Determining Proteins Expression is Ralstonia bacteria that Survive in Ultra Pure Water
EMSL Project ID
15896
Abstract
Pure water free from bacteria is essential to many industries, particularly the semiconductor and pharmaceutical industries, where a single bacterium can be detrimental to the quality of a semiconductor product or a health risk if present in medicine. There have been many studies performed on distilled and ultra-pure water (UPW) that have demonstrated that oligotrophic bacteria survive in these extreme environments even though the water purification systems contain various unit operations specifically designed to remove bacteria. These unit operations include: carbon adsorption, ion exchange, reverse osmosis, small molecular weight cut-off filtration devices, UV light systems, and ozone apparatus. UV light systems in particular are part of an ultra-pure water system to prevent bacteria from reproducing. Most UPW systems contain at least two UV systems. Literature suggests that UV light causes the formation of cyclobutane pyrimidine dimers and cytotoxic photoproducts(pyrimidine (6-4) pyrimidone) that deactivate DNA. An enzyme known as photolyase can then bind to the UV damaged DNA and upon adsorbing a blue-light photon (wavelength 350 to 450 nm) facilitates repair. The focus of the overall research project is to understand how microorganisms survive in UPW and determine how to eliminate bacteria from the system. The University of Arizona has both bench-scale and pilot-plant-scale UPW systems. The organisms that survive in UPW were isolated from the pilot-plant-scale system, identified using 16S RNA sequencing, and some of their nutrient sources were determined.1 The identified strains of bacteria are ubiquitous to UPW systems around the world.2 One of the bacterial strains found throughout the system (and after the both UV light apparatus) is Ralstonia pickettii. Due to its prevalence, it was the species used for further study. Ralstonia pickettii bacteria do undergo cryptic growth (ability to use dead microorganisms as nutrient sources) and survive in UPW for periods of at least 9 months without any loss in viability.3 They can also utilize a wide variety of amino acids and carbohydrates as sole nutrient and energy sources. Preliminary studies suggest that Ralstonia pickettii adapt to UV light and adhere to a wide variety of metal and plastic surfaces. These bacteria are not known pathogens for humans, however Ralstonia eutropia is a plant pathogen. The DOE Joint Genome Institute is interested in this species of bacteria because of its ability to survive in a variety of matrices (soil, water) and in the presence of high concentrations of metals.
The goal of this proposed collaboration with the EMSL group is to determine what proteins are up and down regulated to allow Ralstonia pickettii to survive in ultra pure water and adapt to UV light treatment. In the first set of proposed experiments, the variations in protein expression are to be determined. A precise quantitative amount of all changes in protein expression is not necessary at this time until the types of proteins are known. Then, after a more thorough understanding of the function of these proteins is researched through the literature, more studies may be performed under varying growth and environmental conditions to gain further insight into bacterial survival mechanisms.
Concentrated cells (either frozen or suspended in UPW) will be sent to EMSL for ion trap high performance mass spectroscopy proteomic analysis of the whole cell lysates. Since the methodology for preparing the lysates affects the results greatly, it is desirable to send cells as opposed to lysates. No special handling of the cells (heme characterization etc.) is anticipated at this time. The following cell samples will be sent for analysis:
1) Ralstonia pickettii isolated from after the first UV 254 light source, grown in R2A media (a common media used in environmental/water purification studies) until late exponential growth, washed with phosphate buffer and frozen.
2) The same species of Ralstonia pickettii as #1, but after it has been resuspended in UPW for 2 weeks.
3) Ralstonia pickettii isolated from after the second UV light source, grown in R2A media until late exponential growth, washed with phosphate buffer and frozen.
4) The same species of Ralstonia pickettii as #3, but after it has been resuspended in UPW for 2 weeks.
5) Ralstonia pickettii bacteria (#3) that have been exposed to UV light and have developed resistance to this treatment.
The number of replicate analysis is uncertain. The initial estimate is 2 to 3. The standard automated data analysis performed at EMSL should be sufficient.
In a broad sense, this work is related to a current Fast Program that Professor Ogden is part of with the APEL bio-group at PNL. This group of 4 faculty and 2 students as well as 4 PNL researchers (S. K. Sundram, Tom Weber, Brian Riley, and Athena ) is investigating the use of FTIR to detect changes in mammalian cell culture when exposed to oxidative stress. Some of the issues studies include: determining better methods of adhering mammalian cells to surfaces (Could proteins expressed by Ralstonia pickettii facilitate adhesion since these bacteria adhere to a wide variety of surfaces?) and determining mammalian cell responses to bacterial pathogens that survive in a wide variety of environments (Could some proteins expressed by Ralstonia pickettii in UPW induce immune responses in mammalian cells?). Although linking the proteins to mammalian cell response requires significant amounts of research, there is a “big picture” link between the projects.
This work has broader applications than understanding and eventually eliminating bacteria found in UPW systems used by the semiconductor industry. UPW is also used in the pharmaceutical and biotechnology industries. On the research side, almost every biological and chemical laboratory uses “distilled water”. When water contains unwanted microbes experimental results are not repeatable. Furthermore, a better understanding of adaptation of bacteria is beneficial to Homeland security. There is an increased fear of bioterrorism, including the addition of pathogens to water supplies. If a further understanding of the survival and ability of cells to adapt to new environments is gained, all people who use water will benefit.
1McAlister, M., L. A. Kulakov, M. J. Larkin, and K. L. Ogden. Analysis of Bacterial Contamination in Different Sections of an Ultrapure Water System. Ultrapure Water 18:18-26 (2001).
2Kulakov, L. A., M. B. McAlister, K.L. Ogden, M. J. Larkin, and J. F. O’Hanlon. Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems. App. Env. Micro. 68(4):1548-1555 (2002))
3McAlister, M. B., L. A. Kulakov, J. F. O’Hanlon, M. J. Larkin, and K.L. Ogden. Survival and Behavior of Bacteria in Ultrapure Water J. Ind. Micro Biotech. 29:75-82 (2002).
Project Details
Project type
Exploratory Research
Start Date
2005-08-01
End Date
2007-03-20
Status
Closed
Released Data Link
Team
Principal Investigator