Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
BACTERIAL PROTEINS AND ACTIVITIES OF AGRICULTURAL INTEREST
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005042
Grant No.
(N/A)
Project No.
CA-D-MMG-7235-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 9, 2014
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Parales, R.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Microbiology & Molecular Genetics
Non Technical Summary
Sustainable agriculture requires the availability of pollutant-free soils and water sources. A primary goal of this research is to characterize bacteria that degrade chemical pollutants relevant to California. In this study we will characterize the metabolic pathways and sensory systems used by bacteria to detect and degrade natural and man-made chemicals.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1334010110075%
5114010110025%
Knowledge Area
133 - Pollution Prevention and Mitigation; 511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
A primary goal of this research is to characterize bacteria that degrade chemical pollutants relevant to California. As part of this study, we will characterize the metabolic pathways used by bacteria to break down natural and man-made chemicals, including aromatic hydrocarbons (components of petroleum and gasoline) and nitroaromatic compounds (used as solvents and in the production of dyes, pigments, polymers, pesticides, herbicides, and explosives). In addition, we will evaluate and characterize the ability of specific biodegradative bacterial strains to sense and respond to specific chemicals using chemotaxis. This ability could enhance bacterial degradation of pollutants in the environment, and the identified chemotaxis proteins that are capable of binding specific pollutants can be used to develop useful biosensors to detect environmental contamination and contamination in food. Chemicals to be tested as attractants include aromatic hydrocarbons, nitroaromatic compounds, and structurally-related naturally-occurring aromatic compounds.Specific objectives for the project include: 1) generating bacterial strains capable of growing on multiple nitroaromatic contaminants; 2) characterizing the regulation of genes responsible for the degradation of nitroaromatic compounds; 3) initiating studies to examine how bacteria sense multiple chemicals at one time and determining how they prioritize which chemicals to respond to; 4) identifying the chemicals recognized by cell-surface sensory proteins from environmentally-relevant bacteria and using this information to develop biosensors for the detection of environmental pollutants.
Project Methods
Objective 1. Development of bacterial strains that degrade multiple nitroaromatic pollutants. Our laboratory studies natural bacterial strains from contaminated environments that are capable of degrading a variety of toxic chemical pollutants. One bacterial strain, Acidovorax sp. JS42 grows on the toxic industrial chemicals 2-nitrotoluene and nitrobenzene, but is not capable of growth on the similar chemicals 3-nitrotoluene or 4-nitrotoluene. Since contaminated sites typically contain mixtures of contaminants, it is desirable to obtain bacteria capable of eliminated a wide range of toxic pollutants. We have evolved JS42 in the laboratory to grow on 3-nitrotoluene or 4-nitrotoluene, and the resulting strains retain the ability to grow on 2-nitrotoluene and nitrobenzene. We identified the genetic changes in these newly evolved strains that allow growth on these additional chemicals. We have recently taken a variant strain that is capable of growth on 4-nitrotoluene and evolved it to grow on 3-nitrotoluene. We propose to characterize several of these newly evolved strains that are capable of growth on all four nitroaromatic pollutants by determining the mutations present, the activity of the evolved enzymes, and most importantly for environmental remediation applications, the ability of the strains to grow on mixtures of nitroaromatic substrates.Objective 2. Characterize the regulation of genes for nitroarene degradation.We have characterized the regulation of the genes encoding the initial enzyme of the 2-nitrotoluene degradation pathway in Acidovorax sp. strain JS42. We propose to characterize the regulation of the remaining genes required for 2-nitrotoluene degradation, which are located in a single cluster. Two predicted regulatory genes are present in the cluster and we will determine if one or both of the encoded regulatory proteins control gene expression, and under what conditions they do so. We will also follow up on preliminary evidence indicating that the genes are under catabolite control- that is they are not expressed when an alternative easily degraded carbon source is present. This is a critical question when deciding to use a bacterial strain for environmental clean-up, as the strains may not be efficient for bioremediation if they preferentially use other carbon sources before degrading the pollutants.Objective 3. Determine how bacteria 'choose' which chemical to follow when presented with multiple chemical attractants.Bacteria are capable of sensing chemicals by use of specific cell surface proteins. These cell-surface receptors allow bacteria to