insights into infection mechanisms
Journal of General Plant Pathology 04/2012; 72(6):360-368. · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 01/2012; 78(8):2888-95. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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-N(9)-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.
PLoS ONE 01/2012; 7(2):e31855. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.
[show abstract][hide abstract] ABSTRACT: 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.
Antimicrobial Agents and Chemotherapy 10/2011; 56(1):36-43. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 06/2011; 77(11):3809-18. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in the model plant rice, possesses a hypersensitive response and pathogenicity (hrp), hrp-conserved (hrc), hrp-associated (hpa) cluster (hrp-hrc-hpa) that encodes a type III secretion system (T3SS) through which T3SS effectors are injected into host cells to cause disease or trigger plant defenses. Mutations in this cluster usually abolish the bacterial ability to cause hypersensitive response in nonhost tobacco and pathogenicity in host rice. In Xanthomonas spp., these genes are generally assumed to be regulated by the key master regulators HrpG and HrpX. However, we present evidence that, apart from HrpG and HrpX, HrpD6 is also involved in regulating the expression of hrp genes. Interestingly, the expression of hpa2, hpa1, hpaB, hrcC, and hrcT is positively controlled by HrpD6. Transcriptional expression assays demonstrated that the expression of the hrcC, hrpD5, hrpE, and hpa3 genes was not completely abolished by hrpG and hrpX mutations. As observed in analysis of their corresponding mutants, HrpG and HrpX exhibit contrasting gene regulation, particularly for hpa2 and hrcT. Other two-component system regulators (Zur, LrpX, ColR/S, and Trh) did not completely inhibit the expression of hrcC, hrpD5, hrpE, and hpa3. Immunoblotting assays showed that the secretion of HrpF, which is an HpaB-independent translocator, is not affected by the mutation in hrpD6. However, the mutation in hrpD6 affects the secretion of an HpaB-dependent TAL effector, AvrXa27. These novel findings suggest that, apart from HrpG and HrpX, HrpD6 plays important roles not only in the regulation of hrp genes but also in the secretion of TAL effectors.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of bacteriology 01/2011; 193(8):2076-7. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Microbiology 01/2011; 2011.
[show abstract][hide abstract] ABSTRACT: 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 T(d) (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 T(d) 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.
PLoS ONE 01/2011; 6(8):e23191. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 11/2010; 77(1):156-62. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.
[show abstract][hide abstract] ABSTRACT: Cyclic diguanylate (c-di-GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c-di-GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c-di-GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c-di-GMP, RpoN and T3SS and the potential role of c-di-GMP in T3SS regulation among a wide range of bacterial pathogens.
[show abstract][hide abstract] ABSTRACT: The type III secretion system (T3SS) is considered one of the major virulence factors in many bacterial pathogens. This report demonstrates that RssB, ClpXP, and RpoS play a role in T3SS regulation of Dickeya dadantii 3937. ClpP is a serine-type protease which associates with the ClpX chaperone to form a functional Clp proteolytic complex for degradation of proteins. With the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor. RpoS positively regulates the expression of the rsmA gene encoding an RNA-binding regulatory protein. By interacting with the hrpL mRNA, RsmA reduces HrpL production and downregulates the T3SS genes in the HrpL regulon. In addition, ClpXP, RssB, and RpoS affect pectinolytic enzyme production in D. dadantii 3937, probably through RsmA. The ClpXP and RssB proteins are essential for bacterial virulence.