Progress 10/01/06 to 10/01/11
Outputs
OUTPUTS: Most pathogenic bacteria including plant pathogens produce multiple factors whose functions are required in various pathogenic stages, namely colonization, multiplication and symptom production. Such multi-factorial pathogenicity demands that pathogenicity/virulence determinants must be produced in an orderly manner. Pathogens use global as well as gene specific regulators that ensure their production in a temporal sequence. One of the best studied example of multi-factorial pathogenicity and its regulation comes from Erwinia carotovora ssp. Carotovora (Ecc), which causes soft-rotting disease in plant organs. Ecc produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. These extracellular proteins and bacterial motility are required for full virulence. In a series of studies we have shown that exoprotein and flagellar genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone (AHL), plant signals, an assortment of transcriptional factors/regulators (FlhDC, GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). As in the past, the focus of our research during the year was on regulatory systems that control virulence factors in plant pathogenic Erwinia species. We have filled in some of the major gaps that have remained in the regulatory network that controls extracellular protein/enzyme production and locomotion in the soft-rotting bacterium, Erwinia carotovora. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Most pathogenic bacteria including plant pathogens produce multiple factors whose functions are required in various pathogenic stages, namely colonization, multiplication and symptom production. Such multi-factorial pathogenicity demands that pathogenicity/virulence determinants must be produced in an orderly manner. Pathogens use global as well as gene specific regulators that ensure their production in a temporal sequence. One of the best studied example of multi-factorial pathogenicity and its regulation comes from Erwinia carotovora ssp. Carotovora (Ecc), which causes soft-rotting disease in plant organs. Ecc produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. These extracellular proteins and bacterial motility are required for full virulence. In a series of studies we have shown that exoprotein and flagellar genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone (AHL), plant signals, an assortment of transcriptional factors/regulators (FlhDC, GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). As in the past, the focus of our research during the year was on regulatory systems that control virulence factors in plant pathogenic Erwinia species.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Most pathogenic bacteria including plant pathogens produce multiple factors whose functions are required in various pathogenic stages, namely colonization, multiplication and symptom production. Such multi-factorial pathogenicity demands that pathogenicity/virulence determinants must be produced in an orderly manner. Pathogens use global as well as gene specific regulators that ensure their production in a temporal sequence. One of the best studied example of multi-factorial pathogenicity and its regulation comes from Erwinia carotovora ssp. Carotovora (Ecc), which causes soft-rotting disease in plant organs. Ecc produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. These extracellular proteins and bacterial motility are required for full virulence. In a series of studies we have shown that exoprotein and flagellar genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone (AHL), plant signals, an assortment of transcriptional factors/regulators (FlhDC, GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). As in the past, the focus of our research during the year was on regulatory systems that control virulence factors in plant pathogenic Erwinia species. We have filled in some of the major gaps that have remained in the regulatory network that controls extracellular protein/enzyme production and locomotion in the soft-rotting bacterium, Erwinia carotovora. Research findings were presented nationally and internationally at meetings and symposia. Invited lectures were presented in several universities and research institutes. Several manuscripts have been prepared or are in preparation for publication of the findings in peer reviewed journals of international reputation. The published papers are distributed free of charge to our colleagues upon request. The mutant strains, DNA preparations and other biological resources produced in our laboratory are freely made available to the scientific community at large. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Most pathogenic bacteria including plant pathogens produce multiple factors whose functions are required in various pathogenic stages, namely colonization, multiplication and symptom production. Such multi-factorial pathogenicity demands that pathogenicity/virulence determinants must be produced in an orderly manner. Pathogens use global as well as gene specific regulators that ensure their production in a temporal sequence. One of the best studied example of multi-factorial pathogenicity and its regulation comes from Erwinia carotovora ssp. Carotovora (Ecc), which causes soft-rotting disease in plant organs. Ecc produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. These extracellular proteins and bacterial motility are required for full virulence. In a series of studies we have shown that exoprotein and flagellar genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone (AHL), plant signals, an assortment of transcriptional factors/regulators (FlhDC, GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). As in the past, the focus of our research during the year was on regulatory systems that control virulence factors in plant pathogenic Erwinia species. We have filled in some of the major gaps that have remained in the regulatory network that controls extracellular protein/enzyme production and locomotion in the soft-rotting bacterium, Erwinia carotovora. Research findings were presented nationally and internationally at meetings and symposia. Invited lectures were presented in several universities and research institutes. Several manuscripts have been prepared or are in preparation for publication of the findings in peer reviewed journals of international reputation. The published papers are distributed free of charge to our colleagues upon request. The mutant strains, DNA preparations and other biological resources produced in our laboratory are freely made available to the scientific community at large.
