Characterization of a Growth-Stimulatory Protein with Prospective Use in the Repair of Metabolically-Injured Food-Borne Pathogenic Bacteria

NON-TECHNICAL SUMMARY: When microbial cells are subject to different physical or chemical treatments, many cells suffer metabolic injury resulting in their inability to form colonies on selective media. The project aims to obtain a good understanding, at the biochemical and molecular level, of a protein with a prospect of being used as a repair agent for the metabolically- injured foodborne pathogenic bacteria.

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APPROACH: <BR>
THE ROLE OF THE STRAIN MMM18 GROWTH-STIMULATING PROTEIN AS A REPAIR AGENT: A group of foodborne pathogenic bacteria that is subject to metabolic injury under certain stresses will be gathered. This group of bacteria will include at least one strain of the following species: S. aureus, S. typhimurium, E. coli, and L. monocytogenes. We will concentrate on at least two types of sublethal metabolic injury. The potential role of the growth-stimulatory protein as a repair agent will be investigated by incorporating the culture supernatants of the strain MMM18 into recovery media. The repair of sublethal injury will be followed using a differential plating method. Bacterial cells will be plated on a nonselective medium and a selective medium during exposure to an injurious treatment. The decrease in colony forming units on a nonselective medium will represent the true lethality, while the difference between the values obtained on each medium will be defined as injury. After exposure to an injurious treatment, cells will be incubated in recovery medium (broth medium containing the growth-stimulatory protein of the strain MMM18) and plated at intervals on nonselective medium to determine the total number of viable cells and selective medium that allows growth of uninjured cells. During repair, resistance to selective agents will be regained and the value obtained on the selective medium will approach that of the nonselective medium. The difference between the two counts will give the proportion of injured cells. The completion of repair is when all injured cells regain ability to grow on the selective medium. The experiment will be repeated using a recovery medium that does not contain the growth-stimulatory protein of the strain MMM18. Some of the metabolic requirements for repair of injury will be studied by measuring the incorporation of radiolabeled precursors into cell components in the lag preceding multiplication. <BR>
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CHARACTERIZATION OF THE GROWTH-STIMULATORY PROTEIN: The growth-stimulatory protein will be purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and preparative gel electrophoresis. N-terminal amino acid sequence of the purified protein will be determined. A nucleic acid probe will be designed, synthesized, and labeled. A genomic library of the strain MMM18 will be constructed in E. coli. Colony hybridizations will be carried out. The plasmids isolated from the hybridization-positive colonies will be subject to restriction enzyme analysis and DNA sequencing. The culture supernatants of the hybridization-positive colonies will be used to determine the expression of the growth-stimulating protein in E. coli background. Northern blots will be performed using a labeled DNA probe and RNA samples isolated during different phases of growth of the hybridization-positive colonies. Specific growth rate of the wild-type and the recombinant E. coli will be determined. The gene encoding the growth-stimulatory protein will be over-expressed using QIAexpressionist system. <BR>
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MICROBIAL IDENTIFICATION: The strain MMM18 will be identified by Midi Labs using full-length 16S rRNA gene sequence analysis.