move towards or away from specific beneficial or detrimental chemicals, using a process called chemotaxis. We have shown that the bacterial strains under study for their ability to degrade pollutants are also chemotactic to specific pollutants. We have identified the functions of 8 of the 27 predicted cell-surface receptor proteins in the aromatic hydrocarbon degrading strain Pseudomonas putida F1; these receptors mediate chemotaxis to >35 different chemicals. What has not been studied in any microorganism with such a complex sensory system is the mechanism by which the cells discriminate between competing chemical attractants and 'choose' which chemical to follow. This study will evaluate the role of metabolic preferences in the chemotaxis decision-making process by challenging cells with two different types of nutrients that serve as attractants. These studies will lay the groundwork for predicting cellular behavior in complex environments such as soils or sediments. We will subject cells to gradients of two different attractants in microfluidic chambers and evaluate population behavior by time-lapse microscopic imaging of the cells. For these studies we have teamed with Prof. William Ristenpart, in the Department of Chemical Engineering and Materials Science at UC Davis, who is an expert in microfluidics and microscopic image analysis.Objective 4. Functional characterization of bacterial chemoreceptors and development of biosensors.P. putida F1 is a bacterial strain that is capable of degrading a wide range of chemicals, and it is chemotactic to most of these chemicals. As described above, bacterial chemotaxis involves binding of chemicals to specific cell surface receptors called methyl-accepting chemotaxis proteins (MCPs). Two-component regulatory systems, which mediate changes in gene expression in response to specific chemicals, are similar to MCPs in molecular architecture and mechanism of action. We propose to construct hybrid sensors containing the sensory (input) domain of bacterial MCPs and the signaling domain (output) of a 2-component regulator as a strategy to identify the functions of the uncharacterized MCPs in P. putida F1. A major focus will be on the identification of the receptor(s) for the toxic aromatic hydrocarbons toluene, benzene, and ethylbenzene, as the hybrid MCPs are expected to be useful as biosensors. This project will ultimately allow us to: i) identify the chemoreceptor for aromatic hydrocarbons and the functions of the uncharacterized MCPs in P. putida F1; and ii) construct useful biosensors for the detection of aromatic hydrocarbons and other pollutants - one important application could be the detection of oil-contaminated shellfish.As proof-of-concept, we will first generate a series of hybrid MCP sensors using a functionally characterized P. putida F1 MCP in order to identify the optimal fusion points for obtaining functional and sensitive signal transduction proteins. We will use the recently characterized P. putida F1 receptor PcaY, which detects several aromatic acids as attractants, and the nitrate-responsive E. coli NarX and NarQ proteins, which are studied by Professor Valley Stewart in my department, will serve as the output domains. The Stewart laboratory will provide an E. coli reporter strain in which the signal can be detected by a simple colorimetric enzyme assay. Using this strategy, we will generate a series of similar fusions with the genes encoding the functionally uncharacterized P. putida F1 MCPs and screen a wide range of chemicals to identify the attractants and repellants detected by each. Identification of the aromatic hydrocarbon receptor(s) will provide the basis for developing simple and sensitive biosensors for hydrocarbon contamination using alternative reporters such as light production (using luminescence genes) fluorescence (e.g. using green fluorescent protein), or other simple colorimetric enzyme assays.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Target audiences include the general public (by providing scientific education contributing to science literacy), high school and undergraduate students (by providing microbiology research opportunities), and the State of California (by providing new research findings regarding the role of bacteria in the degradation of pollutants that affect our soil and groundwater). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the review period I hosted a visiting professor who wanted to gain experience in molecular microbiology and chemotaxis methods (1-year visit), as well as a visiting undergraduate student from BYU Idaho (2 month visit). Each year, approximately 35 graduate students, 25 postdoctoral fellows and 6 undergraduates attend the Annual Meeting of the West Coast Bacterial Physiologists, which I co-organize. In a typical year, approximately one-third of the undergraduates and at least half of the graduate students and postdoctoral fellows give oral research presentations at the meeting. The meeting is also attended by ~20-25 faculty members each year, who provide constructive feedback on research presentations to students at all levels and which allows the highly motivated undergraduate attendees the opportunity to discuss potential graduate training