Publications
- Chatterjee, A, H. Hasegawa, Y. Cui, P. Chakrabarty, and A. K. Chatterjee 2009/2010. GacS and GacA, members of a two component system, positively control virulence factors of the fire-blight pathogen, Erwinia amylovora by modulating the levels of rsmB RNA. Journal of Mycopathological Research, In press
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Our research involved analysis of global regulatory systems of soft-rotting Erwinia carotovora ssp. carotovora and plant pathogenic fluorescent Pseudomonas syringae pathovars. We studied the effects of an RNA binding protein, the quorum sensing signal (N-acylhomoserine lactone; AHL) and AHL receptors on bacterial motility and expression of flagellar genes. The goal was to ascertain if motility and extracellular protein production are co-regulated in the soft-rotting bacterium. We also examined the genetic basis for the lack of motility in a wild type strain of E. carotovora. As previously reported, we have discovered a fourth gene for an RNA-binding protein (RsmA4) in Pseudomonas syringae pathovars. We compared its structural and functional characteristics with three other RNA-binding proteins, RsmA1, RsmA2 and RsmA3. Research findings were presented nationally and internationally at meetings and symposia. Invited lectures were presented in several universities and research institutes. Several manuscripts have been prepared or are in preparation for publication of the findings in peer reviewed journals of international reputation. The published papers are distributed free of charge to our colleagues upon request. The mutant strains, DNA preparations and other biological resources produced in our laboratory are freely made available to the scientific community at large. 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
Erwinia carotovora ssp carotovora (Ecc) causes soft-rotting (tissue macerating) disease in many plants and plant organs. While pectinases are the primary determinants of virulence, several ancillary factors that augment bacterial virulence have also been identified. One such factor is bacterial motility. Flagellum formation and bacterial movement are regulated in many enterobacteria including Ecc by FlhDC, the master regulators of flagellar genes and FliA, a flagellum specific sigma factor. flhD and flhC products are known to form an FlhD4C2 hexamer complex. Both FlhD and FlhC subunits are essential for effective transcription. The FlhDC complex binds promoter regions of genes for proteins for the basal body and the hook of the flagellum as well as two regulators, FliA and FliM. FliA is an alternative sigma factor specific for the flagellar regulon and FlgM acts as an anti-sigma factor. In addition to the flagellar genes, FlhDC in Ecc controls an array of traits including virulence, type III secretion system and extracellular enzyme production. We documented that the pleiotropic effect of FlhDC deficiency results from altered expression of four downstream regulators. Expression of flhDC genes is controlled by several regulators. One such regulator in E. coli is CsrA; a homolog of Erwinia RsmA. It is a RNA-binding protein, generally responsible for translational repression and RNA decay. Surprisingly, it was shown to positively regulate flhDC expression. In Ecc, this post transcriptional regulator, designated RsmA (= CsrA), plays a central role in the production of AHL and extracellular enzymes/proteins and virulence. In a series of studies, we have determined that RsmA, thus far without any exception, negatively controls gene expression. Thus, the observation in E. coli regarding the positive regulation of flhDC by CsrA (RsmA) was unexpected. This apparent anomaly prompted us to examine the role of RsmA on FlhDC expression in Ecc. Since the quorum sensing signal, N-acylhomoserine lactone (AHL) is directly responsible for rsmA expression, we also examined the role of AHL on motility. We determined that motility of Ecc is positively regulated by AHL, and negatively regulated by RsmA. Our data show that RsmA negatively regulates flhDC and fliA expression. This is indicated by higher levels of transcripts of flhDC, fliA, fliC and fliE genes in an RsmA- mutant than in the RsmA+ parent. Moreover, the chemical stabilities of transcripts of these genes are greater in an RsmA- mutant than in RsmA+ bacteria. These observations contrast with positive regulation of flhDC and motility by CsrA (= RsmA) in E. coli. In the absence of AHL, the AHL receptors, ExpR1/ExpR2 (= AhlR) in Ecc, negatively regulate motility and expression of flhDC and fliA by activating RsmA production. In the presence of AHL regulatory effects of ExpR1/ExpR2 are neutralized resulting in reduced levels of rsmA expression and enhanced motility. Thus, our work for the first time demonstrated that RsmA and AHL control, in addition to secreted enzymes and effectors, bacterial movement and produced an understanding of the underlying regulatory mechanism.
Publications
- Cui, Y., Chatterjee, A., Yang, H., and Chatterjee, A.K. 2008. Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression. Journal of Bacteriology, 190:4610-4623.