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PROGRESS: 2002/09 TO 2007/06<BR>
OUTPUTS: The most significant dissemination activities for the entire life of the projects are as follows: 1. One peer-review publication (2005) in a journal (Journal of Applied Microbiology) with a high impact factor 2. Three abstracts (2002, 2003, and 2004) published in abstract books for the American Society for Microbiologists General Meeting (2002 and 2003) and the Institute of Technologists Annual Meeting (2005) 3. Three poster presentations at the American Society for Microbiologists General Meeting (2002 and 2003) and the Institute of Technologists Annual Meeting (2005) 4. One M.S. thesis (2004) authored by Mansour Nasser Alotaibi, M.S., who was trained in the Department of Food Science and Toxicology at the University of Idaho <BR>
PARTICIPANTS: The following individuals directly participated in the project: 1. Dr. Gulhan Unlu (Faculty) 2. Dr. Galina Dimitrieva-Moats (Research Support Scientist I) 3. Mansour Nasser Alotaibi, M.S. (Former Graduate Student). An M.S. student was trained in the area of food microbiology and microbial food safety during the course of the project. Dr. Ron Crawford, Director of the Environmental Biotechnology Institute at the University of Idaho, was a collaborator on the project and a co-author on one peer-review publication (2005).<BR>
TARGET AUDIENCES: An efficient repair agent that can successfully be used in the accurate detection and enumeration of heat-injured foodborne pathogens was thoroughly studied during the course of the project. The relevant agencies of the US government can utilize the findings of the project to make recommendations on the integration of this agent to methodologies that deal with detection and enumeration of select foodborne pathogens. The use of more accurate detection and enumeration methodologies would help reduce the number of foodborne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade, benefiting the US government, the food industry, and the consumer. <BR>
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IMPACT: 2002/09 TO 2007/06<BR>

An estimated 76 million cases of foodborne illness occur each year in the US, costing between $6.5 and $34.9 billion in medical care and productivity. Incidence of foodborne illness is documented through FoodNet, a reporting system used by the US public health agencies that captures foodborne illness in over 13% of the population. The presence of food-borne pathogenic microorganisms in foods without further treatment is considered unacceptable, especially where susceptible groups in a population are at risk or there is a chance of further microbial growth in the food. In such cases, it is imperative to detect even severely injured microbial cells. Metabolically injured food-borne pathogenic bacteria fail to grow on selective media generally used in their detection and enumeration. Therefore, there is a great need for improved resuscitation methods to recover metabolically injured foodborne pathogenic bacteria so that their accurate counts can be determined. In many cases, microbial cells injured by physical or chemical stresses recover best on rich medium containing blood, catalase, or pyruvate to avoid problems of oxidative stress. As previous investigations have shown that pyruvate and known catalases are effective in repair of only a selected group of metabolically injured microorganisms, there is a great need for alternative repair agents that are cost-effective and can be used efficiently in repair of variety of metabolically injured food-borne pathogenic bacteria. Marine bacteria are the sources of many biologically active compounds. The extracellular catalase from the marine bacterium P. porphyrae strain MMM18 associated with marine algae has been studied in detail at the University of Idaho. The enzyme was purified and characterized, and was evaluated as a repair agent for select heat-injured foodborne pathogenic bacteria. Incorporation of the enzyme into selective media resulted in an increased recovery of 1075%, 204%, 213%, and 9911% of the heat injured Staphylococcus aureus, E. coli O157:H7, L. monocytogenes, and S. Typhimurium, respectively. Statistical analyses revealed significantly higher percent increased recoveries with the catalase from P. porphyrae strain MMM18 than with bovine liver catalase, except in the case of E. coli O157:H7 where the efficacy of the control and experimental catalases were found to be statistically equal. The overall results of the study indicated that the catalase from P. porphyrae strain MMM18 can successfully be used in the repair/detection of heat-injured S. aureus, L. monocytogenes, E. coli O57:H7, and particularly S. Typhimurium. Roughly two to four million cases of foodborne salmonellosis occur annually in the US, and the estimated 1.3 million cases that occurred in 2000 cost $2.4 billion in medical costs and lost productivity. The prevention of even a very small percentage of these salmonellosis cases via accurate detection and enumeration of the pathogen would help reduce the number of foodborne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade.

