Progress 10/09/09 to 10/08/14
Outputs
Target Audience: a. Person’s with Burkholderia infections: Our research efforts with regard to the Burkholderia cepacia complex (Bcc), that are capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis. Our focused is on the characterization of broad host high molecular weight bacteriocins that may be useful in treatment of infection caused by members of the Bcc and other Burkholderia species. b. Grape growers: Virulent podophages and siphophages phages of Xylella were characterized and evaluated in greenhouse trials. The characterized phages may be useful in the treatment of Pierce’s Disease of grapevines. c. c. Students: See reports 2009-2014 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Burkholderia studies-Graduates students and undergraduate students and Postdocs were given the opportunity to conduct independent research related to the project. Xylella studies-Graduates students and undergraduate students and Postdocs were given the opportunity to conduct independent research related to the project. How have the results been disseminated to communities of interest? See publications and abstracts for period September 2009 to October 2014 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the period of September 2009 to October 2014, we have conducted research targeting the cross-domain bacterial pathogen Burkholderia and the xylem-limited bacterium Xylella. Our long-term goal with regard to Burkholderia has been to understand the genetic basis for pathogenicity in members of the Burkholderia cepacia complex (Bcc), to determine how the genes are regulated, and to develop strategies for therapeutic use of bacteriophage (phage) and/or phage component proteins for infections caused by members of the Bcc. During the five year period we confirmed our hypothesis that the two type IV secretion systems (T4SSs), identified by our laboratory, in Burkholderia cenocepacia perform two different fundamental functions related to pathogenesis: the delivery of an effector molecule(s) to eukaryotic target cells and genetic exchange. The results, in cooperation with the LiPuma laboratory at the University of Michigan–Medical School, showed that the intracellular processing of B. cenocepacia was similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells. Experiments using mutants in the plasmid encoded type IV secretion system showed that the chromosomal system functioned in the mobilization of plasmid DNA. We also tested, in cooperation with the LiPuma and Young (Texas A&M) laboratories, the therapeutic potential of bacteriophages in a mouse model of acute Burkholderia cenocepacia pulmonary infection. It was determined that systemic phage administration was more effective than inhalational administration. Our findings suggested that circulating phages have better access to bacteria in lungs than do topical phages. Additionally, our laboratory identified and characterized an R2-like high molecular weight bacteriocin (tailocin) with board host range against 15 species of Burkholderia. Electron microscopy studies confirmed the phage tail-like structure of tailocin Bcep0425.We determined that the receptor site was a core component of the bacterial lipopolysaccharide. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes and that the tailocin cassette was integrated into a tRNA. Two putative transcriptional regulators were identified to be involved in the induction of tailocin Bcep0425. The second long-term goal of our laboratory is to develop an understanding of Xylella phage biology and to explore the feasibility of phage therapy (the use of phage that kill only the targeted bacteria) that does not hurt the grapevine or beneficial bacteria. The search for new ways to combat microbial pathogens is an ongoing process in both medicine and agriculture and the use of bacteriophages (phages; bacterial viruses) offers a novel strategy not previously pursued for the control of Pierce’s Disease (PD), because methods to isolate and propagate viable phages were not available. During the five year period, we established methods that allows for the isolation, purification and propagation of X. fastidiosa phages. Using the methods, we isolated and characterized the first functional lysogenic phages of X. fastidiosa. Expanding on the information developed for the detection and isolation of lysogenic phage, we isolated, purified and characterized the first virulent phages for Xylella. Efficacy studies to determine protection of grapevines in greenhouse experiments showed that a cocktail composed of four virulent phages can protect grapevines both prophylactically and therapeutically. Our laboratory has developed phage therapy as a potential treatment for PD.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: a. Person’s with Burkholderia infections: Our research efforts with regard to the Burkholderia cepacia complex (Bcc), that are capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis. Our focused is on the characterization of broad host high molecular weight bacteriocins that may be useful in treatment of infection caused by members of the Bcc and other Burkholderia species. b. Grape growers: Virulent podophages and siphophages phages of Xylella were characterized and evaluated in greenhouse trials. The characterized phages may be useful in the treatment of Pierce’s Disease of grapevines. c. Students: During the Spring semester of 2014, I taught 15 students in a writing intensive seminar course as well as mentored 5 students in independent research projects. During the Summer of 2014, I mentored a three students in an independent research projects. During the Fall of 2014, I taught Environment Microbiology (61 students) and mentored two students in independent research projects. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Burkholderia studies. Undergraduate students were given the opportunity to conduct independent research to identify high molecular weight bacteriocins. Xylella studies. Undergraduates, graduate students and postdocs conducted independent research projects. How have the results been disseminated to communities of interest? Xylella research was presented at the American Phytopathological Society Annual Meeting-August 2014 What do you plan to do during the next reporting period to accomplish the goals? New project will be submitted.
Impacts
What was accomplished under these goals?
Objectives 1a-c: Essentially all members of the Bcc demonstrate broad-spectrum antibiotic resistance in vitro. There is a need to develop new antimicrobial therapies against CF-associated Bcc infection. Under objective 1c, we conducted relevant studies to develop high molecular weight bacteriocins. We further characterized broad host range high molecular weight bacteriocin (tailocin) from Burkholderia cenocepacia that exhibits biocidal activity against Bcc isolates tested. The specific mechanism of action and high bactericidal potency indicate that tailocin particles can be developed as single agents or as synergistic combinations of single agents to enhance antibiotic therapy to control antibiotic resistant bacteria. Objective 2 a-c. Xylella fastidiosa is the causal agent of Pierce’s Disease (PD) of grapes. Our research is focused on the development of virulent phage as a biocontrol agent for the treatment and prevention of PD. We have isolated and characterized a pool of virulent podo and siphophages phages for Xylella that were tested for efficacy in greenhouse experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ahern S.J., Das M., Bhowmick T.S., Young R., Gonzalez C.F. (2014). Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas J. Bacteriol. January 196(2) 459-471
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Coutinho, B.G., Das M., Su�rez-Moreno Z. R. , Gonzalez C.F. and Venturi. 2014. In T. Coenye and E. Mahenthiralingam (ed) Burkholderia: From Genomes to Function. Horizon Scientific Press. Chapter 8
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: a. Person’s with Burkholderia infections: During this fiscal year our research efforts with regard to the Burkholderia cepacia complex (Bcc) that are capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis focused on the identification of broad host high molecular weight bacteriocins that may be useful in treatment of infection caused by members of the Bcc. b. Grape growers: With regard to our work with phages of Xylella we were able to characterize several virulent phages that may be useful in the treatment of Pierce’s Disease of grapevines. c. Students: During the Spring semester of 2013 I taught 16 students in a writing intensive seminar course. During the Summer of 2013, I was the Director of an NSF sponsored REU in which 10 students (4 non-minorities and 6 minorities) conducted independent research for 10-weeks. Additionally I mentored a one student in an independent research project. During the Fall of 2013 I taught Environment Microbiology (56 students) and mentored two students in independent research projects. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Burkholderia studies. Undergraduate students were given the opportunity to conduct independent research to identify high molecular weight bacteriocins. Xylella studies. Graduate students and Postdocs conducted independent research projects and presented research at professional meetings. How have the results been disseminated to communities of interest? Burkholderia research was presented at International Burkholderia Cepacia Working Group – April, 2013, Ann Arbor, MI Xylella research was presented at the American Society for Microbiology Annual Meeting-May 2013-Denver, the American Phytopathological Society Annual Meeting-August 2013-Austin. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives 1a-c: Members of the Burkholderia cepacia complex (Bcc) cause plant disease and life-threatening respiratory tract infections in persons with cystic fibrosis (CF). It is the first long-term goal of my laboratory to understand the genetic basis for pathogenicity in members of the Bcc, to determine how the genes are regulated, and to develop strategies for phage (bacterial virus) therapy for infections caused by members of the Bcc. Due to lack of funding of a grant, objectives1a and 1b were not addressed in this fiscal year. However, under objective 1c, we conducted relevant studies to develop antimicrobial phage related therapy. Since essentially all members of the Bcc demonstrate broad-spectrum antibiotic resistance in vitro. The clinical observations emphasize the essential need to develop new antimicrobial therapies against CF-associated Bcc infection. Our laboratory has identified an unusually broad host range high molecular weight bacteriocin (tailocin) from Burkholderia cenocepacia that exhibits biocidal activity against 70% of Bcc isolates tested. The specific mechanism of action and high bactericidal potency indicate that tailocin particles can be developed as single agents or as synergistic combinations of single agents to enhance antibiotic therapy to control antibiotic resistant bacteria. Objective 2 a-c. Pierce’s Disease (PD) of grapes, caused by X. fastidiosa, is a limiting factor in the cultivation of high quality wine grapes in areas of the US, such as Texas and California. There are currently no effective control methods to prevent infection or manage the disease once it has established itself in the vineyard short of aggressive culling of the infected vines. Understanding the biology of Xylella phage (bacterial virus) will aide in the development and implementation of a phage based control for plant diseases caused by Xylella. We have isolated and characterized the first virulent phages for Xylella and tested their efficacy in greenhouse experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ahern S.J., Das M., Bhowmick T.S., Young R., Gonzalez C.F. Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas J. Bacteriol. January 2014 196:2 459-471; published online ahead of print 8 November 2013