- Chatterjee, A. Cui,Y., and Chatterjee, A. K. 2009. RsmC of Erwinia carotovora subsp. carotovora Negatively Controls Motility, Extracellular Protein Production, and Virulence by Binding FlhD and Modulating Transcriptional Activity of the Master Regulator, FlhDC. Journal of Bacteriology, 191: 4582-4593.
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Progress 01/01/07 to 12/31/07
Outputs
To accomplish some of the objectives listed in my project proposal we have made several key bacterial mutants: FlhDC-, GacA-, HexA-, RsmC-, RsmB-, RsmA- and few others. In addition, we have constructed various double mutants where two genes have been inactivated as deemed necessary for the confirmation of the results obtained with single site mutants. Mutants were constructed by transposon insertion followed by allele exchange. Mutants were rigorously characterized both genetically and physiologically. The regulatory mutants were examined for target gene expression and alterations in pathological and physiological properties. Several genes (flhDC, flhD, flhC, fliA) were cloned either by screening gene libraries or by polymerase chain reaction (PCR) technology. Sequence data for several genes were determined. Promoter/regulatory regions of various genes were amplified by PCR and their sequences were confirmed. Such DNA sequences were used to construct gene
(transcriptional) fusions to assay for promoter activity and regulatory protein/regulatory RNA interactions in vivo in desired bacterial mutants and their parent strains. In all instances, lacZ was used as the reporter gene. Segments of the coding regions of an assortment of genes were PCR amplified and the DNA segments were used as probes in Northern blot analysis of transcripts. Research data were discussed orally at meetings, seminar and journal club presentations. Several manuscripts have been or are being prepared for the dissemination of our findings through peer reviewed journals of international reputation. The mutant strains, DNA preparations and other biological resources developed in my laboratory are freely made available for use by the scientific community at large. We also exchange unpublished data of our studies with our colleagues worldwide upon request.
Impacts
Global regulation by FlhDC, the master regulator of flagellar operon was studied in Erwinia carotovora ssp. carotovora (Ecc). This bacterium produces an array of extracellular proteins (= exoproteins) including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators and post-transcriptional regulators (RsmA, rsmB RNA; Rsm = regulator of secondary metabolite production). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (= PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in E. coli), and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of
GacA regulation, we discovered that a locus, bearing strong homology to the flhDC operon of E. coli, also controls extracellular enzyme production and virulence. Sequence analysis and transcript assays disclosed that the flhD operon of Ecc, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. We have determined that FlhDC positively regulates gacA, rsmC and fliA (gene for flagella specific sigma factor), and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA; these genes, in turn, control rsmB RNA production. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in Ecc. Our observations establish a regulatory connection between FlhDC, HexA, GacA and rsmB RNA in the context of exoprotein production and virulence of Ecc. We have produced genetic
evidence for RsmC-FlhDC interaction in controlling motility and extracellular protein production. RsmC is a global negative regulator controlling extracellular proteins/enzymes secreted by types I, II and III secretion systems, motility and plant virulence of Ecc. Structural analysis of RsmC had suggested that it may function as a component of Ecc transcriptional machinery but not as a DNA-binding transcriptional repressor. We have discovered that the expression of all three targets (i.e., gacA, rsmC and fliA), positively regulated in Ecc by FlhDC, is affected by RsmC. The RsmC-mediated inhibition of expression of gacA, rsmC and fliA were confirmed by examining the expression of lacZ fusions. The results of in vivo studies in these bacteria revealed that RsmC interacts with FlhDC leading to the inhibition of its activator function. Our findings for the first time establish modulation of FlhDC transcriptional activity by a novel protein-protein interaction and now provide us with a
clearer picture of the regulatory network controlling exoprotein production in Ecc.
Publications
- 1, Cui, Y., A. Chatterjee, H. Hasegawa, and A.K. Chatterjee. 2006. Erwinia carotovora subspecies produce duplicate variants of ExpR, LuxR homolog that activate rsmA transcription but differ in their interactions with N-acylhomoserine lactone signals. Journal of Bacteriology 188, 4715-4726.
- 2, Chatterjee, A, Y. Cui, H. Hasegawa, and A.K. Chatterjee. 2007. PsrA, the Pseudomonas sigma regulator, controls regulators of epiphytic fitness, quorum-sensing signals, and plant interactions in Pseudomonas syringae pv. tomato strain DC3000. Applied and Environmental Microbiology 73, 3684-3694.
- 3, Cui, Y., A. Chatterjee, H.L. Yang, and A.K. Chatterjee. 2008. Regulatory network controlling extracellular proteins in Erwinia carotovora ssp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory sRNA production by affecting gacA and hexA (lrhA) expression. Provisionally accepted in Journal of Bacteriology.
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