opportunities. How have the results been disseminated to communities of interest?Education and Public Awareness. As part of my outreach to the community, I participate in the Department of Microbiology's Picnic Day demonstration. The demonstration has interactive displays about the beneficial and harmful aspects of microorganisms. I contributed a display about bacteria that are capable of degrading toxic components of petroleum and explosives. Integration of research findings into the undergraduate curriculum. I regularly update my lecture material in undergraduate courses with recent research findings from my laboratory, including bacterial degradation of toxic pollutants, evolution of bacterial enzymes that function on man-made chemicals, and mechanisms used by bacteria to sense pollutants and man-made chemicals. In my undergraduate Microbial Diversity Laboratory, students participate in ongoing research projects to identify new environmental bacteria with beneficial activities. For the past eight years I have co-organized the Annual Meeting of the West Coast Bacterial Physiologists. The primary goal of this meeting is to provide postdoctoral, graduate and undergraduate student trainees a venue at which to gain experience and confidence in formal research presentations, and to interact with other trainees and senior scientists who share similar research interests. I obtain external funding to subsidize the registration fees for postdoctoral, graduate and undergraduate student trainees. Each year my students contribute to the set up of the biotechnology and/or the microbiology demonstrations during "Picnic Day", which is the annual UC Davis open house event. These demonstrations highlight to the public the importance of bacteria in our daily lives, not only those the cause disease, but those that have beneficial or useful properties, like breaking down pollutants or producing industrially useful enzymes. What do you plan to do during the next reporting period to accomplish the goals?During the next grant period, we will plan to complete and publish studies of bacterial chemotaxis to alcohols by Pseudomonas putida F1, and we will continue studies of chemotaxis to caffeine and methanol by the new Methylobacterium isolate that can grow on caffeine as sole carbon and energy source. I am also beginning collaborations with visiting scientists from China who will be studying bacterial chemotaxis to nicotine, aromatic hydrocarbons, and a variety of man-made herbicides.

Impacts
What was accomplished under these goals? My laboratory and others have shown that chemotaxis plays an important role in bacterial biodegradation. We have been continuing to characterize the functions of the 27 putative chemoreceptor proteins in Pseudomonas putida F1 and document the growing list of chemicals that are sensed by this organism. P. putida F1 is capable of growth on and complete degradation of a wide range of both toxic and non-toxic chemicals (naturally occurring sugars, amino acids, carboxylic acids, alcohols, and aromatic acids, as well as man-made aromatic hydrocarbons, etc.). Work in our laboratory indicates that this bacterial strain is capable of sensing most (if not all) of the chemicals it grows on using either metabolism-independent chemotaxis or metabolism-dependent energy taxis. We have expanded our bacterial chemotaxis studies to include chemotaxis by a newly isolated Methylobacterium strain to alcohols (specifically methanol and ethanol) and caffeine. This organism was isolated in my undergraduate Microbial Diversity laboratory for its ability to grow on caffeine as sole carbon, nitrogen, and energy source. We have obtained a draft genome sequence of the strain and identified putative genes for caffeine degradation pathway and for chemotaxis, and are characterizing its chemotaxis responses. We have begun a collaboration with Professor Michael Savageau to study the connections between the degradation of aromatic acids and the ability to sense these chemicals using chemotaxis in the model organism Pseudomonas putida F1. The study has been funded by the National Science Foundation and we are in the process of hiring postdoctoral fellows to participate in the research. The project has both wet lab (experimental) and computational (modeling) aspects, and our two groups will be working together to further our understanding of this complex sensory and signaling process, while also developing Prof. Savageau's phenotype-centric modeling methods. Characterization of bacterial chemoreceptor function. During this review period we characterized the chemotactic responses of Pseudomonas putida F1 to p-coumaric acid, ferulic acid, and caffeic acid. These chemicals are lignin monomers that are abundant in the soil environment due to the breakdown of plant cell walls. They serve as good carbon and energy sources for P. putida, and we identified genes required for their catabolism. We showed that the chemotactic response is inducible and metabolism dependent, and is mediated by the energy taxis receptor Aer2, which we previously reported is required for taxis to phenylacetic acid. This study was published in the journal Microbiology. My undergraduate students in Microbial Diversity Laboratory isolated a novel Methylobacterium