An official website of the United States government.
Our research on PMF and ATP leads to the hypothesis that the ability to regulate F0F1ATPase activity, which balances energy reserves, plays a critical role in L. monocytogenes' response to adverse conditions. <P>
Objective 1 elucidates the physiological mechanisms by which L. monocytogenes regulates F0F1ATPase: a.) at the activity level: by demonstrating that membrane fluidity regulates F0F1ATPase activity, b.) through physiological adaptation: by confirming that the Acid Tolerance Response results in nisin resistance due to alterations in the abundance of F0F1ATPase c subunit, and c.) through mutation by demonstrating that F0F1ATPase abundance determines the acid sensitivity of nisin resistant mutants. <P>Objective 2 genetically characterizes the role of the F0F1ATPase: a.) in two well-studied acid-sensitive/nisin resistant L. monocytogenes mutants, and b.) by creating specific F0F1ATPase mutants and correlating their phenotypes to preservative tolerance.
NON-TECHNICAL SUMMARY: Listeria monocytogenes kills over 500 people per year and is the leading bacterial cause of food recalls. It is especially dangerous to pregnant women, the immunocompromised. These bacteria grow or survive in harsh environments, making it difficult to control in ready-to-eat meats. This unique ability must be related to energy generation and conservation. However, little is known about how L. monocytogenes regulates its two major energy stores. This project examines how L. monocytogenes energy regulation (bioenergetics) controls its ability to grow under adverse conditions. We hypothesize that the ability of L. monocytogenes to balances its two energy currencies plays a critical role in response to adverse conditions. This is demonstrated by elucidating the physiological mechanisms by which L. monocytogenes regulates its energy interconverting enzyme: and genetically characterizing the role of this F0F1ATPase enzyme. This research identifies new targets for L. monocytogenes control and addresses a fundamental factor (i.e. bioenergetics) that influences colonization, multiplication, and the types of treatment/antimicrobials necessary to reduce listerial cell numbers. This foundational understanding of listerial biology provides a platform from which to develop new intervention strategies. <P>
APPROACH: Objective 1a Membrane properties correlate with nisin resistance and antimicrobial sensitivity. ATPase activity is measured in proteoliposomes having different fluidities and fixed protein:lipid ratios. ATPase activity is also determined in proteoliposomes having different fluidities but fixed protein:lipid ratios. We also alter the protein (i.e. amount of ATPase):lipid ratios in proteoliposomes of fixed lipid composition and observe the effect of increased ATPase protein levels on ATPase activity and membrane fluidity. Objective 1b We confirm the identity of the 7.4 kDa down-regulated protein as the ATPase c subunit to verify that its downregulation causes nisin resistance during the Acid Tolerance Response. The proteins that Surface-Enhanced Laser Desorption Ionization, Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) characterizes as differentially-regulated are identified based on comparisons to the L. monocytogenes genome. Protein identities are confirmed using peptide sequencing of in-gel digestion fragments generated after polyacrylamide gel electrophoresis. Objective 1c Proteomic analysis of proteins from L. monocytogenes mutants. The relative abundance of the ATPase c subunit is determined using SELDI-TOF MS. . Objective 2a We hypothesize that the nisin-resistant strain NR30 has a mutation in the ATPase c subunit. To test this, strain NR30's nisin-resistance determinant(s) are cloned, expressed, and analyzed for the levels of ATP, PMF and ATPase activity. In addition, we create ATPase mutants and demonstrate the relationship between the gene and the acid tolerance/nisin resistance phenotype. Total DNA from L. monocytogenes nisin-resistant (NR30) cells is isolated and fragments are amplified using the pTV3 shuttle vector, in the E. coli MB2159 cells. The L. monocytogenes NR30 DNA library is used to transform the L. monocytogenes Lmdd cells with selection for the nisin resistance. The cloned L. monocytogenes NR30 DNA fragments containing determinant(s) for nisin resistance are sequenced and analyzed by comparing with the whole Listeria genome sequence. Objective 2b. To confirm that the c-subunit of the F0F1ATPase is responsible for acid-tolerance/nisin- resistance, degenerate PCR primers are used to generate four classes of mutants. The first class of mutants are designed using PCR primers based on conserved regions of ATPase subunits. The second set are generated using primers targeting the non-conserved regions. The third set is specifically designed using primers that target the residue(s) involved in proton transport. The fourth mutant class comprises deletions in genes coding for ATPase subunits. The constructed deletions are introduced into the L. monocytogenes chromosome. Mutants from each class are first tested for nisin-resistance by replicate plating, with positive clones subsequently screened for acid sensitivity and the ability to generate an Acid Tolerance Response. The mutations which give rise to acid-sensitive/nisin-resistant clones and those causing the ATR/nisin-resistant phenotype are verified by sequencing.
<P>
PROGRESS: 2007/09 TO 2008/08<BR>
OUTPUTS: The first objective of the project is to elucidate the mechanism(s) which regulate FOF1ATPase activity. Part of this has been accomplished by demonstrating that the membrane fluidity is regulated in wild-type cells but not mutant strains which are incapable of making branched chain fatty acids (BFAs); the ATPase activity was correlated with membrane fluidity. However, when the BFA precursors were added to the mutant, normal fluidity was restored without the corresponding change in ATPase activity. This led to an analysis using realtime PCR, which demonstrated that the regulation occurs at the transcriptional level. These results have been disseminated through publication and discussions at national meetings which have led to collaborations with scientists at Memorial Sloan Kettering Hospital and Cornell University. <BR>
PARTICIPANTS: Dr. Thomas Montville, PD Dr. Michael Tchikindas, Co-PD Dr. Ruth Wirwan, responsible for genetic studies Mr. Mohamed Badaoui Najjar, responsible for physiological studies Ms. Shanta Addeeb, USDA National Need Fellow Dr. Martin Weidman, Cornell University, informal collaboration on membrane fluidity of Listeria mutants Dr. Michael Glickman, Memorial Sloan Kettering Cancer Center, informal collaboration on membrane fluidity of Mycobacteria <BR>
TARGET AUDIENCES: Targets include, members of the food industry who wish to control listeria in foods, scientists who study bioenergetics as an underlying theme to microbial physiology, and researchers interested in the dynamics of the microbial membrane and its use as a model system. <BR>
<BR>
IMPACT: 2007/09 TO 2008/08<BR>
These findings have resulted in several changes in knowledge. 1. In contrast to the generally accepted dogma, we have demonstrated that the membrane composition is not the sole determinant of its fluidity. 2. Although the BFA mutant and wild-type had the expected differences in fluidity and ATPase activity, complimenting the mutation reversed the former, but not the later. 3. The F0F1 appears to be regulated at the transcriptional level.
<P>
PROGRESS: 2006/09/01 TO 2007/09/01<BR>
In the two months since the initiation of this project, team members have been selected, oriented to laboratory protocols, and are beginning studies on membrane fluidity.
<BR>
IMPACT: 2006/09/01 TO 2007/09/01<BR>
We expect this research to provide mechanisms which will reduce the growth of listeria in foods and thereby reduce listeriosis and its associated mortality