Chill and Osmotic Tolerance of Listeria Monocytogenes

NON-TECHNICAL SUMMARY: Listeria monocytogenes is a food poisoning bacterium that can grow in the refrigerator or under other stressful conditions. To grow under stress, the organism accumulates protective molecules from the food in which it lives by the action of 3 transport enzymes. We will find what causes these enzymes to be synthesized and activated, and how they are activated. Our goal is to prevent infections. The ultimate impact is to reduce the viability of Listeria monocytogenes, particularly persistent strains, by understanding the mechanism of resistance to environmental stress and refrigeration. Reducing the viability will decrease the loss of life and economin burden of product recalls due to Listeria monocytogenes. <P>APPROACH: Our previous project involved a study of the expression of the genes/operons encoding the three osmolyte transporters of Listeria monocytogenes. Our studies indicate that the Gbu transporter is the most important,and that it is activated by hyperosmotic stress chill. It is comprised of three proteins, which we have purified for characterization, for functional experiments and for reconstitution experiments.A major contribution to the chill tolerance of LM can be traced to its membrane, the lipids of which contain highly saturated fatty acids. The fluidity of the membrane at low temperature is maintained because the membrane contains branched-chain fatty acids, either iso or anteiso. The proportion of shorter-chain and the anteiso-branched fatty acids in the membrane incrases when the cells are grown at 4 degrees, compared to 30. We have mutants that are unable to make precursors of the branched-chain fatty acids, and are chill sensitive. Precursors or certain other molecules restore the chill-tolerant phenotype. We will analyze the membrane lipids of these mutants to determine the nature of the fatty acids that confer chill tolerance. We will also prepare large amounts of membrane lipids from cells grown at 4 and 37 degrees, and from the mutants grown in the presence of various supplements for use in reconstitution experiments.Liposome vesicles can be prepared by dispersing phospholipids through a stainless steel filter. We will prepare liposomes from the LM lipids described above, and characterize them.GbuC is tethered to the membrane by a diacylglycerol moiety attached via a thioether bond to its N-terminal cysteine. We have expressed this protein in E. coli and in LM; the nature of the lipids can be determined by mass spectrometry. We will determine the identity of the attached fatty acids and whether they change when the organism is grown at a lower temperature. We have also prepared a mutant lacking the site of the lipid tether and a deletion mutant lacking gbuC. The growth and transport kinetics of these mutants and the wild type will be measured to determine the function of the lipid tether.We will attempt functional reconstitution of the Gbu transport system in liposomes prepared from LM membrane lipids. The functional assay will require that we incorporate an ATP-generating system, such as the creatine kinase system.If functional reconstitution can be attained, we will submit the system to cryoelectron microscopy to determine the organization of GbuABC in the membrane. We will repeat the study using lipids enriched in the shorter-chain fatty acids, which would form a thinner membrane that could alter the protein interactions within the complex. We will obtain preliminary NMR data on GbuA and GbuC to determine the feasibility of a structural determination of these subunits in solution. Dimerization of GbuA is of particular interest.The design of strategies to defeat the stress resistance mechanisms if L. monocytogenes will depend on the results of our studies. Such strategies could include the use of transport inhibitors or depletion of certain foods of glycine betaine and/or carnitine.<P>
PROGRESS: 2007/01 TO 2007/12 <BR>
OUTPUTS: When stressed by high salt concentration or refrigeration temperature L. monocytogenes (LM) cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region of the genes encoding the three stress-related transporters. The new emphasis is to determine whether presence of the substrates glycine betaine, carnitine or other factors alter the expression, and if so, by what mechanism. We previously characterized the membrane lipids of LM and found them to contain highly saturated, often branched-chain fatty acids, largely C17:0anteiso at higher temperatures, shifting to C15:0anteiso at lower temperature. Wilkinson showed mutants unable to make branched chain fatty acids are cold sensitive. We have provided alternate fatty acid primers to Wilkinson's mutants, and found that linoleic, oleic, vaccenic, isolaleric and t-butylactic acids reestablished chill tolerance to the level of the wild type. The properties and compositions of the membranes synthesized with these primers and under various stress conditions are being determined. <BR>PARTICIPANTS: Pachanee Yasurin, Graduate Student, Biotechnology Program, The Assumption University of Thailand. Mun Hua Tan, Undergraduate Student, UC Davis. Yinghua Xiao, Graduate Student, Wagenengin University, The Netherlands <BR>TARGET AUDIENCES: This work will be presented to microbiology and microbial food safety professionals via publication in professional journals and in food science literature <BR>PROJECT MODIFICATIONS: All changes involved changes in methodology to enhance the probability of success.
