Progress 10/01/06 to 09/30/12
Outputs
OUTPUTS: Three non-flagellar (PAI1, PAI2 and PAI3) and two flagellar (Fla1 and Fla2) type III secretion systems (T3SSs) exist in the genome of Erwinia amylovora strains. Phylogenetic trees constructed using conserved protein indicated that the three non-flagellar T3SSs belong to the Hrp1 and Inv/Spa groups, respectively. Concatenated phylogenetic tree constructed from 14 conserved flagellar proteins suggested that both flagellar systems were originated from enterobacterial branch of the gamma-proteobacteria. We have adopted an easy and fast mutagenesis method and have generated deletion mutants for three T3SS PAIs, type II secretion system (T2SS) and the amylovoran (ams) operon, including delta T3SS (33.35kb deletion), delta T2SS (12.5 kb deletion), delta AMS (15.8kb deletion), delta EPI1 (23.45kb deletion), delta EPI2 (25.7kb deletion), delta EPI1/EPI2 double mutant, and Fla2, flhDC1, fliA1 etc. We have demonstrated that the PAI2 and PAI3 are not involved in virulence. Gene expression analysis revealed that the regulatory genes of the PAI2 and PAI3 were differentially expressed in rich medium at high temperature, and regulated by PhoPQ system. In addition, deletion of the whole Fla2 system did not affect swarming motility and flagellar formation, and no gene expression was detected for flhDC2 gene; whereas deletion of a single flhDC1 or fliA1 gene in Fla1 system completely abolished flagellar formation and swarming motility. Using genomic and bioinformatics techniques, we have identified about 45 two-component signal transduction (TCSTs) genes in the genome of E. amylovora including 20 sensor kinases (HK), 19 response regulators (RR), one hybrid, four sensory box proteins and one serine-threonine kinase. We have generated a total of 59 TCST mutants and screened for their contribution to virulence and other phenotypes including amylovoran biosynthesis and swarming motility. Four groups of mutants were identified exhibiting varying levels of amylovoran production in vitro. Three different swarming phenotypes were observed. Classification of TCSTs based on domain structure and domain combinations was performed. Multiple sequence alignment has classified the HKs and RRs of E. amylovora into four groups or families, respectively. We have conducted a comparative genomic analysis of TCSTs in 53 genomes of 16 enterobacterial species. Comparative genomic analysis revealed that enterobacteria contain eight pairs of core TCSTs. Phylogenetic trees showed that most TCST protein trees in the Enterobacteriaceae or in species of the gamma- Proteobacteria agreed well with that of the corresponding 16S rRNA gene. Several core TCSTs such as EnvZ were quite ancient and universal based on phylogenomic analysis of protein structures. In addition, we also systematically characterized TCST systems including Rcs, PhoPQ, and PmrAB systems. Furthermore, we have systematically characterized various environmental conditions such as pH, temperature, carbon and nitrogen sources in regulating amylovoran production in vitro. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
One goal is to define the evolution origin of both the flagellar and non-flagellar type III secretion systems in E. amylovora, and to determine whether these systems are functional in normal growth conditions or during infection of host plants. Our results indicated that both flagellar systems may be originated from enterobacterial branch of the gamma-proteobacteria, and PAI2, PAI3 and Fla2 may be acquired from a similar enterobacterial source through horizontal gene transfer. These results also suggest that PAI2, PAI3 and Fla2 might function in other hosts of E. amylovora, other than the plant hosts. A type III secretion system is a critical component of virulence for many gram negative plant pathogenic bacteria and has been the target for disease control. The mutants we generated will provide us with basic tools to study gene function and signal transduction in E. amylovora. For the first time, we have demonstrated that T3SS genes in plant pathogenic bacteria are expressed in rich medium at high temperature. These results suggest that bacteria with different lifestyles may have developed different regulatory systems in response to environmental cues to regulate gene expression and survival. The next goal is to systematically identify and characterize TCSTs in E. amylovora. Comparative genomic studies indicate that core TCSTs are relatively conserved, and suggest that these enterobacteria may have maintained their ancient core TCSTs and might acquire specific new TCSTs for their survival in different environments or hosts, or may have evolved new functionalities of the core TCSTs for adaptation to different ecological niches. This is, for the first time, we provide evidence that the core set of TCSTs exist before bacterial speciation. The significance to characterizing TCSTs is to define what roles TCSTs play during bacterial pathogenesis and to identify new virulence factors or regulators. TCSTs are potential targets for antimicrobial therapy and studies of TCSTs may lead to the development of improved disease management strategies. Our results clearly indicate that TCSTs in E. amylovora play a major role in its pathogenesis and survival and suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora. The new knowledge will help us to better understand how E. amylovora interacts with its host and environmental conditions, and will lead to the development of improved disease management strategies. The final goal is to characterize how various environmental conditions such as pH, temperature, and carbon sources affect amylovoran production in vitro. Our results indicate that regulation of amylovoran production is very complicated in E. amylovora and suggest the presence of regulatory networks governing the expression of amylovoran biosynthesis genes. These regulatory genes may respond to different environmental conditions.
