Progress 09/15/02 to 08/14/06
Outputs
Listeria monocytogenes is a foodborne bacterial pathogen that is responsible for the disease listeriosis that has a high fatality rate. Contamination of food with L. monocytogenes leads to expensive product recalls. This grant was focused on understanding the molecular mechanisms involved in the psychrotolerance of L. monocytogenes. L. monocytogenes has the unusual ability to grow at refrigeration temperatures, and this is a highly significant factor in its role as a foodborne pathogen. The roles of selected genes in the low temperature growth of L. monocytogenes were studied. The genes interrupted in 20 cold-sensitive mutants induced by transposon mutagenesis were identified. Upstream and downstream chromosome fragments around the transposon insertion were cloned and sequenced and compared with L. monocytogenes database genome sequences. The transposon was inserted in a variety of genes in the mutants, and three of the mutants were studied in considerable detail. In
mutant cld-14 transposon Tn917 was inserted into a locus pgpH encoding a putative protein PgpH. PgpH contained a number of hydrophobic transmembrane regions, and it is proposed that the N-terminal end extends outside the membrane where it may act as cold sensor, and the C-terminal end may extend into the cytoplasm. The mutant accumulated higher levels of highly phosphorylated guanosine nucleotides (ppGpp and pppGpp) than its parent strain, and PgpH is proposed to have (p)ppGpp hydrolase activity. These nucleotides play significant roles in the cold shock and cold acclimation responses. Two other cold-sensitive mutants, cld-1 and cld-2, were studied in detail. In both mutants the Tn917 insertion was in the branched-chain α-keto acid dehydrogenase (bkd) cluster. This locus is critical in the biosynthesis of branched-chain fatty acids, in particular fatty acid anteiso C15:0, that play a critical role in the ability of L. monocytogenes to grow at refrigeration temperatures. Studies
with strains cld-1 and cld-2 led to significant insights into how L. monocytogenes produces high amounts of anteiso C15:0, and how this is further increased at low temperatures. The increase in anteiso C15:0 that occurs in L. monocytogenes when grown at low temperatures is a results of two factors: (i) an increase in the anteiso: iso fatty acid ratio, i.e., switching of branching and (ii) fatty acid shortening. In addition L. monocytogenes genome DNA microarrays were used to study gene expression by the organism under cold shock and cold acclimation conditions. Genes related to the regulation of purine and pyrimidine biosynthesis, branched-chain fatty acid biosynthesis, transport, and flagellum biosynthesis were overexpressed, as well as genes encoding a number of alternative sigma factors.
Impacts
Listeriosis is a disease with a high mortality rate. It is estimated that 500 people die from L. monocytogenes infection per year in the U.S. making it the most common cause of death from a foodborne illness. A wide variety of foods have been involved in listeriosis. This has led to a considerable number of expensive product recalls. A key component of the role of L. monocytogenes as a foodborne pathogen is its ability to grow at refrigeration temperatures. Work on cold-sensitive mutants has led to significant insights into how L. monocytogenes grows at low temperatures. Studies of mutant cld-14 have raised the possibility of a membrane protein that senses temperature and modulates the levels of a nucleotide that controls gene expression relevant to low temperature growth. Studies of mutants cld-1 and cld-2, where the mutation is in the branched-chain α-keto acid dehydrogenase complex, have led to significant insights into how L. monocytogenes produces high amounts
of fatty acid anteiso C15:0. This fatty acid plays a critical role in growth of the organism in the cold through its membrane fluidizing properties. Our knowledge of L. monocytogenes psychrotolerance has now matured from work carried out in this and other laboratories to the point where it is possible to focus on a critical aspect of psychrotolerance, with the ultimate goal of developing novel and specific methods for control of the growth of L. monocytogenes in foods at refrigeration temperatures. This will be the focus of future work in this laboratory.
Publications
- Refereed Publications Wonderling, L. D., B. J. Wilkinson, and D. O. Bayles. 2004. The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions. Appl. Environ. Microbiol. 70:1935-43.
- Zhu, K., D. O. Bayles, A. Xiong, R. K. Jayaswal, and B. J. Wilkinson. 2005. Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold sensitive mutants with transposon interrupted branched-chain α-keto acid dehydrogenase. Microbiology. 151:615-623.
- Zhu, K., X. Ding, M. Julotok, and B. J. Wilkinson. 2005. Exogenous isoleucine and fatty acid shortening ensure high content of anteiso C15:0 fatty acid required for low-temperature growth of Listeria monocytogenes. Appl. Environ. Microbiol. 71:8002-8007.
- Liu, S., D. O. Bayles, T. M. Mason, and B. J. Wilkinson. 2006. A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane protein, and shows altered (p)ppGpp levels. Appl. Environ. Microbiol. 72:3955-3959.
- Abstracts Four abstracts from the Annual Meeting of the American Society for Microbiology were published 2003-2005.
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Progress 09/15/02 to 09/14/03
Outputs
A critical aspect of the causation of disease and costly food product recalls by Listeria monocytogenes is the ability of the bacterium to grow at refrigeration temperatures. Membrane fatty acid composition and membrane fluidity play important roles in the growth of the organism at low temperatures. We studied the effects of temperature and membrane fluidity on the susceptibility of L. monocytogenes to inhibitors of fatty acid biosynthesis and membrane active compounds, which may ultimately have a role in control of the growth of the organism. Compound minimum inhibitory concentrations (MICs) for 7 strains were determined by agar dilution. A branched chain fatty acid-deficient mutant (cld-1) with lower membrane fluidity than its parent strain (10403S) was used to assess the impact of fluidity on susceptibility to the compounds. Growth temperature had little impact on the MICs of fatty acid biosynthesis inhibitors, lipids, parabens, surfactants, and plant derived
compounds. Of the inhibitors of fatty acid biosynthesis, triclosan was the most potent inhibitor with an MIC of 4 micrograms /ml that was unaffected by temperature. However, cerulenin, also a fatty acid biosynthesis inhibitor, showed an MIC change from 256 to 32 micrograms/ml from 37 to 5 degrees celsius. There was little difference in MIC values for various compounds between strain cld-1 and the parent strain at 37 degrees celsius. Overall, growth temperature, membrane fatty acid composition, and membrane fluidity did not appear to have great impact on the susceptibility of L. monocytogenes to various compounds affecting the membrane.
Impacts
These studies will extend our kowledge of potential agent for chemical contol of the growth of L. monocytogenes, and reveal the impact of refrigeration temperatures on the resonse of the organism to compounds targeting the membrane.
Publications
- Carnahan, D. W. and B. J. Wilkinson. 2003. Influence of temperature and membrane fluidity on the susceptibility of Listeria monocytogenes to inhibitors of fatty acid biosynthesis and membrane active compunds. Abstract of the Annual Meeting of the American Society for Microbiology.
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