strain that is capable of growth on caffeine. Like other members of this species, it is a methylotroph capable of growth on methanol. Interestingly, we have been unable to find reports of bacterial chemotaxis to alcohols, so we began investigating chemotaxis to methanol, ethanol (also a good carbon and energy source for the strain), and also to caffeine in this strain. We have documented strong chemotaxis to alcohols and weak but clear chemotaxis to caffeine. We obtained a draft genome sequence and annotated genes putatively involved in caffeine catabolism and chemotaxis. We have preliminary evidence that we can genetically manipulate the strain and obtained plasmids that are used to generate mutants of the model methylotroph Methylobacterium exorquens AM1. Our next step will be to generate mutants to confirm the function of caffeine pathway genes and attempt to identify the chemoreceptors for alcohols and caffeine. Since we demonstrated chemotaxis to alcohols in Methylobacterium, we began wondering whether P. putida F1 was capable of chemotaxis to alcohols, and if so, which receptor(s) were involved. We knew that ethanol is a good carbon and energy source for P. putida F1, and we found that linear alcohols with from three to at least twelve carbons are also good carbon and energy sources (doubling times < 3 hr), and all are sensed as attractants by P. putida F1. We screened our library of receptor mutants and found that a single receptor, which we designated McfP, is responsible for chemotaxis to ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and 1-dodecanol. The response to alcohols was inducible. We are currently setting up to test whether the mcfP gene itself is inducible. Once the last few experiments are complete, we will submit a manuscript reporting these data to Applied and Environmental Microbiology.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: J.G. Hughes, X. Zhang, J.V. Parales, J.L. Ditty and R.E. Parales. 2017. Pseudomonas putida F1 uses energy taxis to sense hydroxycinnamic acids. Microbiology 163:1490-1501.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: R.E. Parales, R. Navara, R. Gettys and J. Huang. 2017. Genome sequence of Pseudomonas putida ASAD, an acetylsalicylic acid degrading bacterium. Genome Announc. 5:e01169-17.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: R.E. Parales and J.L. Ditty. 2017. Substrate transport. In: Handbook of Hydrocarbon and Lipid Microbiology. T. Krell, (ed.) Springer-Verlag, Berlin.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: R.E. Parales and J.L. Ditty. 2017. Chemotaxis. In: Handbook of Hydrocarbon and Lipid Microbiology. T. Krell, Ed. Springer-Verlag, Berlin.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: R.E. Parales and J.L. Ditty. 2017. Chemotaxis to atypical attractants by soil bacteria. Methods in Molecular Biology. M. Manson, Ed. Springer-Verlag, Berlin. (Invited review in press).


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audiences include the general public (by providing scientific education contributing to science literacy), high school and undergraduate students (by providing microbiology research opportunities), and the State of California (by providing new research findings regarding the role of bacteria in the degradation of pollutants that affect our soil and groundwater). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I have hosted 3 undergraduate researchers in my laboratory during the grant period. Each year, approximately 35 graduate students, 25 postdoctoral fellows and 6 undergraduates attend the Annual Meeting of the West Coast Bacterial Physiologists, which I co-direct. In a typical year, approximately one-third of the undergraduates and at least half of the graduate students and postdoctoral fellows give oral research presentations at the meeting. The meeting is also attended by ~20-25 faculty members each year, who provide constructive feedback on research presentations to students at all levels and which allows the highly motivated undergraduate attendees the opportunity to discuss potential graduate training opportunities. How have the results been disseminated to communities of interest?Education and Public Awareness. As part of my outreach to the community, I participate in the Department of Microbiology's Picnic Day demonstration. The demonstration has interactive displays about the beneficial and harmful aspects of microorganisms. I contributed a display about bacteria that are capable of degrading toxic components of petroleum and explosives. Integration of research findings into the undergraduate curriculum. I regularly update my lecture material in undergraduate courses with recent research findings from my laboratory, including bacterial degradation of toxic pollutants, evolution of bacterial enzymes that function on man-made chemicals, and mechanisms used by bacteria to sense pollutants and man-made chemicals. What do you plan to do during the next reporting period to accomplish the goals?During the next grant period, we will finalize the remaining experiments for publication of the hybrid receptor work and will expand this work for the analysis of receptors from other bacteria and even fro receptors encoded in metagenomic data.