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Coutinho, B.G., Das M., Su�rez-Moreno Z. R. , Gonzalez C.F. and Venturi. 2013. In T. Coenye and E. Mahenthiralingam (ed) Burkholderia: From Genomes to Function. Horizon Scientific Press. Chapter 8 Publication date: February 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Duarte, I, G. Wang, J. J. LiPuma, R. F. Young and C. F. Gonzalez. Characterization of a broad host range tailocin from Burkholderia. International Burkholderia Cepacia Working Group April, 2013, Ann Arbor, MI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Ahern, S. J. M. Das, T. Bhowmick, R. F. Young, III, C. F. Gonzalez. Virulent Phages that Attack Xylella fastidiosa and Other Members of the Xanthomonadaceae. American Society for Microbiology Annual Meeting-May 2013-Denver
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Das, M., T. Bhowmick, S. J. Ahern, R. F. Young, III, C. F. Gonzalez. Application of Phages for Control of Pierces Disease. American society for Microbiology Annual Meeting-May 2013-Denver
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Bhowmick, T. S., M. Das, K. M. Heinz, P. C. Krauter and C. F. Gonzalez. Transmission of phage by glassy-winged sharpshooter. American Phytopathological Society Annual Meeting-August 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Das, M., T. S. Bhowmick, S. J. Ahern, R. Young and C. F. Gonzalez. Therapeutic and prophylactic application of phages to control Pierces Disease. American Phytopathological Society Annual Meeting-August 2013
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Members of the Burkholderia cepacia complex (Bcc) are opportunistic human pathogens that exhibit broad-spectrum antibiotic resistance capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis. Because of innate resistance, the effective treatment with commonly used antibiotics is limited and in many cases, a regiment of multiple antibiotics is necessary for effective infection control. Phages and phage-tail like bacteriocins (tailocins) offer promising alternative treatment strategies. High-molecular weight (HMW) bacteriocins like the well-studied R- and F-type pyocins (tailocins) from Pseudomonas aeruginosa closely resemble phage tails. Tailocins are attractive candidates for novel antibacterial strategies because they can be genetically manipulated like bacteriophages and can be deployed with defined dosage, since they do not propagate in therapeutic use. Using an 18 X 18-test matrix of B. cenocepacia clinical isolates was used to identify tailocin Bcep0425. Tailocin proteins were separated by SDS-PAGE and the four major protein bands were excised for N-terminal analysis. Proteins were subjected to LC-MS/MS analysis. BC0425 genome was sequenced by 454 pyrosequencing. Protein-coding regions were initially predicted using GeneMark, refined by manual analysis, and annotated using BLAST. In-frame deletion mutants were generated in tailocin for both holin and antiholin genes and complemented in-trans. Bcep0425 exhibited broad host range activity against members of the Bcc. Electron microscopy studies confirmed the phage tail-like structure of Bcep0425. Genes encoding for Bcep0425 were identified in contig 77 of a draft BC0425 genome. The tailocin cassette was integrated into a tRNA. The gene adjacent to gene1 encoded an integrase with a conserved C-terminal integrase domain and had significant sequence similarity to phage P22 integrase. Pierce's Disease (PD) of grapes, caused by Xylella fastidiosa, is a limiting factor in the cultivation of high quality wine grapes in areas of the US, such as Texas and California. There are currently no effective control methods to prevent infection or manage the disease once it has established itself in the vineyard short of aggressive culling of the infected vines. The search for new ways to combat microbial pathogens is an ongoing process in both medicine and agriculture and the use of bacteriophages (phages; bacterial viruses) offers a novel strategy not previously pursued for the control of PD, because methods to isolate and propagate viable phages were not available. Our laboratory has developed the technology to overcome this problem so that it is now feasible that phage therapy (involves the use of phage that kill only the targeted bacteria) can be developed as a treatment for PD; while not hurting the vine or beneficial bacteria. We have isolated several virulent phages and are characterizing. PARTICIPANTS: Research in my laboratory has both a basic and applied component. The post-doctoral fellows and students present their data at scientific meetings and publish their results in peer-reviewed journals. Current Post docs and students: Mayuhk Das(Post Doc), Tushar Bhowmick (Post Doc), Guichun Wang(Res Assoc),Iris Duarte (Graduate Student) Stephen Ahern (Graduate Student) Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan. TARGET AUDIENCES: The Burkholderia phage and tailocin research is aimed at the development of potential therapeutic agents. This research is important in cystic fibrosis research. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to the grape growing industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ongoing research is characterizing a broad host range tailocin that could be used as an antibacterial agent. The identification of virulent phages for Xylella is a first step in the development of a potential treatment for PD.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The Burkholderia cepacia complex (Bcc) is a group of opportunistic bacterial pathogens capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis. Effective treatment of these infections is limited due to the microorganisms' high level of resistance to most of the commonly used antibiotics. These clinical difficulties have led to increased interest in finding more effective therapeutic agents to combat Bcc infections. Phages and phage-tail like bacteriocins (tailocins) offer promising alternative treatment strategies. We have previously reported on the identification of tailolcin Bcep0425 which exhibits broad host range activity against members of the Bcc. Targeted mutagenesis of genes involved in the biosynthesis of the bacterial lipolysaccharide (LPS) was conducted to determine the putative receptor site of Bcep0425. A deletion mutant in the recA gene resulted in no lysis of an induced host indicating a role for the recA gene in the induction process. A mutation in the putative activator gene delayed lysis. Further genetic analysis of the tailocin encoding genes is needed to determine their role in regulation and production. The search for new ways to combat microbial pathogens is an ongoing process in both medicine and agriculture. Bacteriophages (phages) were widely used for antibacterial therapy during the pre-DNA era of microbiology, but their use died out in the West due to the lack of a firm scientific basis and the growing availability of antibiotics based on natural products. Phages are highly specific to one or another bacterial species; they are non-toxic to animals and plants or non-target bacteria that may be beneficial to plants. Phages also increase in titer as they infect, multiply in, and kill their target hosts. The development of a phage-based biocontrol strategy offers a novel approach for the control of Pierce's Disease caused by Xylella fastidiosa (XF). The overall objective of our research project is to develop phages with biocidal activity against X. fastidiosa without harming the vine. There are currently no effective control methods to prevent infection or manage the disease once it has established itself in the vineyard short of aggressive culling of the infected vines. We have developed an assay system that allows our laboratory to detect, isolate and propagate XF phages and have in fact isolated, propagated and characterized the first XF temperate phage. Ongoing research is focused on identifying virulent phage for Xylella. It is imperative that temperate phages that are capable of integrating into the host chromosome not be used (a common mistake) and that the only virulent phage be used when developing phages for preventative or therapeutic purposes to control disease, since temperate phage can lead to resistance to phage-based control and transfer of DNA. PARTICIPANTS: Research in my laboratory has both a basic and applied component. The post-doctoral fellows and students present their data at scientific meetings and publish their results in peer-reviewed journals. Current Post docs and students: Jason Gill(Post Doc) Mayuhk Das(Post Doc), Guichun Wang(Res Assoc),Iris Duarte (Graduate Student) Stephen Ahern (Graduate Student) Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan. TARGET AUDIENCES: The Burkholderia phage and tailocin research is aimed at the development of potential therapeutic agents. This research is important in cystic fibrosis research. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to the grape growing industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
There is a substantial need to develop new strategies for antimicrobial therapy since the development of new antibiotics is at an all time low. Tailocins offer a potential anti-bacterial agent against antibiotic resistant members of the Bcc. The development of phage offers an alternative strategy for control of Pierce's Disease since there are currently no effective control methods to prevent infection or manage the disease once it has established itself in the vineyard short of aggressive culling of the infected vines.