Publications
- Triplett, L., Zhao, Y.F. and Sundin, G. W. 2006. Genetic differences among blight-causing Erwinia species with differing host specificities identified by suppression subtractive hybridization. Appl. Environ. Microbiol. 72:7359-7364.
- Zhao, Y.F. and Sundin, G.W. 2007. Preliminary molecular characterization of two novel Erwinia amylovora type III secretion pathogenicity islands (T3SS-PAIs). Phytopathology 97:S129.
- Nakka, S. and Zhao, Y.F. 2008. Molecular characterization of the PhoP/PhoQ two-component signal transduction system in Erwinia amylovora. Phytopathology 98:S111.
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2008. Biofilm formation in Erwinia amylovora: Implications in pathogenicity. Acta Horticulturae. 793:67-71.
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2007. Biofilm formation in Erwinia amylovora: Implications in pathogenicity. Phytopathology 97:S59.
- Zhao, Y.F., Wang, D., Nakka, S., Sundin, G.W. and Korban, S.S. 2009. A systems-level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics 10:245.
- Wang, D., Korban, S.S. and Zhao, Y.F. 2009. The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora. Mol. Plant Pathol. 10:277-290.
- Zhao, Y.F., Sundin, G.W. and Wang, D.P. 2009. Construction and analysis of pathogenicity island deletion mutants in Erwinia amylovora. Can. J. Microbiol. 55:457-464.
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2009. The contribution of the exopolysaccharide amylovoran and levan to biofilm formation: Implication in pathogenicity. Phytopathology 99:1237-1244.
- Zhao, Y.F. 2007. Will genomics help find the silver bullet for fighting the Anthrax of Fruit Trees Biotechnology News, page 1-3, 2007. The Roy J. Carver Biotechnology Center of UIUC.
- Zhao, Y.F. and Sundin, G.W. 2008. A simple genetic tool for studying gene function and signal transduction in Erwinia amylovora. Acta Horticulturae. 793:171-178.
- Zhao, Y.F., Wang, D. and Nakka, S. 2008. Two-component signal transduction systems play a major role in Erwinia amylovora pathogenesis and survival. Phytopathology 98:S180.
- Wang, D. and Zhao, Y.F. 2008. The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora. Phytopathology 98:S165.
- Berry, M., McGhee, G.C., Zhao, Y.F. and Sundin, G.W. 2009. Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora. FEMS Microbiol. Lett. 291:80-87.
- Wang, D. and Zhao, Y.F. 2009. Molecular signature of Erwinia amylovora virulence. Phytopathology 99:S137.
- Nakka, S., Qi, M. and Zhao, Y.F. 2010. The PmrAB system in Erwinia amylovora renders the pathogen more susceptible to polymyxin B and more resistant to excess iron. Res. Microbiol. 161:153-157.
- Nakka, S., Qi, M. and Zhao, Y.F. 2010. The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems. Microbiol Res. 165:665-673.