Impacts
What was accomplished under these goals? A reporter assay was developed in collaboration with Professor Valley Stewart (Department of Microbiology and Molecular Genetics, UC Davis) that allows in vivo screening for the direct binding of chemical attractants to bacterial chemoreceptor proteins. The method can also be used to identify chemicals sensed by bacterial chemoreceptors of unknown function. To demonstrate the validity of this reporter system and to show that the aromatic acid chemoreceptor PcaY directly binds a wide range of aromatic attractants, we fused the ligand-binding domain of PcaY to the signaling domain of the nitrate receptor protein NarQ from Escherichia coli. The gene encoding this fusion protein was expressed in an Escherichia coli reporter strain developed by Professor Stewart, which allows one to detect the output of PcaY binding to chemical ligands as a simple colorimetric enzyme assay (LacZ). We showed that the fusion protein sensitively and specifically detected known attractants that are sensed by PcaY in a concentration dependent fashion. Structurally similar aromatic compounds that are not sensed as chemoattractants by PcaY in P. putida were not capable of signaling. Our collaborators in China used isothermal titration calorimetry to demonstrate that the purified ligand-binding domain of PcaY could bind directly to aromatic acid attractants. The strength of binding to various aromatic chemicals correlated well with the strength of the chemotactic responses in the native bacterial strain as well as to the level of LacZ activity in Escherichia coli expressing the hybrid PcaY-NarQ protein. We also showed that a hybrid receptor constructed using the chemoreceptor for malate, succinate and fumarate (McfR) from Pseudomonas putida functioned as expected, sensitively and specifically detecting these chemicals, but not detecting the structurally similar dicarboxylic acid citrate, which is not sensed by McfR in the native host. Ten of 14 additional hybrid receptors constructed using the same junction point to NarQ were shown to be capable of signaling in Escherichia coli, indicating that they are functional. Using the screening assays that we developed, we identified four hybrid receptors that detect propionate. We verified that propionate is a good attractant for Pseudomonas putida, and verified the function of these four receptors in propionate chemotaxis by constructing and analyzing the appropriate mutant strains. We are writing a manuscript describing this work.

Publications

  • Type: Book Chapters Status: Published Year Published: 2016 Citation: W.A. Tan and R.E. Parales. 2016. Dioxygenases and their applications. In: Biocatalysis: Green Technology. R. Patel, Ed. John Wiley & Sons.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: S.G. Pati, H.-P. E. Kohler, A. Pabis, P. Paneth, and R.E. Parales, T.B. Hofstetter. 2016. Substrate and enzyme specificity of the kinetic isotope effects associated with the dioxygenation of nitroaromatic contaminants. Environ. Sci. Technol. 50:6708-6716.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Z. Huang, B. Ni, C.-Y. Jiang, Y.-F. Wu, Y.-Z. He, R.E. Parales, and S.-J. Liu. 2016. Direct sensing and signal transduction during bacterial chemotaxis towards aromatic compounds in Comamonas testosteroni. Mol. Microbiol. 101:224-237.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: J.L. Ditty, N.N. Nichols and R.E. Parales. 2017. Protocols for the measurement of hydrocarbon transport in bacteria. In: Protocols for Hydrocarbon Microbiology K.N. Timmis, (ed.) Springer-Verlag, Berlin.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: J.L. Ditty and R.E. Parales. 2017. Protocols for the measurement of hydrocarbon chemotaxis in bacteria. In: Protocols for Hydrocarbon Microbiology K.N. Timmis, (ed.) Springer-Verlag, Berlin.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: C.A. Rabinovitch, J.L. Ditty and R.E. Parales. 2017. Motility and Chemotaxis of Acidovorax. In: Plant Pathogenic Acidovorax species. S. Burdman and R. Walcott, Eds. American Phytopathological Society Press (Invited review in press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: R.A. Luu, R. Truong, C. Brunton, M. Lee, J.V. Parales, V. Stewart, and R.E. Parales. Biosensors for the Identification of Chemoattractants. Nara Institute of Science and Technology (NAIST) Workshop and Symposium on Stress adaptation/Environmental responses, Nara, Japan. November 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: R.A. Luu, R. Truong, C. Brunton, M. Lee, J.V. Parales, V. Stewart, and R.E. Parales. Biosensors for the Identification of Chemoattractants. Abstr. Annual Meeting of the West Coast Bacterial Physiologists, Pacific Grove, CA, December 2015.