Publications
- Gill, J.J, E. J. Summer, W. K. Russell, S. M. Cologna, T. M. Carlile, A. C. Fuller, K. Kitsopoulos, L. M. Mebane, B. N. Parkinson, D. Sullivan, L. A. Carmody, C. F. Gonzalez, J. J. LiPuma, R. Young. (2011). Genome and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia. J. Bacteriol.193:5300-5313.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The Burkholderia cepacia complex (Bcc) consists of at least 17 phenotypically similar but genotypically distinct species of non-fermenting, Gram-negative bacteria that are found in a diverse set of niches. Members of the Bcc are opportunistic pathogens, capable of causing disease in plants, animals and particularly causing life-threatening respiratory infections in persons with cystic fibrosis. Essentially all Bcc isolates demonstrate in vitro broad-spectrum antibiotic resistance. In fact, many clinical isolates are resistant to all currently available antibiotics, rendering antibiotic therapy ineffective. There is a substantial need to develop new antimicrobial therapies. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", as alternative anti-bacterial agents against Bcc is being investigated in our laboratories. A 20 X 20 test matrix of B. cenocepacia clinical isolates was used to identify a tailocin designated Bcep0425. Bcep0425 exhibits broad host range activity against members of the Bcc. The putative receptor site for Bcep0425 was determined to be on the core component of the LPS. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes. Two putative transcriptional regulators were found to be involved in the induction of Bcep0425. Pierce's Disease of grapes, caused by the xylem-limited, fastidious bacterium Xylella fastidiosa, is a limiting factor in the cultivation of high quality wine grapes. Pierce's Disease has become a major threat to the nation's most valuable wine producing regions in California and Texas with the potential of having a large economic impact. Bacteriophages (phages) are highly specific bacterial viruses that have been used in antibacterial therapy in human and plant systems. The formulation of a semi-solid medium that is conducive to even dispersal and confluent growth of X. fastidiosa, a technique which is required for the efficient manipulation and study of phages and high molecular weight (HMW) bacteriocins (defective phage), has not been previously reported in the literature. We have established such techniques and developed an efficient plate assay for detection of phage and HMW-bacteriocins. Using this novel technique my laboratory has isolated and characterized the first functional lysogenic phage of X. fastidiosa. This is a significant step forward in understanding the biology of X. fastidiosa and its phages, which will allow us to study the phage-Xylella interaction and their potential use as a biocontrol agent. PARTICIPANTS: Research in my laboratory has both a basic and applied component. The post-doctoral fellows and students present their data at scientific meetings and publish their results in peer-reviewed journals. Current Post docs and students: Jason Gill(Post Doc) Mayuhk Das(Post Doc), Guichun Wang(Res Assoc),Iris Duarte (Graduate Student) Stephen Ahern (Graduate Student) Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan. TARGET AUDIENCES: The Burkholderia phage and tailocin research is aimed at the development of potential therapeutic agents. This research is important in cystic fibrosis research. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to the grape growing industry. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Essentially, all Bcc clinical isolates demonstrate broad-spectrum antibiotic resistance in vitro. Even with combined antibiotic therapy, clearance of the members of the Bcc is not normally observed. There is a substantial need to develop new strategies for antimicrobial therapy. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", is a potential anti-bacterial agent against Bcc. There are currently no effective control methods to prevent infection or manage the Pierce's Disease once it has established itself in the vineyard short of aggressive culling of the infected vines. The propagation and genomic analysis of Xfas53is a first step in understanding Xylella phage biology.
Publications
- Summer, E. J., C. J. Enderle, S. J. Ahern, J. J. Gill, C. P. Torres, D. N. Appel, M. C. Black, R. Young, and C. F. Gonzalez. 2010. Genomic and Biological Analysis of Phage Xfas53 and Related Prophages of Xylella fastidiosa. J Bacteriol 192:179-190.
- Carmody, L. A, J.J. Gill, E.J. Summer,U. S. Sajjan,C. F. Gonzalez, R. F. Young, and J. J. LiPuma. 2010. Efficacy of Bacteriophage Therapy in a Model of Burkholderia cenocepacia Pulmonary Infection. J Infect Dis 201:264-271
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have confirm our hypothesis that the two type IV secretion systems (T4SSs) identified by our laboratory in Burkholderia cenocepacia perform two different fundamental functions related to pathogenesis: the delivery of an effector molecule(s) to eukaryotic target cells, and genetic exchange. The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92-kb plasmid is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS of Agrobacterium tumefaciens. In contrast to the Ptw T4SS, we found that the bc-VirB/D4 T4SS is dispensable for Ptw effector(s) secretion. However, the bc-VirB/D4 T4SS is involved in plasmid mobilization. We also found that the fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid. Additionally we have isolated a tailocins (defective phage) which exhibits broad host range activity against members of the Burkholderia cepacia complex. Analysis of the tailocin proteins determined that the sheath, core, and tail fiber (major proteins) have a mass of 43, 17, and 100 kD, respectively. Using LPS isolated from a sensitive isolate it was determined that the attachment site is the bacterial LPS. We are currently characterizing the attachment site and are focused on identifying genes encoding for the components of the tailocin. The therapeutic potential of bacteriophages in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed in cooperation with the laboratories of Ry Young and John LiPuma (Co-PIs). Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. We found that systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages. My laboratory has also isolated and characterized the first functional lysogenic phage (designated Xfas53) of X. fastidiosa which we have physically characterized. Transmission electron microscopy of purified phage revealed that phage Xfas53 possesses a 55-nm head and a short, 12-nm-diameter, non-contractile tail, typical of podophage morphology. Clones from a shotgun library were sequenced to eight-fold coverage, and the sequence reads were assembled into a single contig of 36,674 bp. The genome has an average GC content of 57%, which is slightly higher than the 53% GC content of Xylella. The 36.7-kb genome is predicted to encode 45 proteins. PARTICIPANTS: Research in my laboratory has both a basic and applied component. The post-doctoral fellows and students present their data at scientific meetings and publish their results in peer-reviewed journals. Current Post docs and students: Elizabeth J. Summer (Post Doc) Jason Gill(Post Doc), Guichun Wang(Res Assoc),Iris Duarte (Graduate Student) Stephen Ahern (Graduate Student) Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University and John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan. TARGET AUDIENCES: The Burkholderia T4SS research is aimed at determining the role secretion systems in pathogenicity and identifying effector(s) molecules. This research is important in cystic fibrosis research. The Burkholderia bacteriophage and tailocin research is aimed at developing a potential method to treat or augment treatment for infection caused by B. cenocepacia. This research is important to cystic fibrosis research. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to the grape growing industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Secretion of proteins across bacterial membranes and genetic adaptation are of fundamental importance in bacterial virulence. Our research has provided information on the role of two T4SSs in Burkholderia cenocepacia, an important opportunistic pathogen of patients with cystic fibrosis. Experiments to determine the therapeutic potential of phages in a mouse model of acute Burkholderia cenocepacia pulmonary infection is a first step in developing phage therapy to treat Burkholderia infections. The propagation of a functional phage for Xylella is a first step in understanding Xylella phage biology.
Publications
- Durate, I., G. Wang, R. F. Young, J. J. LiPuma, and C. F. Gonzalez. 2009. Characterization of a broad host range tailocins from Burkholderia. Phytopathology 99, No. 6 (Supplement) S30.