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PROGRESS: 2006/01/01 TO 2006/12/31<BR>
The objective of this study was to clone and characterize the gene that encodes the catalase enzyme from the strain MMM18. Electrocompetent cells of Escherichia coli UM2 (katE2 katG15), a double catalase mutant, were prepared using standard protocols. A partial genomic library of the strain MMM18 was constructed using pJDC9 vector DNA. The Sau3AI-digested genomic DNA was ligated with the BamHI-digested and dephosphorylated pJDC9 DNA. The ligation mixtures were electroporated with electrocompetent E. coli UM2 cells. The resulting transformants were selected on Luria Bertani medium containing Erythromycin, X-Gal (5-Bromo-4-Chloro-3-Indolyl-â-D-Galactopyranoside), and IPTG (Isopropyl-â-D-Thiogalactoside). Approximately 1,000 Em-resistant white-color E. coli UM2 derivatives were subjected to phenotypic confirmation in the form of determining catalase activity. Initially, 60 catalase-positive colonies were detected based on the formation of oxygen gas bubbles upon exposure to 0.87 M hydrogen peroxide. The presence of pJDC9-based plasmid DNA and the size of genomic insert in the plasmid were confirmed in selected catalase-positive E. coli UM2 derivatives. Comparisons were made among selected catalase-positive E. coli UM2 derivatives with respect to their catalase activities. Some strains revealed an enzymatic reaction in about 15-30 minutes after being exposed to hydrogen peroxide. Since the P. porphyrae catalase is an excreted enzyme, growth of selected E. coli UM2 derivatives and the amount of total protein in their supernatants were monitored as a function of time. Three cultures, namely the clones 6, 17, and 40, demonstrated peaks in protein excretion between 43-45 hours of growth, which corresponds to the beginning of the stationary growth phase in the presence of Em. Other strains, namely the clones 35 and 39, excreted proteins in a steadier manner after 43 hours of growth. Selected catalase-positive E. coli UM2 derivatives were grown to an optical density where the maximum amount of protein was detected in cell free supernatant (CFS). Catalase activity was measured in both cell extract (CE) and CFS. E. coli UM2 and E. coli DH5-alpha were used as controls. As expected, the double catalase mutant E. coli UM2 did not display any catalase activity neither in CE or CFS. The catalase activity was detected in CE from E. coli DH5-alpha but not detected in CFS. The clones 35 and 36 displayed catalase activity comparable to the other clones in CE. However, catalase activity observed in CFS was much less than that of observed for other clones. It seems that the cloned catalase is an excreted enzyme in clones 6, 17, 20, 39, 40, and 64. Our overall results indicate that we have cloned P. porphyrae strain MMM18 catalase gene(s) that encode catalase enzyme(s). With the goal of further genotypic confirmation, our current work deals with sequencing of selected genomic inserts.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
The CDC estimates that at least 6.5 million and as many as 76 million cases of food-borne disease occur in the US each year, with 325,000 hospitalizations. Estimates of the annual number of deaths caused by food-borne disease in the US range as high as 5,000-9,000. Food-borne disease incurs substantial costs to ill people, food companies, and the US economy, a cost estimated at $7.7 to 8.8 billion annually. Clearly, the presence of highly infective pathogens, even injured ones, in foods that are to be consumed without further treatment is considered unacceptable, particularly where susceptible groups in population are at risk or there is a chance of further microbial growth in the food. In such cases, it is very important to detect even severely injured cells. Current resuscitation methods do not allow full recovery of injured cells that require strictly anaerobic conditions for repair or those needing very long repair times or growth-stimulatory substances. Therefore, the importance of repair processes in recovering injured bacteria and significance of severely injured cells need clarifying. Our current research aims to obtain a good understanding of a growth-stimulatory protein that has the prospect of being used as a repair agent for the metabolically-injured food-borne pathogenic bacteria. Our research has the prospect of ensuring safety of foods and thus helping reduce the number of food-borne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade.

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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
Evaluation of the growth-stimulatory protein from Pseuodoalteromonas porphyrae strain MMM18 as a potential repair agent for the culturing of metabolically-injured food-borne pathogenic bacteria (Objective 1): Heat-injured Staphylococcus aureus RN4220, Escherichia coli O157:H7 ATCC 43890, Salmonella typhimurium ATCC 19585 and Listeria monocytogenes CWD1002 were subjected to this study. The number of heat-injured cells was determined by plate counts on appropriate nonselective and selective media. The number of repaired cells was determined by plate counts on appropriate selective medium containing either a commercial catalase from bovine liver or a partially-purified freeze-dried catalase preparation from P. porphyrae strain MMM18. For incorporation into agar media, catalase was added to the tempered (45-50 degrees Celsius) media. The activity of commercial catalase from bovine liver was 1927 units/mg solid and was used at a final concentration of 1mg/ml of media. The partially-purified freeze-dried catalase preparations were also used at a final concentration of 1mg/ml of media, with an assumption that the preparation is 100% catalase. Incorporation of bovine liver catalase and the experimental catalase into Baird Parker Agar (BPA) medium resulted in an increased recovery of 547% and 1075% of the heat-injured S. aureus RN4220 cells, respectively. When using Tryptic Soy Agar (TSA) containing 10% NaCl (TSAS), the increased recovery of the heat-injured S. aureus RN4220 cells was 525% and 1105% with bovine liver catalase and the catalase of interest, respectively. Percent increased recovery of the heat-injured E. coli O157:H7 ATCC 43890 cells was observed at levels of 280% and 204% with bovine liver catalase and the catalase of interest, respectively. When Oxford Listeria Agar (OXA) medium was used, a 27% increased recovery of the heat-injured L. monocytogenes CWD1002 cells was observed with bovine liver catalase while a 213% increased recovery was observed with the catalase of interest. The increased percent recovery of the heat-injured L. monocytogenes CWD1002 cells on TSAS containing 4% NaCl was 31% and 373% with bovine liver catalase and the catalase of interest, respectively. The percent increased recovery of the heat-injured Sal. typhimurium ATCC 19585 cells was 5641% and 9911% with bovine liver catalase and the catalase of interest, respectively, on selective Xylose Lysine Tergitol 4 Agar (XLT4) medium. Statistical analyses revealed significantly higher percent increased recoveries with the catalase from P. porphyrae strain MMM18 than with bovine liver catalase, except in the case of E. coli O157:H7 ATCC 43890 where the efficacy of the control and experimental catalases were found to be statistically equal. Our overall results indicate that the catalase from P. porphyrae strain MMM18 can successfully be used in the repair/detection of heat-injured S. aureus, L. monocytogenes, Sal. typhimurium, and E. coli O157:H7. Our present research deals with the characterization of the catalase from P. porphyrae strain MMM18 at the biochemical and molecular level (Objective 2).