Progress 10/09/14 to 09/30/15

Outputs
Target Audience:Target audiences include the general public (by providing scientific education contributing to science literacy), high school and undergraduate students (by providing microbiology research opportunities), and the State of California (by providing new research findings regarding the role of bacteria in the degradation of pollutants that affect our soil and groundwater). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I actively participate in the UC Davis Young Scholars Program, which brings top high school students to the campus for summer research experience. I have hosted seven summer YSP students since 2009, as well as a Davis High School student. I have also hosted 13 undergraduate researchers in my laboratory during the grant period. Each year, approximately 35 graduate students, 25 postdoctoral fellows and 6 undergraduates attended the Annual Meeting of the West Coast Bacterial Physiologists, which I co-direct. In a typical year, approximately one-third of the undergraduates and at least half of the graduate students and postdoctoral fellows give oral research presentations at the meeting. The meeting is also attended by ~20-25 faculty members each year, who provide constructive feedback on research presentations to students at all levels and which allows the highly motivated undergraduate attendees the opportunity to discuss potential graduate training opportunities. How have the results been disseminated to communities of interest?Education and Public Awareness. As part of my outreach to the community, I participate in the Department of Microbiology's Picnic Day demonstration. The demonstration has interactive displays about the beneficial and harmful aspects of microorganisms. I contributed a display about bacteria that are capable of degrading toxic components of petroleum and explosives. Integration of research findings into the undergraduate curriculum. I regularly update my lecture material in undergraduate courses with recent research findings from my laboratory, including bacterial degradation of toxic pollutants, evolution of bacterial enzymes that function on man-made chemicals, and mechanisms used by bacteria to sense pollutants and man-made chemicals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Bacterial sensing of aromatic compounds. 1. We identified a bacterial chemoreceptor protein for aromatic compounds in Pseudomonas putida. This cell-surface receptor (PcaY) is a methyl-accepting chemotaxis protein (MCP) that mediates the detection of two hydroaromatic compounds and five aromatic acids all of which serve as good carbon and energy sources for P. putida, as well as 9 other substituted benzoates that are not degraded by the strain. We showed that the gene encoding the MCP is coordinately regulated with genes for aromatic acid catabolism. The same transcriptional activator (PcaR) and intermediate in aromatic compound degradation (beta-ketoadipate) are required for expression the gene encoding PcaY and expression of aromatic acid catabolism genes. We also elucidated long-standing mystery of the role of the 4-hydroxybenzoate (4HBA) transport protein PcaK in the chemotactic response to 4HBA. PcaK is required to allow entry of sufficient 4HBA for production of the inducer beta-ketoadipate. This study was published in Molecular Microbiology. 2. In collaboration with Professor Valley Stewart in the Department of Microbiology and Molecular Genetics, we developed a reporter system that allows 1) in vivo testing of direct binding of chemoattractants to MCP ligand binding domains, and 2) identification of ligands detected by MCPs of unknown function. To demonstrate the validity of this reporter system and to show that PcaY directly binds a wide range of aromatic attractants, we fused the ligand-binding domain of PcaY to the signaling domain of the nitrate receptor protein NarQ. The gene encoding this fusion protein was expressed in an Escherichia coli reporter strain developed by Professor Stewart, which allows one to detect the output of PcaY binding to