- Zhang, R., J. J. LiPuma, and C. F. Gonzalez. 2009. Two type IV secretion systems with different functions in Burkholderia cenocepacia K56-2. Microbiology 155:4005-13.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In my laboratory we have identified two type IV secretion systems (TFSSs) in Burkholderia cenocepacia of the epidemic ET12 lineage; one system is plasmid encoded (designated the Ptw TFSS) whereas the other is chromosomally encoded (designated the VirB/D TFSS) and shows homology to the Agrobacterium tumefaciens VirB/D4 TFSS. Insertion mutants of the ptwD4 gene do not produce the watersoaking phenotype. We have obtained non-polar deletion mutants that have been complemented. The complemented mutants produce the watersoaking phenotype. Our studies have shown the Ptw-TFSS is involved in the translocation of a protein effector(s) involved in the expression of the watersoaking phenotype exhibited by strains of the ET12 lineage. Our hypothesis is that the chromosome II, VirB/D TFSS found in ET12 strains is involved in the trafficking of DNA. We have constructed a deletion mutation in the chromosome b/c VirD4 gene and shown that mobilization of plasmid DNA is affected. Ongoing will test complements for the ability to restore the phenotype. The development of a phage/bacteriocin-based biocontrol strategy offers a novel approach for the control of Pierce's disease caused by Xylella fastidiosa. The overall objective of the research project in my laboratory is to develop phages and/ or bacteriocins with biocidal activity against Xylella without harming the vine. We have developed an assay system that allows our laboratory to detect Xylella phages and bacteriocins. We are currently characterizing an identified bacteriophage. Studies to assess the potential role of Burkholderia bacteriophage in abrogating bacterial infection caused by B. cenocepacia in a lung mouse model have been initiated. Preliminary studies show promising results. PARTICIPANTS: Research in my laboratory is multi-faceted and will give the students and post-doctoral fellows' training that contains components of both basic and applied science. The post-doctoral fellows and students present their data at scientific meetings and publish their results in peer-reviewed journals. It is the policy of our laboratories to have weekly meetings in which students and the postdoctoral fellow discuss current results and research strategy. Current Post docs and students Elizabeth J. Summer- Post Doc Jason Gill- Post Doc Ruifu Zhang- Post Doc Iris Duarte- Graduate Student Stephen Ahern-Graduate Student Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan TARGET AUDIENCES: The Burkholderia research is aimed at determining the role secretion systems in pathogenicity. This research is important to patients with cystic fibrosis and is funded by the Cystic Fibrosis Foundation. The Burkholderia bacteriophage research is aimed at developing a potential method to abrogate bacterial infection caused by B. cenocepacia. This research is important to patients with cystic fibrosis and is funded by NIH. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to person in the grape growing industry and is funded by USDA-APHIS PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Virulence factors as well as the transfer of DNA are important in disease development and genetic diversity. Our research will provide information on the role that Type IV secretions systems play in the elaboration of virulence factors and transfer of genetic material. Our bacteriophage research in both Burkholderia and Xylella addresses the basic biology of phage and to the use of phage as potential treatments.
Publications
- Sajjan, S. U., Carmody, L. A. Gonzalez, C.F. and LiPuma, J. J. 2008. A Type IV Secretion System Contributes to Intracellular Survival and Replication of Burkholderia cenocepacia. Infect. Immun. 76:5447-5455.
- Orchard,R. C., Summer, E. J.,Gill, J, Savva,C.,Cohen,N., Gonzalez, C. F. and Young, R. F. (2008) Analysis of phage BcepNY3 and Rhodococcus equi bacteriophages. Texas A&M AgriLife Conference
- Carmody, L. A., Gill, J. J., Gonzalez, C. F. Sajjan U.S., Young, R. and LiPuma, J. J (2008) Bacteriophage therapy in a mouse model of Burkholderia infection. Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), October 2008
- E. J. Summer, Enderle, C. J., Torres, C. P. Appel, D. N., Black, M. C., Young, R. F. and Gonzalez, C. F. (2008)Isolation and Characterization of Xylella fastidiosa phage Xfas53. 2008 Pierces Disease Conference, San Diego, Ca
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: As previously reported, in my laboratory, we have identified two type IV secretion systems (TFSSs) in Burkholderia cenocepacia of the epidemic ET12 lineage; one system is plasmid encoded (designated the Ptw TFSS) whereas the other is chromosomally encoded (designated the VirB/D TFSS) and shows homology to the Agrobacterium tumefaciens VirB/D4 TFSS. Our studies indicate the Ptw-TFSS is involved in the translocation of a protein effector expressed as a watersoaking phenotype in the onion plant model. Insertion mutants of the ptwD4 gene do not produce the watersoaking phenotype. However, these mutants appear to be polar since complementation is not possible. Deletion mutants are currently being constructed. Additionally, we hypothesize that the chromosomal VirB/D TFSS found in ET12 strains is involved in the trafficking DNA. A deletion mutant of the chromosomal VirD4 gene has been constructed and is currently being evaluated. We have also initiated studies to assess the
potential role of Burkholderia bacteriophage in abrogating bacterial infection caused by B. cenocepacia in an in vitro airway epithelial cell culture model. Preliminary studies show promising results. Bacterial viruses, or bacteriophage (phage), are by far the most numerous biological entities on the planet and play a significant role in the ecology and evolution of bacteria in all environments. The search for novel approaches to combat microbial pathogens is an ongoing process in both medicine and agriculture. The development of a phage-based biocontrol strategy offers a novel approach for the control of Pierce's disease caused by Xylella fastidiosa. The complete genome sequence of several X. fastidiosa isolates is now available and our analyses of this information indicate that each of the sequenced strains carries several prophages (integrated bacterial viruses). The long-term objective of this research project is to develop phage with biocidal activity against Xylella without
harming the vine. We have initiated research to develop culture methods for Xylella and a plate assay for phage detection, which are not currently available and to validate these methods. Our current research is the first phase in the development of a novel non-antibiotic approach for the control of Xylella, and will provide basic knowledge on genetic adaptation by Xylella as a plant pathogen.
PARTICIPANTS: Current Post docs and students Elizabeth J. Summer- Post Doc Jason Gill- Post Doc Ruifu Zhang- Post Doc Iris Duarte- Graduate Student Collaborators: Ryland F. Young III, Professor, Department of Biochemistry and Biophysics, Texas A&M University John J. LiPuma, M.D., Professor, Associate Chair for Research, Department of Pediatrics University of Michigan Grants funding current research: Phages of Agronomic Bacteria: a student based genomic project", National Science Foundation. Co-PIs: R. Young, C. F. Gonzalez, D. Adelson, and A Holzenburg $266,966; 10/1/05 - 9/30/08 Translocation of Virulence Proteins by Burkholderia cenocepacia", Cystic Fibrosis Foundation, PI: C.F. Gonzalez. Total Amount of Award: $194,400, Period Covered by Award: 5/1/06 - 4/31/08 Bacteriophages of Xylella fastidiosa: A potential control for Pierce's disease" USDA-APHIS- Texas Pierce's Disease Research and Education Program. PI:C.F. Gonzalez. Total Amount of Award: $59,750, Period covered by Award:
4/1/07 -3/31/08. Phages of Burkholderia cepacia: Biology and Therapeutics, NIH R01,Co-PIs R. Young, C. F. Gonzalez, and J.J. LiPuma.).Total Amount of Award: $1,342,065. Period Covered by Award:(4/1/06 - 3/31/10)
TARGET AUDIENCES: The Burkholderia research is aimed at determining the role secretion systems in pathogenicity. This research is important to patients with cystic fibrosis and is funded by the Cystic Fibrosis Foundation. The Burkholderia bacteriophage research is aimed at developing a potential method to abrogate bacterial infection caused by B. cenocepacia. This research is important to patients with cystic fibrosis and is funded by NIH. The development of a potential treatment for Pierce's disease caused by Xylella fastidiosa is of importance to persons in the grape growing industry and is funded by USDA-APHIS
Impacts
Since many virulence factors are translocated via secretion systems our research will provide information on the role that Type IV secretions systems play in the elaboration of virulence factors. Our bacteriophage research in both Burkholderia and Xylella are potential treatments for both pathogens.
Publications
- Summer, E.J., Gill, J.J., Upton, Gonzalez, C.F. and Young, R.F. 2007. Role of phages in the pathogenicity of Burkholderia, or, where are the toxin genes in Burkholderia phages? Current Opinion in Microbiology 10: 410-17.
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Progress 01/01/06 to 12/31/06
Outputs
In my laboratory we have identified two type IV secretion systems (TFSSs) in Burkholderia cenocepacia of the epidemic ET12 lineage; one system is plasmid encoded (designated the Ptw TFSS) whereas the other is chromosomally encoded (designated the VirB/D TFSS) and shows homology to the Agrobacterium tumefaciens VirB/D4 TFSS. Our studies indicate the Ptw-TFSS is involved in the translocation of a protein effector. Our hypothesis is that the chromosomal VirB/D TFSS found in ET12 strains is involved in the trafficking of additional substrates likely to be involved in DNA transfer. We have also determined that strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like TFSS (designated the BcVirB/D TFSS) and we have identified at least two putative effector proteins associated with this system that have C-terminal transport signals and sequences different from the effector proteins of the Ptw TFSS system. Current efforts in my laboratory are
focused on: (1) identifying Ptw effector protein (PtwE1) and (2) characterizing the chromosomal BcVirB/D TFSS of PHDC lineage strains and determining its potential role in pathogenesis in relevant models of infection.