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IMPACT: 2007/01 TO 2007/12
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The results are too new to have produced an impact. <P>
PROGRESS: 2006/01/01 TO 2006/12/31<BR>
When stressed by high salt concentration or refrigeration temperature L. monocytogenes (LM) cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region of the genes encoding the three stress-related transporters. Our fusion for the opuC operon proved to be faulty, and we generated a new mutant. All three systems are expressed to some extent during balanced growth without added salt stress. Expression is enhanced by the addition of NaCl. Enhancement of expression by chill is more difficult to determine because all processes are slower. The new emphasis is to determine whether presence of the substrates glycine betaine or carnitine alter the expression, and if so, by what mechanism. We previously characterized the membrane lipids of LM and found them to contain highly saturated, often branched-chain fatty acids, largely C17:0anteiso at higher temperatures, shifting to C15:0anteiso at lower temperature. Wilkinson showed mutants unable to make branched chain fatty acids are cold sensitive. We have provided alternate fatty acid primers to Wilkinson's mutants, and found that some of them conferred cold tolerance. We are now analyzing the lipid composition in these cells.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
The ultimate impact is to reduce the viability of Listeria monocytogenes, particularly persistent strains, by understanding the mechanism of resistance to environmental stress and refrigeration. Reducing the viability will decrease the loss of life and economin burden of product recalls due to Listeria monocytogenes.<P>
PROGRESS: 2005/01/01 TO 2005/12/31<BR>
When stressed by high salt concentration or low (e.g., refrigeration) temperature L. monocytogenes cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. The activities of the transport systems are regulated by the presence of the stress. Previous work in which genes encoding the transport proteins were deleted showed the relative importance of each transport system and the conditions under which it functions. Besides regulation of transport activity at the biochemical level, it is likely that L. monocytogenes regulates the amount of each transport system by controlling synthesis of the proteins at the genetic level. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region of the gbu gene, and similar constructs involving two other stress-related transporters, BetL and OpuC. We grow the cells in the asence of stress and the absence of glycine betaine and carnitine, and test the expression of the reporter gene when the cells are subjected to salt stress, salt stress not involving sodium ion, sugar-mediated osmotic stress and chill stress. We are also testing whether the presence or absence of substrates glycine betaine and carnitine affect expression. Assays using the chromogenic substrate OMPG proved not to be sufficiently sensitive, and we have begun using the fluorogenic substrate MUG. Preliminary results indicate that the transporters are at least partially constitutive, and are present even in rich media unsupplemented with salt. <P>

IMPACT: 2005/01/01 TO 2005/12/31<BR>
The ultimate impact is to reduce the viability of Listeria monocytogenes, particularly persistent strains, by understanding the mechanism of resistance to environmental stress and refrigeration. Reducing the viability will decrease the loss of life and economin burden of product recalls due to Listeria monocytogenes.<P> PROGRESS: 2004/01/01 TO 2004/12/31<BR>
Our objectives were to measure expression of the three compatible solute transporters (Gbu, BetL and OpuC) of Listeria monocytogenes using reporter gene fusions, express green fluorescent protein (GFP)-labeled proteins of the Gbu transport system, and purify histidine-tagged GbuA, GbuB and GbuC. The expression of Gbu and BetL was measured in response to salt stress, osmotic stress (sucrose) and chill stress in the presence and absence of glycine betaine and carnitine. The OpuC label proved to be faulty and we are currently preparing a new strain. E. coli strains harboring plasmids containing genes encoding hexahistidine-tagged GbuA, GbuB and GbuC under the control of a Lac promoter have been prepared and the proteins are being purified using zinc affinity chromatography. The gene encoding GFP has been cloned into a plasmid suitable for our purposes.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
The ultimate impact is to reduce the viability of Listeria monocytogenes, particularly persistent strains, by understanding the mechanism of resistance to environmental stress and refrigeration.
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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
LISTERIA MONOCYTOGENES is a foodborne pathogen that survives under hyperosmotic stress and grows at refrigeration temperatures. Solute transport systems aid in the stress-hardiness by importing the protective molecules glycine betaine and carnitine, which are found in the food. Deletion experiments showed that there are three separate transport systems that are responsible for stress-activated transport of these solutes; they are encoded by the gbu and opuC operons and the betL gene. Gbu and BetL are primarily glycine betaine transporters and OpuC is a carnitine transporter, although Gbu and OpuC show some transport of the other solute. These two transporters are activated by chill or by hyperosmotic stress, whereas BetL is activated by hyperosmotic stress. Operon fusions of a reporter gene show that expression of gbu and betL is enhanced under stress; the fusion mutant involving opuC is defective and it is being re-designed.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
Knowledge of the mechanism of osmotic tolerance and of growth In the refrigerator should lead to food formulations that do not support growth of L. MONOCYTOGENES under osmotic or chill stress, and to the design of better cleaning regimens and handling methods to reduce the possibility of contamination of food by equipment or from the processing environment.