- Anocon, V., Wang, D.P. and Zhao, Y.F. 2012. Dynamics and environmental regulation of virulence gene expression in Erwinia amylovora. Phytopahtology 102:S4.5.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Genome sequencing reveals that three non-flagellar (PAI1, PAI2 and PAI3) and two flagellar (Fla1 and Fla2) type III secretion systems (T3SSs) exist in the genome of Erwinia amylovora strains. Phylogenetic trees constructed using conserved HrcV or InvA protein indicated that the three non-flagellar T3SSs belong to the Hrp1 and Inv/Spa groups, respectively. Concatenated phylogenetic tree constructed from 14 conserved flagellar proteins suggested that both flagellar systems were originated from enterobacterial branch of the gamma-proteobacteria. Interestingly, among these phylogenetic trees, one clade each containing non-flagellar (PAI2 and PAI3) and the tightly-clustered flagellar (Fla2) T3SSs, respectively, were both clustered together with those from Sodalis glossinidius. We have previously demonstrated that the PAI2 and PAI3 are not involved in virulence on host plants. Gene expression analysis revealed that the regulatory genes of the PAI2 and PAI3 were differentially expressed in rich medium at high temperature. In addition, our results showed that deletion of the whole Fla2 system did not affect swarming motility and flagellar formation, and no gene expression was detected for flhDC2 gene; whereas deletion of a single flhDC1 or fliA1 gene in the Fla1 system completely abolished flagellar formation and swarming motility. The requirement of the exopolysaccharide amylovoran for Erwinia amylovora pathogenesis is well documented. We have systematically characterized various environmental conditions such as pH, temperature, carbon and nitrogen sources in regulating amylovoran production in vitro. Our results showed that E. amylovora strains produced more amylovoran at high temperature or pH (7.0) than that at low temperature or acidic pH (5.5). PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our first goal is to define the evolution origin of both the flagellar and non-flagellar type III secretion systems in E. amylovora and related species, and to determine whether these systems are functional in normal growth conditions or during infection of host plants. Our results indicated that both flagellar systems may be originated from the enterobacterial branch of the gamma-proteobacteria, and PAI2, PAI3 and Fla2 may be acquired from a similar enterobacterial source through horizontal gene transfer. Our results also suggest that PAI2, PAI3 and Fla2 might function in hosts of E. amylovora other than the plant hosts. Our second goal is to characterize how various environmental conditions such as pH, temperature, and carbon sources affect amylovoran production in vitro. Our results indicate that regulation of amylovoran production is very complicated in E. amylovora and suggest the presence of regulatory networks governing the expression of amylovoran biosynthesis genes. These regulatory genes may respond to different environmental conditions.
Publications
- Zhao, Y.F., Qi, M. and Wang, D. 2011. Evolution and function of flagellar and non-flagellar type III secretion systems in Erwinia amylovora. Acta Hortic. 896:177-184.
- Wang, D., Korban, S.S., Sundin, G.W., Clough, S., Toth, I. and Zhao, Y.F. 2011. Regulatory genes and environmental regulation of amylovoran biosynthesis in Erwinia amylovora. Acta Hortic. 896:195-202.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The PhoPQ system confers resistance to antimicrobial peptides (AMPs): The PhoPQ system is a pleiotropic TCST that controls many pathogenic properties in several mammalian and plant pathogens. Our results showed that phoPQ mutants were more resistant to strong acidic conditions (pH 4.5 or 5) than that of the wild-type (WT) strain, suggesting that this system in E. amylovora may negatively regulate acid resistance gene expression. In addition, survival of PhoPQ mutants was about 10-fold lower than that of WT when treated with cecropin A at pH 5.5, suggesting that the PhoPQ system renders the pathogen more resistant to cecropin A. The PhoPQ system negatively regulated gene expression of two novel type III secretion systems (T3SS) in E. amylovora. These results are in contrast to those reported for the PhoPQ system in Salmonella and Xanthomonas, where it positively regulates type III secretion system and acid resistance. The PmrAB system confers susceptibility to polymyxin B: The PmrAB system responds to extracellular iron and acidic pH. Our results showed that the survival rate of the pmrAB mutants was much higher than that of WT when treated with polymyxin B. However, pmrAB mutants were more sensitive to extracellular iron than WT strain. These results demonstrated that the PmrAB system in E. amylovora renders the pathogen more susceptible to polymixin B, but more resistance to excess iron. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the first time, we have demonstrated that T3SS genes in plant pathogenic bacteria are expressed in rich medium at high temperature (28C). Our results clearly demonstrate that the PhoPQ system functions differently in regulating genes responsible for acid resistance and type III secretion. These results suggest that bacteria with different lifestyles may have developed different regulatory systems in response to environmental cues to regulate gene expression and survival. Contrary to other bacterial pathogens, we found that PmrAB renders the pathogen less resistance to polymyxin B. Though the E. amylovora PmrAB system shares similarity with their counterparts in Salmonella in resistance to high levels of iron, our results demonstrate that this system functions differently in terms of regulating genes responsible for resistance to AMPs.
Publications
- Zhao, Y.F., Qi, M. and Wang, D. 2010. Evolution and function of flagella and non-flagella type III secretion systems in Erwinia amylovora. 12th International Workshop on Fire Blight, August 16-20, Warsaw, Poland.