Impacts
Our research will provide information on the translocation of effector molecules that are virulence factors of bacterial plant and human pathogens.
Publications
- Summer, E.J., Gonzalez, C. F., Bomer, M., Carlile, T., Embry, A., Kucherka, A. M. , Lee, J., Mebane, L., Morrison, W. C., Mark, L., King, M. D., LiPuma, J.J., Vidaver, A. .K., and Young, R. 2006. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex. J. Bacteriol. 188:255-268.
- Gonzalez, C.F., Venturi, V., and Engledow, A.E. 2006. The Phytopathogenic Burkholderia. In T. Coenye and P. Vandamme (ed) Burkholderia: Molecular Microbiology and Genomics. Horizon Scientific Press. Chapter 8, pp154-176.
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Progress 01/01/05 to 12/31/05
Outputs
Our laboratory recently identified two Type IV secretion systems (TFSSs) in Burkholderia cenocepacia of the epidemic ET12 lineage; one system is plasmid encoded (designated the Ptw TFSS) whereas the other is chromosomally encoded (designated the VirB/D TFSS) and shows homology to the Agrobacterium tumefaciens VirB/D4 TFSS. We determined that the plasmid encoded Ptw system is a chimeric TFSS composed of VirB/D4-like elements and F-specific subunits. We also found that this system translocates protein effectors (PtwE1 and PtwE2) that are cytotoxic to plant cells and induce an inflammatory response in respiratory epithelial cells in vitro. Additionally we determined that an extracellular factor(s) produced by cultured epithelial cells can upregulate the Ptw TFSS and effector proteins. Our preliminary studies indicate that the chromosomal VirB/D TFSS found in ET12 strains is involved in the trafficking of additional substrates likely to be involved in virulence. We have
also determined that strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like TFSS (designated the BcVirB/D TFSS) and we have identified at least four putative effector proteins associated with this system that have C-terminal transport signals and sequences different from the effector proteins of the Ptw TFSS system. Thus, we have identified three TFSSs in epidemic B. cenocepacia lineages. From two of these we have identified effector protein(s) that have cytotoxic effects on eukaryotic cells. Our preliminary studies also indicate that a factor(s) secreted from respiratory epithelial cells can upregulate the Ptw TFSS and effectors in B. cenocepacia.
Impacts
Our research will provide important new information for mechanisms involved in the translocation of virulence factors by bacterial pathogens.
Publications
- Engledow, A. E.,. Sajjan U., LiPuma, J.J., and Gonzalez, C.F. 2005. Burkholderia cenocepacia effectors cause plant tissue watersoaking and proinflammatory response in respiratory epithelial cells. International Burkholderia cepacia Working Group Meeting, Oklahoma City, OK.
- Engledow, A. S., J. J. LiPuma, and C. F. Gonzalez. 2005. Type IV Secretion System in Burkholderia cenocepacia of the PHDC Lineage. Phytopathology 95:S28 July 30- August 3, 2005 Austin TX
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Progress 01/01/04 to 12/31/04
Outputs
Our laboratory has determined that the plant watersoaking (ptw) phenotype common in B. cenocepacia strains of ET12 lineage is caused by a protein(s) that are translocated via a plasmid-encoded type IV secretion system. In addition we have identified a second chromosomally encoded type IV secretion system. The chromosomally encoded VirB-VirD-like type IV secretion system is not responsible for the translocation of the protein(s) involved in expression of the ptw phenotype. We are investigating its potential role in plasmid transfer. Using a genomic and proteomics approach along with the plant protoplast assay system we have identified putative structural gene(s) for the cytotoxic protein(s) translocated by the plasmid encoded system. Mutational analysis and complementation experiments will be used to verify the role of the putative gene(s) in producing the ptw phenotype. We will conduct time course studies to determine temporal production of the bioactive protein(s)
using the LacZYA reporter and Western blot analysis.
Impacts
Our research will lead to a better understanding of the mechanism by which virulence proteins are translocated by members of the B. cepacia complex.
Publications
- Summer, E., C. F. Gonzalez, T. Carlisle, L. M. Mebane, A. M. Cass, C.G. Savva, J. J. LiPuma and R. Young. 2004. Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. J. Mol. Biol. 340: 49-65.
- Engledow, A. S., E.G. Medrano, E. Mahenthiralingam, J. J. LiPuma and C.F. Gonzalez. 2004. Involvement of a Plasmid Encoded Type IV Secretion System in the Plant Tissue Watersoaking Phenotype of Burkholderia cenocepacia. J. Bacterol.186:6015-6024.
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Progress 01/01/03 to 12/31/03
Outputs
My laboratory is continuing our studies towards understanding the role of a cytotoxic protein(s) produced by Burkholderia cenocepacia and the secretion system responsible for its export. Our study is fundamental to efforts to understand the pathogenic process. We have identified two potential proteins that appear be involved in the watersoaking phenotype observed in B. cenocepacia. Mutants in genes designated ptw D, F, and G have been obtained using site-directed mutagenesis. Gene ptwD appears to be an ATPase, whereas genes ptwF and ptwG are involved in pilus formation. Using a vector developed in our laboratory we have also complemented the mutants in trans. Using plasposon mutagenesis we have now obtained watersoak-negative mutants of B. cepacia. Sequence analysis of the cloned sequences should help to identify potential genes involved. Our laboratory has now completed the entire sequence of four B. cepacia complex phages.
Impacts
Our research will determine genes and their products that are important to disease caused by members of the B. cepacia complex.
Publications
- Engledow,A.S. , E. G. Medrano , M. B. Willett , E. Mahenthiralingam , and C. F. Gonzalez . 2003 Plant tissue watersoaking: a type IV secretion system associated trait in Burkholderia cepacia strain K-56-2. 8th Annual International Burkholderia cepacia Working Group Meeting (Abstract)
- Summer, E.J., LiPuma, J.J., Young, R.F. and Gonzalez, C.F. 2003. Identification Of A Novel Bacteriophage Family For Burkholderia Cepacia Genomovars I And III 2003 North American CF Conference in Anaheim, California, (Abstract)
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Progress 01/01/02 to 12/31/02
Outputs
The search for new ways to combat microbial pathogens is an ongoing process in plant and animal pathogenesis. The understanding how a system functions and its role in the pathogenic process could lead to insight about potential targets to interfere a basic mechanism involved in the infection process and virulence. Understanding the role of a cytotoxic protein(s) produced by Burkholderia cepacia and the secretion system responsible for its export is fundamental to efforts for finding new targets to control virulence. Using plant protoplast our laboratory has identified a cytotoxic protein(s) that causes watersoaking (electrolyte leakage) in plant tissue and plasmolysis of plant protoplast. The protein(s) is exported from genomovar III B. cepacia cells via a type IV secretion system. We have determined that this watersoaking phenotype of common in B. cepacia of both plant and clinical origin. Using a genomic and proteomics approach along with the plant protoplast assay
system we will conduct experiments to identify the structural gene(s) for the cytotoxic protein(s) and purify the protein for analysis. We have identified the role of three of 11 gene in the identified type IV. Further analysis will determine the role of the remaining genes. Mutants in the plant watersoaking phenotype have now been obtained in the a plant strain and sequence analysis will determine if the genes involved in the plant pathogen are the same as those found in the clinical isolate. Our laboratory has also isolated from soil samples B. cepacia bacteriophage with an inter-genomovar host range. Complete sequence analysis of one phage has been completed and others are in progress.
Impacts
Our research will determine common themes in pathogenicity and determine new ways to combat microbial pathogens.
Publications
- LiPuma, J. J, T. Spilker, T. Coenye and C. F. Gonzalez.2002. Identification in soil of an epidemic Burkholderia cepacia complex strain. Lancet 359:2002-2003.
- Gonzalez, C. F., E.M. Provin, L. Zhu, and D. J. Ebbole. 2002. Independent and synergistic activity of synthetic peptides against thiabendazole resistant Fusarium sambucinum. Phytopathology 92: 917-924.
