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Listeria monocytogenes is a food-borne pathogen that is second only to Salmonella as a leading cause of death due to food-borne illness. It is responsible for more than 25% of the deaths caused by food poisoning. There are three known transporters for the compatible solutes glycine betaine and carnitine. Understanding osmotic regulation in L. monocytogenes requires that we know which solute permeases are important, i.e., which ones are expressed and which ones are necessary for survival under a particular set of environmental conditions. To determine which transport systems are functional and/or required under which environmental stresses, we are constructing strains containing in-frame deletions of each transporter, of pairs of transporters and of all three transporters. These will be used in growth and transport experiments. Regulation of transport is accomplished at the genetic level and at the biochemical level. We will explore the genetic level regulation of transport by using operon fusions with a promoterless lacZ gene. The mechanism of biochemical activation of transport by hyperosmotic shifts or by chill must be examined using physical and biochemical methods. We will focus on the mechanism of chill activation of transport by glycine betaine porter II (GbuABC). Our initial approach involves two aspects: purification, characterization and reconsititution of the transport system, and characterization of the state of and phase transitions in the membrane. With a better understanding of chill and osmotic tolerance in L. monocytogenes, we will design approaches to reducing its proliferation and reduce the likelihood of food-borne illness and economic loss due to food product recalls.
The contribution of each of the three stress-related transport systems to chill and osmotic tolerance will be assessed by using three deletion mutants, one containing genes enclding only glycine betaine porter I, one containing genes encoding only glycine betaine por ter II, and one containing gnes encoding only the carnitine transporter. The mutants are designed to be in-frame deletions of the major portion of each gene or operon. Expression of each of the three transport systems will be determined by the insertion of a promoterless IacZ gene immediately before the structural gene of the transporter. Each mutant will contain an intact copy of the promoter region and structural gene, as well, so that the mutants will be stress tolerant. Gene expression will be determined by measurement of the production of beta-galactosidase after onset of stress. Preliminary studies indicate that the Gbu transporter (glycine betaine porter II) is the most important of the transporters, and that it is activated by hyperosmotic stress or by chill. It comprises three proteins, an ATPase (GbuA), a channel (GbuB) and an extracellular solute-binding protein (GbuC). We will purify these for characterization, for functional experiments and for future reconstitution experiments. For purification, we will insert nucleotides encoding six histidine residues at the beginning or end of the structural gene, then purify the expressed protein using metalloaffinity chromatography n a Ni++ column. Characterization will include MALDI-tof mass spectrometry to determine if GbuC contains a lipid moiety to anchor it to the membrane. If so, the lipid will be identified using mass spectrometry. Functional experiments will include a study of potential reversible demerization of GbuA in response to ATP binding, hydrolysis or release to test a hypothesis concerning the mechanism of action of Gbu. Dimerization will be observed using gel filtration chromatography, native polyacrylamide gel electrophoresis and MALDI-tof mass spectrometry. The phase transition and changes in the thickness of the cell membrane seems to be involved in the activation of Gbu. We will use fluorescently labeled lipids and proteins in conjunction with fluorescence anisotrophy to investigate the phase behavior (sequestering of lipids and proteins) of the membrane. Fluorescent green protein conjugates of the Gbu proteins may be used. Fluorescence resonant energy transfer between donor-acceptor pairs of labeled molecules will be used to determine changes in relative locations of membrane proteins and lipids within the membrane. We can prepare vesicles of purified Listeria lipids. With purified Gbu proteins, we will attempt to reconstitute glycine betaine transport activity into such vesicles. Various strategies are reported in the literature. We will include either ATP or a renewable source of ATP in the vesicles in order to power transport. The design of strategies to defeat the stress resistance mechanisms of 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.
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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. 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.
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PROGRESS: 2004/01 TO 2004/12<br>
During the first year of this project, 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 TO 2004/12<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. <p>
PUBLICATIONS: 2004/01 TO 2004/12<br>
No publications reported this period