- Nakka, S., Qi, M. and Zhao, Y.F. 2010. The PmrAB system in Erwinia amylovora renders the pathogen more susceptible to polymyxin B and more resistant to excess iron. Res. Microbiol. 161:153-157.
- Nakka, S., Qi, M. and Zhao, Y.F. 2010. The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems. Microbiol Res. 165:665-673.
- Wang, D. and Zhao, Y.F. 2009. Molecular signature of Erwinia amylovora virulence. Phytopathology 99:S137.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Classification of HKs and RRs based on domain structure and domain combinations was performed. Multiple sequence alignment has classified the HKs of E. amylovora into four groups. We, for the first time, have classified HrpX as a type III HK. Similarly, 20 RRs belong to four major families of RRs found in prokaryotes, which account for about 64% of all RRs identified. Analysis of the domain architecture of E. amylovora HKs has revealed that all HKs in E. amylovora, except for HrpX and NtrB, contain one to three hydrophobic transmembrane (TM) helices within the N-terminal sensor region. This indicates that these HKs are periplasmic sensors. HrpX and NtrB contain PAS domain (initially found in PER, ARNT, and SIM proteins) within the N-terminal sensor region, indicating that HrpX and NtrB are soluble cytoplasmic proteins, and may sense intracellular signals. Phenotypes of the deletion mutant strains were evaluated by measuring amylovoran biosynthesis in vitro. In this screening, four groups of mutants were identified exhibiting varying levels of amylovoran production in vitro. Group I: three mutants showed no amylovoran production; Group II: six mutants showed a dramatic increase in amylovoran production, about 20-fold, compared to that of the WT; Group III mutants showed increased production of amylovoran, from 2- to 8-fold, compared to that of the WT. Group IV mutants did not show much difference in amylovoran production as compared to that of WT. Our study suggests that the pathogen has developed a system to control this major pathogenicity factor. Phenotypes of the deletion mutant strains were further evaluated using a swarming plate assay. Swarming represents a complex lifestyle adaptation in response to medium conditions rather than merely a form of locomotion. WT cells can swarm via the combined effects of flagellar motility, chemotaxis, and growth, thus creating a circular colony. Defects in cell motility, chemotaxis or growth can produce alterations in swarming size or density. Three different swarming phenotypes were observed. Two mutants exhibited substantially larger and with lower density swarms as compared to the WT. In addition, three mutants showed dramatic reduction in swarm size, but with an increase in density. All five mutants exhibited circular swarming. A third group of mutants showed smaller swarms (less dramatic), and colonies exhibited an irregular circular pattern. As negative controls, all flagella-deficient mutants (flhDC and fliA) were non- motile. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Objective Four of this Hatch project was eliminated and will be conducted in a USDA-AFRI funded project "Transcriptome of Erwinia amylovora signal transduction networks". The reason is that Objective Four partially overlapped with Objective One of the new project.
Impacts
The significance is to define what roles TCSTs play during bacterial pathogenesis and to identify new virulence factors or regulators. TCSTs are potential targets for antimicrobial therapy and studies of TCSTs may lead to the development of improved disease management strategies. Conclusions: Our results demonstrated that TCSTs in E. amylovora played major roles in virulence and in regulating amylovoran biosynthesis and swarming motility.
Publications
- Zhao, Y.F., Wang, D., Nakka, S., Sundin, G.W. and Korban, S.S. 2009. A systems-level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics 10:245.
- Wang, D., Korban, S.S. and Zhao, Y.F. 2009. The Rcs system is essential for pathogenicity in Erwinia amylovora. Mol. Plant Pathol. 10:277-290.
- Zhao, Y.F., Sundin, G.W. and Wang, D.P. 2009. Construction and analysis of pathogenicity island deletion mutants in Erwinia amylovora. Can. J. Microbiol. 55:457-464.