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Progress 01/01/01 to 12/31/01
Outputs
Burkholderia cepacia was first identified as a hytopathogen of onions. However, the ability of B. cepacia to act as a pathogen of both plants and humans represents a high degree of adaptability not expressed or observed in most bacterial species. Polygalacturonase negative phytopathogens produce a watersoaking (wts) phenotype in plant tissue. We have reported that the wts phenotype is also common among genomovar III (clinical) isolates. Using transposon mutagenesis we have generated a bank of 5000 mutants that are being evaluated for the wts phenotype to determine genes involved in the production, regulation or transport of the factor. Preliminary evidence indicate that a secretion system is involved in the transport. Our plan to is to continue to characterization of the bank of mutants to determine other genes involved in this phenotype and to extend our studies to determine the role of this phenotype in the pathogenic process of both plant and clinical strains. We
have also isolated from soil samples bacteriophage with inter-genomovar host-range. The primary goal of this project is to demonstrate that stable bacteriophage-mediated horizontal gene transfer can and does occur between species of the B. cepacia complex. Magnaporthe grisea, the causal agent of rice blast disease, is a major threat to rice production. Understanding the mechanisms by which compounds can inhibit appressorium formation is important to efforts to develop safe effective strategies to protect plants from appressorium producing fungi such as Magnaporthe, Colletotrichum ,and Blumeria. In preliminary results, we have found synthetic peptide pools that have significant activity in inhibiting appressorium formation. We have now identified several hexamers from the peptide combinatorial libraries that exhibit inhibitory activity in vitro. The inhibitory effect of the new peptides is bypassed by addition of extracellular cAMP (a known activator of appressorium development).
Several targets can be envisioned for the peptide mode of action.
Impacts
B. cepacia research will determine if a common pathogenicity factor exist in plant and clinical isolates of this pathogen. The use of bioactive peptides offers a novel approach to the control of diseases that are responsible for significant crop losses and the opportunity for the development of a range of non-toxic and non-persistent products to control disease
Publications
- Gonzalez C. F., Mark G. L., Mahenthiralingam, E., and LiPuma, J.J. 2001. Isolation of soilborne Genomovar I, III and VII Burkholderia cepacia and lytic phages with inter-genomovar host range. International B. cepacia Working Group Meeting. Niagara On the Lake.
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Progress 01/01/00 to 12/31/00
Outputs
Burkholderia cepacia has historically been regarded as a plant pathogen causing onion soft rot. However, B. cepacia is now of wider interest in agriculture, biotechnology, and medicine. Members of the taxonomically diverse Burkholderia cepacia complex have also become a major health risk for patients with cystic fibrosis. We have reported that plant isolates cured of a endo-polygalacturonase-encoding plasmid or that have a 35-kb deletion of the plasmid do not macerate plant tissue but still cause a watersoaking (wts) reaction in the tissue. Using transposon mutagenesis we have generated a series of wts-negative mutants in both a plant and clinical isolate. A BLAST search using the translated sequence of a clinical wts -negative mutant has revealed homology to a compoent of a secretion system. Since wts activity has been proposed to result from host cell electrolyte leakage induced by a pathogen effector molecule it is possible that a mutation in the system affects the
secretion of the effector involved in wts. Sequence analysis of several other mutants is being conducted in an effort to elucidate and compare wts symptoms produced by B. cepacia of plant and clinical origin. Magnaporthe grisea, the causal agent of rice blast disease, is a major threat to rice production. We are screening synthetic combinatorial libraries to identify novel peptides with anti appressorium activity. Screening of synthetic combinatorial libraries to identify a single hexapeptide from a pool of over 56 million is continuing. Our goal is to identify the optimal peptide or group of peptides from the peptide libraries that can inhibit appressorium formation and elucidate the mechanism.
Impacts
B. cepacia research will determine if a common pathogenicity factor exist in plant and clinical isolates of this pathogen. The use of bioactive peptides offers a novel approach to the control of diseases that are responsible for significant crop losses and the opportunity for the development of a range of non toxic and non-persistent products to control disease.
Publications
- Gonzalez, C.F., Mark,G.L., Mahenthiralingam, E. LiPuma, J.J. 2000. Isolation of soil-borne Genomovar III Burkholderia cepacia and lytic phages with inter-species host range. Pediatric Pulmonology, Suppl 20:288-289. 2000.
- Medrano, E. G. and Gonzalez, C.F. 2000. Genetic characterization of the plant watersoaking phenotype expressed by plant and clinical Burkholderia cepacia. Pediatric Pulmonology, Suppl 20:291-292. 2000
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Progress 01/01/99 to 12/31/99
Outputs
Burkholderia cepacia was first identified as a phytopathogen of onions. This plant pathogen is now recognized as an important pathogen in patients with cystic fibrosis and has become an important bioremediation agent and biocontrol agent. Recent studies have identified five genomic species (genomovars) of B. cepacia. We have evaluated 33 isolates of B. cepacia representing the five genomovars for plant associated pathogenicity traits. Plant,clinical, and environmental isolates were evaluated using PCR to determine the presence of the PehA gene and by blot analysis to determine plasmid association of PehA. Peh-negative phytopathogens produced a water-soaking(wts) phenotype in plant tissue. We have observed the wts phenotype in 14/15 genomovar III isolates tested. Genomovar II isolates tested did not express a wts phenotype whereas representatives of genomovars I,IV, and V showed variable results. Cultural supernatants of a Peh-negative phytopathogenic isolate indicate
that a inducible extracellular product is involved in the production of the wts phenotype in plant tissue. Magnaporthe grisea, the causal agent of rice blast disease, is a major threat to rice production. The additive costs of preventative chemical treatment and reduced yields from blast make this chronic disease an economically important global problem. A critical stage in the pathogenic process is the production of the infection structure, the appressorium. Chemicals that block appressorium maturation are effective anti blast agents. Recently, it was discovered that the peptide mating pheromone of yeast, alpha-factor, inhibits appressorium formation in M. grisea. This led us to screen synthetic combinatorial libraries to identify novel peptides with enhanced anti appressorium activity. Preliminary screening of synthetic combinatorial libraries led to the identification of peptide pools that efficiently inhibit appressorium formation of M. grisea. Our goal is to identify the optimal
peptide or group of peptides from the peptide libraries that can inhibit appressorium formation.
Impacts
B. cepacia research will determine if a common pathogenicity factor exist in plant and clinical isolates of this pathogen. The use of bioactive peptides offers a novel approach to the control of diseases that are responsible for significant crop losses and the opportunity for the development of a range of non toxic and non-persistent products to control disease.
Publications
- Medrano, E.M., Gonzalez, C.F. 1999. Evaluation of Genomovars I-V isolates of Burkholderia cepacia for plant disease associated traits. American Society for Microbiology Annual Meeting. (Abstract)
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Progress 01/01/98 to 12/31/98
Outputs
Since reporting the first plasmid encoded pectin degrading enzyme in the phytobacterial pathogen Burkholderia cepacia we have now determined that plasmid encoded endopolygalacturonases (Peh) are quite common. We have to date not found any transfer proficient Peh-encoding plasmids. We had reported that elimination of the Peh-encoding plasmid revealed water-soaking phenotype that was previously undetectable. This phenotype appears to be common in B. cepacia of plant and clinical origins. We are presently investigating the role of this phenotype in the pathogenic process. We have previously reported on the development of a hexapeptide with broad antifungal activity. We have now evaluated the potential of the peptide and derivatives to act on fungicide resistant fungal pathogens both independently and synergistically. The hexapeptide (66-10) exhibited an MIC of 10 micrograms(ug)/ml for thiabendazole(TBZ) resistant (MIC of 186 to 241 ug/ml) Fusarium sambucinum strains.
Heptapeptide derivatives 77-3 and 77-12 exhibited MICs of 4 and 7 ug/ml, respectively, against the same F. sambucinum strains. The results indicate that small peptides can act synergistically with TBZ.
Impacts
(N/A)
Publications
- Gonzalez, C.F., E.M. Provin, D.J. Ebbole, and M.T. Longnecker. 1998. Independent and synergistic activity of synthetic peptides against thiabendazole resistant Fusarium sambucinum. American Phytopathological Society Annual Meeting (abstract).