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
The CDC estimates that at least 6.5 million and as many as 76 million cases of food-borne disease occur in the US each year, with 325,000 hospitalizations. Estimates of the annual number of deaths caused by food-borne disease in the US range as high as 5,000-9,000. Food-borne disease incurs substantial costs to ill people, food companies, and the US economy, a cost estimated at $7.7 to 8.8 billion annually. Clearly, the presence of highly infective pathogens, even injured ones, in foods that are to be consumed without further treatment is considered unacceptable, particularly where susceptible groups in population are at risk or there is a chance of further microbial growth in the food. In such cases, it is very important to detect even severely injured cells. Current resuscitation methods do not allow full recovery of injured cells that require strictly anaerobic conditions for repair or those needing very long repair times or growth-stimulatory substances. Therefore, the importance of repair processes in recovering injured bacteria and significance of severely injured cells need clarifying. Our current research aims to obtain a good understanding of a growth-stimulatory protein that has the prospect of being used as a repair agent for the metabolically-injured food-borne pathogenic bacteria. Our research has the prospect of ensuring safety of foods and thus helping reduce the number of food-borne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade.

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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
Evaluation of the growth-stimulatory protein from Pseuodoalteromonas porphyrae strain MMM18 as a potential repair agent for the culturing of metabolically-injured food-borne pathogenic bacteria (Objective 1): Growth curves generated for the foodborne pathogenic bacteria of interest indicated that the following A600 values represent the late exponential-early stationary growth phase: Staphylococcus aureus RN4220: A600 of 3.0-4.0; Listeria monocytogenes CWD1002: A600 of 1.0-1.2; Escherichia coli O157:H7 ATCC 43890: A600 of 0.6-0.7; and Salmonella typhimurium ATCC 19585: A600 of 0.7-1.0. All pathogenic bacterial strains of interest were subjected to experiments where appropriate temperature and time for heat injury was determined. Bacterial cells were appropriately diluted in potassium phosphate buffer and heated in a water bath with pre-adjusted temperature. S. aureus RN4220 was exposed to heat at 45, 52, 57 and 60oC for various time periods of 5, 10, 15, 20, 25 and 30 minutes. L. monocytogenes CWD1002 was exposed to heat at 53 and 57oC for various time periods of 10, 15 and 20 minutes. E. coli O157: H7 ATCC 43890 was heated at 52 and 57oC for 10, 15 and 20 minutes. Sal. typhimurium ATCC 19585 was heated at 50, 54 and 57oC for 10 and 15 minutes. Heated bacterial cell suspensions were plated both on selective and nonselective media in order to determine the number of dead and injured cells. When subjected to several heat treatments (52oC for 5, 10, 15, 20, 25, and 30 minutes), S. aureus RN4220 was injured successfully by a heat treatment at 52oC for 15 minutes. Percent injury at levels of 89% and 87% were detected using TSAS (10% NaCl) and BPA as selective media, respectively. The death percentage of heat-injured S. aureus RN4220 was determined to be 99% using TSA. When using a heat treatment at 54oC for 10 minutes, percent injury at levels of 99.89% and 93.39% were detected for L. monocytogenes CWD1002 using TSAS (4% NaCl) and OXA Listeria Agar as selective media, respectively. With a heat treatment at 54oC for 15 minutes, percent injury at levels of 97.76% and 97.45% were detected using TSAS (4% NaCl) and OXA Listeria Agar as selective media, respectively. The death percentage of heat-injured L. monocytogenes CWD1002 was determined to be 97% using TSA. Upon exposure to various heat treatments (52oC for 10, 15, and 20 minutes), E. coli O157:H7 ATCC 43890 was successfully injured (i.e.% injury of 99.8) by a heat treatment at 51-52oC for 10 minutes. The death percentage of heat-injured E. coli O157:H7 ATCC was determined to be 39% using TSA. When using a heat treatment at 54oC for 10 and 15 minutes, percent injury of Sal. typhimurium ATCC 19585 at levels of 56.3% and 80.5%, respectively, were detected using XLT4 Agar as selective media. Percent death was observed at levels of 93.2% and 99.5% on TSA upon exposure to a heat treatment 54oC for 10 and 15 minutes, respectively. Our present research deals with the recovery of heat-injured foodborne pathogenic bacteria with freeze-dried catalase preparations (incorporated into selective media) from P. porphyrae strain MMM18. As a control, bovine liver catalase will be used.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