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2009. The contribution of the exopolysaccharide amylovoran and levan to biofilm formation: Implication in pathogenicity. Phytopathology 99:1237-1244.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The two-component signal transduction system (TCST), consisting of a histidine kinase (HK) and a response regulator (RR), represents a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions and in bacterial pathogenesis. In this fiscal year, we screened all 59 TCST mutants we generated for their contribution to virulence and other phenotypes including amylovoran biosynthesis and swarming motility. In addition, we also systematically characterized two TCST systems including the Rcs phosphorelay system and the PhoPQ system. The Rcs phosphorelay system (RcsCDB) is a modified two-component signal transduction system (TCST) found exclusively in Enterobacteriaceae. Our results showed that rcsB, rcsC, rcsD, and rcsBD mutants were non-pathogenic on immature pear fruit. Bacterial growth of these mutants was also greatly reduced compared to that of the wild type (WT) strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production; whereas, the rcsC mutant exhibited higher amylovoran production than that of WT. Consistent with amylovoran production, gene expression of the amylovoran biosynthetic gene amsG, using GFP as a reporter, was not detectable in rcsB, rcsD, and rcsBD mutants both in vitro and in vivo. Expression of amsG in vitro was higher in the rcsC mutant than that in WT, while, its expression in vivo was higher in WT than that in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than WT, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. The PhoP/PhoQ system is a pleiotropic two-component signal transduction system that controls many pathogenic properties in several animal and plant pathogens, and it is a master regulator of virulence genes in Salmonella. Three different cues have been proposed to activate the PhoP/PhoQ system: a mild acidic pH, antimicrobial peptides (AMPs), and low Mg2+. The role of the PhoP/PhoQ system in Erwinia amylovora pathogenesis is unknown. Our results showed that, while phoP, phoQ, and phoPQ double mutants were pathogenic on immature pear fruit, these mutants were more resistant to strong acidic conditions than the WT strain. Growth of the mutants was similar to WT at pH 5.5 and 7 at low Mg2+ concentration; however, growth of the mutants at pH 4.5 and 5 was greatly increased. At 24 hours, bacterial numbers of the mutants were about 100-fold higher than that of WT at pH 4.5. At pH 5.5 and low Mg2+ concentration, survival of mutants was about 2- and 10-fold lower than that of WT when treated with AMPs, thionin, and cecropin A, respectively, thus suggesting that the PhoP/PhoQ system renders the pathogen more resistant to AMPs. Further analysis has demonstrated that phoP/phoQ mutants are more sensitive to osmotic stress and iron than those of WT strain at acidic pH. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our results showed that five mutants were non-pathogenic on immature pears. Results from phenotypic characterization and gene expression experiments, using GFP as a reporter, of TCST mutants indicate that TCSTs in E. amylovora not only control gene expression of hrp-type III secretion system and amylovoran biosynthesis, two major pathogenicity factors in E. amylovora, but also regulate gene expression of the two novel T3SS pathogenicity islands (EPI1 and EPI2) and flagella biosynthesis. In addition, we identified positive (non-motile), negative (hypermotile), and intermediate phenotype regulators for swarming motility in E. amylovora. Furthermore, several TCSTs also conferred E. amylovora resistance to antimicrobial peptides, iron, and osmotic stress. Our results clearly indicate that TCSTs in E. amylovora play a major role in its pathogenesis and survival. Our studies on individual TCST systems indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit, whereas the PhoP/PhoQ system plays an important role in the survival of the pathogen under stress conditions, although it does not function in virulence. Some details are found to be specific to E. amylovora, and different from other enterobacteria such as E. coli and Salmonella sp., indicating that gene regulation by the complex Rcs phosphorelay and PhoPQ systems is individually adapted to each distinct species. This knowledge will help us to better understand how E. amylovora interacts with its host and environmental conditions, and will lead to the development of improved disease management strategies.
Publications
- Zhao, Y.F. and Sundin, G.W. 2008. A simple genetic tool for studying gene function and signal transduction in Erwinia amylovora. Acta Horticulturae. 793:171-178.
- Zhao, Y.F., Wang, D. and Nakka, S. 2008. Two-component signal transduction systems play a major role in Erwinia amylovora pathogenesis and survival. Phytopathology 98:S180.
- Wang, D. and Zhao, Y.F. 2008. The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora. Phytopathology 98:S165.
- Nakka, S. and Zhao, Y.F. 2008. Molecular characterization of the PhoP/PhoQ two-component signal transduction system in Erwinia amylovora. Phytopathology 98:S111.
- Berry, M., McGhee, G.C., Zhao, Y.F. and Sundin, G.W. 2008. Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora. FEMS Microbiol. Letters (In Press).
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2008. Biofilm formation in Erwinia amylovora: Implications in pathogenicity. Acta Horticulturae. 793:67-71.