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Progress 01/01/97 to 12/31/97
Outputs
Our laboratory has identified and reported on the first plasmid encoded pectin degrading enzyme in a phytobacterial pathogen. The plasmid was identified in a phytopathogenic strain of Burkholderia cepacia (synonym Pseudomonas cepacia). The endopolygalacturonase (Peh) is encoded by a resident 200-kb plasmid (pPEC320) that is not transfer proficient. Preliminary evidence in our laboratory indicate that plasmid-borne pehA gene may be widely distributed in phytopathogenic B. cepacia. Elimination of the Peh-encoding plasmid has revealed necrosis that was previously undetectable. A systematic evaluation of a synthetic peptide combinatorial library of 400 peptide mixtures for biological activity against selected phytopathogenic fungi has been accomplished. A hexapeptide that exhibits activity against Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani, Pythium ultimum, and Ceratocystis fagacearum has been identified.
Impacts
(N/A)
Publications
- REED, J. D., D. L. EDWARDS, and C. F. GONZALEZ. 1997. Synthetic peptide combinatorial libraries: A method for the identification of bioactive peptides against phytopathogenic fungi. Mol. Plant-Microbe Interactions 10:537-549.
- GONZALEZ, C. F., E. A. PETTIT, V. A. VALADEZ, and E. A. PROVIN. 1997. Mobilization, cloning, and sequence determination of a plasmid encoded polygalacturonase from a phytopathogenic Burkholderia (Pseudomonas) cepacia. Mol. Plant-Microbe Interactions 10:840-851.
- REED, J. D., E. M. PROVIN, D. L. EDWARDS, and C. F. GONZALEZ. 1997. A synthetic peptide causes rapid death of fungal plant pathogens. American Society for Microbiology (abstract).
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Progress 01/01/96 to 12/30/96
Outputs
We have identified a plasmid encoded polygalacturonase (Peh) gene (pehA) from Burkholderia cepacia. The plasmid (pPEC320) in strain ATCC25416 was tagged with Tn5-Mob. The tagged plasmid was mobilized into B. cepacia strains of soil and clinical origin. Trans-conjugants containing pPEC321 expressed Peh and plant macerating activity. A 2.85 kb cloned from pPEC320 containing the plasmid-borne pehA gene was sequenced. Analysis of the protein sequence shows conserved domains that are found in the Peh sequence of Erwinia carotovora subsp. carotovora, Ralstonia solanacearum, and Lycopersicon esculentum. Studies on the potential of synthetic peptides to control plant pathogens have identified a pentapeptide (PPD1) with all five amino acids designated. Peptide PPD1 exhibited an IC50 of 31, 29, 31, and 7 g/ml for Fusarium oxysporum f.sp. lycopersici, Rhizoctonia solani, Pythium ultimum, and Ceratocystis fagacearum, respectively.
Impacts
(N/A)
Publications
- Reed, J. D., C. F. Gonzalez, and D. L. Edwards. 1996. A systematic identification of antifungal peptides. Phytopathology 86:S32
- Reed, J. D., D. L. Edwards, and C. F. Gonzalez. 1996 Synthetic Peptide Combinatorial Libraries: A Method for the Identification of Bioactive Peptides Against Phytopathogenic Fungi. Mol. Plant- Interactions. Submitted
- Gonzalez, C. F., M. A. Angell, and V. A. Valadez. Mobilization, Cloning, and Sequence Determination of a Plasmid Encoded Polygalacturonase From a Phytopathogenic Burkholderia (Pseudomonas) cepacia. In Preparation.
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Progress 01/01/95 to 12/30/95
Outputs
Analysis of the plasmid encoded polygalactoronase gene (pehA) from Pseudomonas cepacia has shown that the gene sequence is not similar to those reported for other phytopathogenic bacteria. The sequence appears to be more similar to that reported for a plant polygalactoronase. Analysis of the gene bank of a plasmid cured derivative, that is Peh-negative derivative and expresses a necrosis positive phenotype, has not yielded the gene responsible for its production. We have recently developed a suicide vector containing trimethoprim resistance flanked by IS50. This new vector will allow for the selection of random mutants at frequency similar to that obtained with Tn5. Studies on the potential of synthetic peptides to control plant pathogens have been initiated. The studies will test potential antimicrobial peptides for their activity against selected plant pathogenic fungi representing diverse groups.
Impacts
(N/A)
Publications
- Gonzalez, C.F., J.D. Reed, and D.L. Edwards. 1995. Synthetic peptide combinatorial libraries: An approach to identifying antimicrobials for the control of plant pathogens. Phytopathology 85:1126.
- White, T.J. and C.F. Gonzalez. 1995. Electroporation of Xanthomonas. In J. A. Nickoloff (ed.) Protocols for electroporation and electrofusion of microorganisms. Humana Press pp. 135-141.
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Progress 01/01/94 to 12/30/94
Outputs
Sequence analysis of the plasmid encoded polygalactoronase gene (pehA)from Pseudomonas cepacia has been completed and sequence comparison to known polygalacturonase genes is underway. A gene bank of the a plasmid cured derivative that is Peh-negative derivative that expresses a necrosis positive phenotype has been completed. Testing of clones to identify chromosomal genes responsible for the necrosis factor is underway. Additionally, transposon mutagenesis is being accomplished to obtain necrosis-negative mutants.
Impacts
(N/A)
Publications
- BROOKS, D. S., C. F. GONZALEZ, D. N. APPEL and T. H. FILER. 1994. Evaluation of Endophytic Bacteria As a Potential Biological Control Agent For Oak Wilt. Biocontrol, in-press.
- WHITE, T. J. and C.F. GONZALEZ. 1994. In "Electrofusion of Microorganisms", Electroporation of Xanthomonas (J. A. Nickloff, ed), Humana Press Inc, In Press.
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Progress 01/01/93 to 12/30/93
Outputs
The cloned structural gene for pectate hydrolase from P. cepacia has been characterized using Tn3-Gus mutagenesis to determine the structural limits of the gene and the direction of transcription. Sequence analysis of the structural genes is underway. In addition, a restriction map of the of the gene was constructed. One of the Tn3-gus mutants has been used in site-directed mutagenesis experiments to obtain Peh(superscript -) mutants of three additional strains of P. cepacia. The construction of the Peh(superscript -) mutants has allowed for the identification of a necrosis factor which is produced by P. cepacia. Present efforts are to clone the genes responsible for the production of the necrosis factor.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
1) Genetics of Pathogenicity. Our laboratory has previously identified a 200 kb plasmid that encodes for polygalacturonase in strain PCO25 of Pseudomonas cepacia. The structural gene has been cloned in a 5.3 Kb Sstl fragment. This fragment has been subcloned as a 3.5 Kb BamHI-Sstl fragment. A series of Tn3-Gus insertions of the 3.5 kb fragment have been obtained. The Tn3-Gus fusions will used to determine the physical limits of the gene and the transcriptional organization. Using the cloned gene as a probe we have identified plasmid encoded polygalacturonase genes in at least 5 other phytopathogenic strains of Pseudomonas cepacia. 2) Beneficial bacteria- plant interactions. Studies to identify potential biological controls for oak wilt are continuing. In cooperation with the forest pathology program that is studying pathogen survival in tree roots, our laboratory is surveying for indigenous bacteria that may act as biocontrol agents. We have isolated Pseudomonas
species that produce phenazine-1-carboxylic acid from root samples. The isolates obtained exhibit a high degree of in vitro inhibition to Ceratocystis fagacearum. The isolates are being evaluated as potential biocontrol agents. Plant colonization studies with phyllosphere bacteria isolated from rice plants are continuing. The isolates being tested show inhibitory activity towards a several fungal rice pathogens.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
1) Genetics of Pathogenicity. We have identified plasmid pPEC320 in P. cepacia that carries the structural gene for the production of a single polygalacturonase with a pl of 8.1. The resident plasmid (pPEC320) in strain PCO25 was labeled with Tn5-Mob. The pPEC320::Tn5Mob (pPEC321) plasmid was mobilized into soil and clinical strains of P. cepacia. Transconjugants containing pPEC321 express a pec(superscript +) phenotype, macerate plant tissue, and can act as donors in conjugal matings. The structural gene(s) for polygalacturonase production by P. cepacia has been cloned and are presently being characterized. 2) Beneficial bacteria - plant interactions: The potential of the microbial population of the rice plant to control or reduce loss due to disease caused by certain plant pathogenic fungi is being explored. We have completed the initial screening of 215 individual bacterial isolates obtained from the rice rhizosphere and phyllosphere of plants. The bacteria were
individually screened for their in vitro ability to inhibit disease causing fungi such as Rhizoctonia solani (Sheath blight), R. oryzae (Sheath spot), Gaeumannomyces graminis var. graminis (Black Stem Rot), and Pyricularia oryzae (Rice Blast). Of the 215 isolates tested, 7 showed broad spectrum inhibition against the pathogens tested. Plant colonization and persistence studies are being conducted.