The CDC estimates that at least 6.5 million and as many as 76 million cases of food-borne disease occur in the US each year, with 325,000 hospitalizations. Estimates of the annual number of deaths caused by food-borne disease in the US range as high as 5,000-9,000. Food-borne disease incurs substantial costs to ill people, food companies, and the US economy, a cost estimated at $7.7 to 8.8 billion annually. Clearly, the presence of highly infective pathogens, even injured ones, in foods that are to be consumed without further treatment is considered unacceptable, particularly where susceptible groups in population are at risk or there is a chance of further microbial growth in the food. In such cases, it is very important to detect even severely injured cells. Current resuscitation methods do not allow full recovery of injured cells that require strictly anaerobic conditions for repair or those needing very long repair times or growth-stimulatory substances. Therefore, the importance of repair processes in recovering injured bacteria and significance of severely injured cells need clarifying. Our current research aims to obtain a good understanding of a growth-stimulatory protein that has the prospect of being used as a repair agent for the metabolically-injured food-borne pathogenic bacteria. Our research has the prospect of ensuring safety of foods and thus helping reduce the number of food-borne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade.
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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
Characterization of the growth-stimulatory protein at the biochemical and molecular level: The biologically active microbial metabolite from Pseudoalteromonas sp. strain MMM18 was purified by precipitation using ammonium sulphate, desalting, gel-filtration, ultrafiltration and anion exchange chromatography. At each step of the purification, biological activity of the microbial metabolite of interest was determined using seed germination of Brassica juncea and confirming the statistical significance of the data. The native PAGE analysis of the purified protein sample indicated the overproduction of one particular protein in the form of a single and intense protein band. Some very minor protein bands were also observed with the native PAGE analysis of the partially purified protein. The exposure of the native PAGE gel to 3% of H2O2 resulted in the release of oxygen gas bubbles where the purified protein sample was located, indicating that the biologically active microbial metabolite of interest is a catalase. An identical bubbling reaction was observed with a protein marker that contains pure bovine liver catalase. The migration patterns of the pure bovine liver catalase and the bacterial catalase from Pseudoalteromonas sp. strain MMM18 were identical. The SDS PAGE analysis of the purified protein sample revealed the presence of a single protein band with a molecular weight of about 82 kDa, suggesting that the catalase from Pseudoalteromonas sp. strain MMM18 has three identical subunits. Similar to the catalase enzyme from Pseudoalteromonas sp. strain MMM18, the catalase from bovine liver also positively influenced seed germination of Brassica juncea, however bacterial enzyme shows a statistically higher biological activity (P<0.05) than the bovine form. The biostimulatory effect on seed germination disappeared when these enzymes were inactivated by exposure to heat (60-90C for 5 minutes). The amount of excreted catalase by the strain MMM18 varied depending on temperature of growth and NaCl concentration in growth media. When a 1.5-L culture supernatant obtained from bacterial growth in SAM medium (a medium specially adapted for marine bacteria) containing 27 g/ml NaCl was subjected to gel-filtration using Sephadex G-150, the total amount of protein in catalase-active fractions was determined to be 3.8 mg. When SAM medium with 2.7 g/l NaCl was used, however, the total amount of protein in catalase-active fractions increased to 6.7 mg. Remarkably, when the strain MMM18 was subjected to incubation at 10-12C prior to cultivation at 30C, the amount of total protein increased to 24.2 mg. Microbial identification: Phylogenetic trees were generated based on the 16S rRNA sequences from Pseudoalteromonas sp. strain MMM18 and other marine bacteria. The analysis of the complete 16S rRNA sequence (1535 bp) from Pseudoalteromonas sp. strain MMM18 indicated a 93-99% homology to the 16S rRNA sequences from other Pseudoalteromonas strains. The highest homology (99.46%) was observed with the 16S rRNA sequence from Pseudoalteromonas porphyrae.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