chemical ligands as a simple colorimetric enzyme assay (LacZ). We showed that the fusion protein sensitively and specifically detected known attractants that are sensed by PcaY in a concentration dependent fashion. Structurally similar aromatic compounds that are not sensed as chemoattractants by PcaY in P. putida were not capable of signaling. We are currently writing a manuscript describing this work. We have also identified putative ligands for P. putida MCPs of unknown function using this system and are in the process of verifying the chemotactic responses to these chemicals in P. putida. These hybrid receptors have the potential to be used as biosensors. We are currently writing a manuscript describing this work. 3. We demonstrated that P. putida is chemotactic to the phenylpropanoid compounds p-coumaric acid, ferulic acid and caffeic acid, which are the major aromatic monomers of lignin. The response to these compounds is metabolism dependent and is mediated by the energy taxis receptor Aer2. We are currently writing a manuscript describing this work.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sampedro, R.E. Parales, T. Krell, and J. Hill. 2015. Pseudomonas Chemotaxis. FEMS Microbiol. Rev. 39:17-46.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: H. Jabeen, S. Iqbal, S. Anwar, and R.E. Parales. 2015. Optimization of profenofos degradation by a novel bacterial consortium PBAC using response surface methodology. Int. Biodeter. Biodeg. 100:89-97.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: C.M. Timm, A.G. Campbell, S.M. Utturkar, S.R. Jun, R.E. Parales, W.A. Tan, M.S. Robeson, T.-Y.S. Lu, S. Jawdy, S.D. Brown, D.W. Ussery, C.W. Schadt, G.A. Tuskan, M.J. Doktycz, D.J. Weston and D.A. Pelletier. 2015. Metabolic functions of Pseudomonas fluorescens from Populus deltoids depend on rhizosphere or endosphere isolation compartment. Front. Microbiol. 6:1118.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: R.E. Parales, R.A. Luu, J.G. Hughes, and J.L. Ditty. 2016. Chemotaxis to xenobiotic chemicals and naturally-occurring analogs. Curr. Opin. Biotechnol. 33:318-326.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: J.L. Ditty, N.N. Nichols and R.E. Parales. 2016. Protocols for the measurement of hydrocarbon transport in bacteria. In: Protocols for Hydrocarbon Microbiology K.N. Timmis, (ed.) Springer-Verlag, Berlin. (In press).
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2016 Citation: J.L. Ditty and R.E. Parales. 2016. Protocols for the measurement of hydrocarbon chemotaxis in bacteria. In: Protocols for Hydrocarbon Microbiology K.N. Timmis, (ed.) Springer-Verlag, Berlin. (In press).
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2016 Citation: W.A. Tan and R.E. Parales. 2016. Dioxygenases and their applications. In: Biocatalysis: Green Technology. R. Patel, Ed. John Wiley & Sons (In press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: R.A. Luu, R. Truong, C.N. Brunton, V. Le, M. Lee, J.V. Parales, V. Stewart, and R.E. Parales. Biosensors for the identification of chemoattractants Abstr. 114th General Meeting of the American Society for Microbiology, New Orleans, LA, May 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: W.A. Tan, J. Santos, I. Haack, C. Ma, and R.E. Parales. Evolution of variants of of Acidovorax sp. strain JS42 that degrade multiple nitroarene compounds Abstr. 114th General Meeting of the American Society for Microbiology, New Orleans, LA, May 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: K.M. Mahan, J.T. Penrod, R. Truong, W.A. Tan, N. Al Kass, J.V. Parales, K.-S. Ju, and R.E. Parales. 2015. Selection for growth of 2-nitrotoluene degrading Acidovorax sp. strain JS42 on 3-nitrotoluene identifies nitroarene dioxygenases with altered specificities. Appl. Environ. Microbiol. 81:309-319.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: R.A. Luu, J. Kootstra, C. Brunton, V. Nersteryuk, J.V. Parales, J.L. Ditty and R.E. Parales. 2015. Integration of chemotaxis, transport, and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY. Mol. Microbiol. 96:134-147.