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Progress 10/01/06 to 09/30/07
Outputs
Erwinia amylovora is a serious pathogen of apples and pears, and the disease it causes resulted in huge losses. Early studies in elucidating the molecular basis for pathogenesis of E. amylovora have identified an essential virulence system - the hrp type III secretion system (hrp-T3SS) which delivers effector proteins into host plants. Subtractive hybridization and genome sequence have revealed two novel T3SS PAIs (Erwinia-pathogenicity islands, EPI1 and EPI2). The two PAIs are closely related to each other, have a significantly lower %G+C content (38.4 and 43.4% mol G+C, respectively, compared to 53.5% mol G+C content in the whole genome), and phylogenetically related to the tsetse fly endosymbiont Sodalis glossinidius SSR-1 and to human pathogens Salmonella SPI-1 and Yersinia Ysa T3SS-PAIs. Deletion mutants for major pathogenicity islands and effectors including three T3SS PAIs, type II secretion system (T2SS) and the amylovoran (ams) operon have been generated using
a novel gene knockout technique - recombineering. Mutants generated include T3SS (33.35kb deletion), T2SS (12.5 kb deletion), AMS (15.8kb deletion), EPI1 (23.45kb deletion), EPI2 (25.7kb deletion), EPI1/EPI2 double mutant, and effector gene yopH and sopA mutants. Pathogenicity assay with immature pear fruit and apple seedlings showed that EPI1 and EPI2 are not involved in virulence in plants. These results indicated that both EPI1 and EPI2 are acquired by E. amylovora through horizontal gene transfer and may function during interaction with insect vectors. We are now working towards understanding the role of EPI1 and EPI2 and their effectors during interaction with insect vectors and how genes are regulated using GFP as a reporter gene. In prokaryotes, two-component signal transduction (TCST) systems play critical roles in sensing and responding to environmental conditions and in bacterial pathogenesis. Recently, we identified several TCST genes including hrpX, grrS, envZ, and kdpE
that are induced during infection of host tissue in E. amylovora, indicating that TCSTs are key players in controlling the expression of virulence factors required for infection. Using genomic and bioinformatics techniques, we have identified about 45 signal transduction regulatory genes in the genome of E. amylovora including 20 sensor kinases, 19 response regulators, one hybrid, four sensory box proteins and one serine-threonine kinase. In order to determine the function and signal transduction pathways in E. amylovora, a total of 59 single, double and triple mutants for these signal transduction genes have been constructed using a PCR-based technique - one step inactivation of chromosomal genes, also referred to as red-cloning. Our results indicated that we have successfully adapted this genetic engineering technique to generate mutants in E. amylovora. Although red-cloning has been widely used in E. coli and other related enterobacteria, to the best of our knowledge this is the
first time that this technique has been used on plant pathogenic enterobacteria. Currently, we are working on selected TCST mutants for their phenotypic and genetic characteristics and their contribution to virulence.
Impacts
Type III secretion system is a critical component of virulence for many gram negative plant pathogenic bacteria and has been the target for disease control. E. amylovora contains three T3SSs. By using a novel technique, we have constructed several dozen single, double, triple, and island deletion mutants in E. amylovora. These mutants will provide us with basic tools to study gene function and signal transduction in E. amylovora. More importantly, the application of red-cloning in E. amylovora will further accelerate our efforts to dissect the genetic information encoded in the genome of E. amylovora. Better understanding of genetics and signaling transduction networks during pathogenesis and practical information on how E. amylovora interacts with its host and environmental conditions will lead to the development of improved disease management strategies. Understanding the genetics, molecular mechanisms of E. amylovora pathogenesis, and signaling will also greatly
enhance the likelihood of developing novel methods of control.
Publications
- Triplett, L., Zhao, Y.F. and Sundin, G.W. 2006. Genetic differences among blight-causing Erwinia species with differing host specificities identified by suppression subtractive hybridization. Appl. Environ. Microbiol. 72:7359-7364.
- Zhao, Y.F. and Sundin, G.W. 2007. Preliminary molecular characterization of two novel Erwinia amylovora type III secretion pathogenicity islands (T3SS-PAIs). Phytopathology 97:S129.
- Koczan, J.M., McGrath, M., Zhao, Y.F. and Sundin, G.W. 2007. Biofilm formation in Erwinia amylovora: Implications in pathogenicity. Phytopathology 97:S59.
- Zhao, Y.F. 2007. Will genomics help find the silver bullet for fighting the Anthrax of Fruit Trees? Biotechnology News, page 1-3, 2007. The Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign(http://www.biotec.uiuc.edu/news/newsletter/).
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