The CDC estimates that at least 6.5 million and as many as 76 million cases of food-borne disease occur in the US each year, with 325,000 hospitalizations. Estimates of the annual number of deaths caused by food-borne disease in the US range as high as 5,000-9,000. Food-borne disease incurs substantial costs to ill people, food companies, and the US economy, a cost estimated at $7.7 to 8.8 billion annually. Clearly, the presence of highly infective pathogens, even injured ones, in foods that are to be consumed without further treatment is considered unacceptable, particularly where susceptible groups in population are at risk or there is a chance of further microbial growth in the food. In such cases, it is very important to detect even severely injured cells. Current resuscitation methods do not allow full recovery of injured cells that require strictly anaerobic conditions for repair or those needing very long repair times or growth-stimulatory substances. Therefore, the importance of repair processes in recovering injured bacteria and significance of severely injured cells need clarifying. Our current research aims to obtain a good understanding of a growth-stimulatory protein that has the prospect of being used as a repair agent for the metabolically-injured food-borne pathogenic bacteria. Our research has the prospect of ensuring safety of foods and thus helping reduce the number of food-borne illness related hospitalizations, deaths, and economical loss due to medical expenses, lost income and productivity, cost of litigation and penalties, and loss of trade.
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PROGRESS: 2002/01/01 TO 2002/12/31<BR>
Bacteria associated with marine macroorganisms excrete a variety of biologically active compounds. Our objective was to characterize one such compound. The bacterium Pseudoalteromonas sp. strain MMM 18 was isolated from the thallus surface of Laminaria japonica of Japan Sea, Russian cost. The biological activity of the metabolic products of MMM 18 was investigated through seed germination, marine algae-bacteria interaction and bacterial growth experiments using young plants of Laminaria japonica, seeds and germs of Raphanus sativus, Triticum durum, Soya ripida, Ovena sativa, Pisum sativum and Brassica juncea, cells culture of ginseng callus R-1, Escherichia coli K-12, Staphylococcus aureus, Pseudomonas aeruginosa, Microccus luteus and Bacillus subtilus. Microbial exo-metabolites were separated by ultra-filtration (pore size of 50 A), gel-filtration and hydrophobic interaction chromatography and PAGE. Research findings indicated that MMM 18 is capable of increasing longevity and improving state of marine algae. Culture supernatants of the actively growing MMM 18 and its purified fractions increased seed germination (up to 100% with initial ability of 40-70%), germ length (up to 230%) and the biomass of ginseng callus cells (up to 48%). The admission of the culture supernatants to selected media provoked stimulatory effect on different phases of bacterial growth. Spectrophotometric scans (200-700 nm) of MMM 18 cultural liquid revealed a single sharp peak with the maximum absorption at 280 nm during the transition stage from the logarithmic to the stationary growth phase. When subjected to heat, MMM 18 cultural liquid did not exhibit any of the biological activities that it exhibited initially. The biologically active compound secreted by MMM 18 was determined to be a water-soluble protein with four subunits and a molecular mass of about 240 kDa. The bacterium Pseudoalteromonas sp. strain MMM 18 excretes a biologically active protein that is a heat-labile secondary metabolite with a large molecular mass. The compound has a stimulatory effect on growth of certain bacterial and plant cells. Its stimulatory effect on growth of plants seems to be similar to those of auxins. The current research aims to obtain a good understanding, at the biochemical and molecular levels, of this growth-stimulatory protein that has the prospect of being used as a repair agent for the metabolically-injured food-borne pathogenic bacteria.