Impacts
(N/A)
Publications
- WHITE, T.J. and GONZALEZ, C.F. 1991. Application of electroporation for efficient transformation of Xanthomonas campestris pv. oryzae. Phytopathology 81:521-524.
- XU, G.W. and GONZALEZ, C.F. 1991. Plasmid, genomic, and bacteriocin diversity in USA strains of Xanthomonas campestris pv. oryzae. Phytopathology. 81:628-631.
- WHITE, T.J. and GONZALEZ, C.F. 1991. Positive selection and isolation of an insertion sequence element of Xanthomonas campestris pv. oryzae. 1991. 91st Meeting of American Society for Microbiology.
- GONZALEZ, C.F. and ANGELL, M.E. 1991. Conjugal transfer of polygalacturonase-encoding plasmid from Pseudomonas cepacia. Annual Meeting American Phytopathological Society. St. Louis, Mo.
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Progress 01/01/90 to 12/30/90
Outputs
Genetics of Pathogenicity: An efficient method for the electroporation of plasmid DNA into Xanthomonas campestris pv. oryzae (Xco) has been developed in our laboratory. Currently we are able to obtain 10 transformants per mu g DNA. This method has been used to introduce a constructed transposon trapping plasmid into Xco. We have isolated an insertion sequence (IS) from Xco strain X37-2 (US strain). The IS will be characterized and evaluated as a potential RFLP probe and will be compared to an IS isolated from an Asian strain of Xco. Beneficial bacteria- plant interactions: Field inoculations to evaluation the survival, distribution and ability of selected bacterial strains to act as potential biocontrol agent for oak wilt have been completed. Preliminary data indicate some survival and random distribution of the introduced bacteria. Partial purification of the antifungal compound produced by strain 1-15 has been accomplished and the biospectrum of the compound has
been determined.
Impacts
(N/A)
Publications
- GERHING, E.H., APPEL, D.N., GONZALEZ, C.F. and FILER, T.H. 1990. Environmental fitness of selected endophytic bacteria: A potential biocontrol for oak wilt. Phytopathology 80:1011 (Abstr.).
- WHITE, T.J. and GONZALEZ, C.F. 1990. Optimizaton of electrical parameters for efficient electroporation of plasmid DNA into xanthomonas campestris pv. oryzae Phytopathology 80:965. (Abstr.).
- GONZALEZ, C.F., XU, G.W., LI, H.L. and COSPER, J.W. 1991. leersia hexandra, an alternative hosts for Xanthomonas campestris pv. oryzae in Texas. Plant Disease 75: (in-press).
- WHITE, T.J. and GONZALEZ, C.F. 1991. Application of electroporation for efficient transformation of Xanthomonas campestris pv. oryzae. Phytopathology 81:(in-press).
- XU, G.W. and GONZALEZ, C.F. 1991. Plasmid, genomic, and bacteriocin diversity in USA strains of Xanthomonas campestris pv. oryzae. Phytopathology 81:(Accepted for Publication).
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Progress 01/01/89 to 12/30/89
Outputs
Genetics of Pathogenicity: Studies to determine the role of extracellular enzymes in pathogenesis of Xanthomonas campestris pv. oryzae (Xco), were conducted. Transposon Tn4431 was used to obtain mutants in the production of extracellular enzymes. Five classes of mutants were obtained. One class included protease mutants which were reduced in pathogenicity. Population dynamics studies in planta showed the mutant population to be 50-100 fold lower than that observed for the parental strain. The results indicated that protease may play an active role in disease caused by Xco. Beneficial bacteria-plant interaction: Studies conducted during the collection of endophytic bacteria to act as potential biological control agents indicate that there is a seasonal fluctuation in the population of endophytic bacteria which are able to show in vitro inhibition to the oak wilt fungus. The population shifts observed appear to be between two genera, Pseudomonas and Bacillus. The shifts
were observed over a one year period in three separate location in Texas. Studies to determine the survival of introduced bacteria into a closed tree vascular system under natural conditions have been initiated.
Impacts
(N/A)
Publications
- BENEDICT, A.A., ALVAREZ, A.M., BERETECKY, J, IMANNKA, W, MIZUMOTO, C.Y., POLLARD, T.W., MEW, & GONZALEZ, C.F. 1989. Relaationship among phytopathogenic bacteria defined with monoclonal antibodies. I. Surface antigens specific for.
- JONES, R.K., BARNES, L.W., GONZALEZ, C.F., LEACH, J.E., ALVAREZ, A.A. and BENEDICT, A.A. 1989. Ident. of low virulence isolates of Xanthomonas campestris pv. oryzae from rice in the 79:984-990.
- XU, G.W. and GONZALEZ, C.F. 1989. Evaluation of TN4431 induced protease mutants of Xanthomonas campestris pv. oryzae for growth in planta and pathogenicity. Annual APS Meetings, Richmond, VA. No. 599 (Abstr.)
- GONZALEZ, G.F., XU, G.W. and LI, H.L. 1989. Leersia hexandra, an alternate host for Xanthomonas campestris pv. oryzae in Texas. Annual APS Meetings, Richmond, Va. No 659(Abstr.).
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Progress 01/01/88 to 12/30/88
Outputs
Genetics of pathogenicity: Analyses of USA strains of Xanthomonas campestris pv. oryzae (Xco) has shown them to be related but distinct from strains isolated from Asia and South America. A semi-selective medium has been developed for the isolation of Xco from plant material and seed. The medium has been used in studies that have identified Leersia hexandra (clubhead grass) as an alternate host for Xco in the USA. Bacteriocin typing studies indicate the presence of 3 production groups in the 1987 isolates and a new, fourth group, in the 1988 isolates. Other studies include the physical analysis of USA-Xco isolates for the presence of plasmid DNA. This study has shown that of 27 isolates analyzed, 24 from Texas and 3 from Louisiana, 20 were observed to harbor resident plasmids. The Xco isolates were divided into three groups based on the size of the resident plasmid(s). Group A consists of 14 isolates and harbors a plasmid of 31.7 kb. Group B contains 3 isolates
and harbors a 24.4 kb plasmid. Group C (3 strains from Louisiana) harbor a 16.4 kb and 15.5 kb plasmid. Beneficial bacteria-plant interaction: Studies indicate that Bacillus spp. and Pseudomonas spp. are able to colonize Spanish oaks and live oaks, respectively. Challenge experiments in live oaks using a pre-treatment of one Pseudomonas isolate has shown that the treatment significantly (P=.05) reduced disease symptoms over the control treatment.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Genetics of pathogenicity: A procedure for obtaining mass quantities of Pseudomonas cepacia plasmid DNA has been developed. Restriction enzyme analysis of plasmid DNA from non-pectolytic mutants using Bg1II has confirmed a deletion of at least 25 MDa1 from the parental 120 MDa1 plasmid. Transformation of a Group W cloning vector into soil P. cepacia has been accomplished at frequencies of 3x10 transformants/ug of DNA. Transformation of plant P. cepacia with P. cepacia-modified DNA has yielded several putative transformants. This DNA will now be purified for further transformation studies. Identification of Xanthomonas campestris pv. oryzae has been confirmed on rice in Texas. A specific bacteriophage and bacteriocin have been identified. Beneficial bacteria-plant interactions: Biological control of oak wilt: Identification of endophytic bacteria isolated from the functional sapwood of oak trees is continuing. Strains producing siderophores, toxic peptides, and
chitinase have been isolated. A group of six isolates have been evaluated for their ability to colonize 1-gallon containerized oak trees. Qualitative studies have shown that two isolates are able to colonize plant tissue.
Impacts
(N/A)
Publications
- BROOKS, D.S., GONZALEZ, C.F., APPEL, D.N. and FILER, T.H. 1988. Isolation and Evaluation of Endophytic bacteria from Live Oaks for In vitro Inhibition to Ceratocystis fagacearum and Colonization of Oak Trees. (Abstr.).
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