An official website of the United States government.
The long-term goal of the research is to develop improved methods for eradicating L. monocytogenes in foods. Toward this end, we are studying the mechanism of action of biological control agents known as class IIa bacteriocins produced by many strains of lactic acid bacteria used in food production.<P> In their attack on Listeria cells, class IIa peptides bind to the principal glucose transport protein in this organism, EIItMan, which is encoded by the mpt operon. The expression of this phosphotransferase system (PTS) transporter is positively regulated by glucose and is likely to vary in different food environments.<P> Currently little is known about how expression of EIItMan and other PTS transporters are regulated by carbohydrate sources in Listeria. Greater knowledge about EIItMan regulation, in particular, will be important for optimum application of class IIa bacteriocins in food preservation. <P>To address the long-term goal of our research, the following short-term objectives will be investigated under this proposal. <P>Objective 1. Inactivation of putative PTS system regulators homologous to the Lin0142 protein in L. monocytogenes. In preliminary research, a novel activator (Lin0142) of the mpt operon was discovered in the non-pathogenic model bacterium, L. innocua. Homologs of this protein are linked to several PTS operons involved in carbohydrate transport in both Listeria species. Under this aim, the genes encoding these proteins in the fructose (lmo2131 & 2132), B-glucoside (lmo0740), and trehalose (lmo1251) PTS operons will be knocked out in L. monocytogenes to obtain mutant strains in which the regulatory effects of gene deletion can be analyzed. <P>Objective 2. Characterization of growth phenotypes and the effects of regulatory protein inactivation on PTS operon transcription. The objectives of this aim are to determine the effects of lin0142 homolog disruption on carbohydrate utilization and transcription of genes in the fructose, B-glucoside, and trehalose PTS operons. Based on the properties of the Lin0142 protein, it is hypothesized that Lin0142 homologs positively regulate transcription of these operons. The experiments also will identify promoters controlled by the Lin0142 homologs. <P>Objective 3. Characterization of the mechanism by which mpt expression is down regulated in the absence of glucose. The objectives of this aim are to 1) determine how expression of the mpt operon is down-regulated when cells are grown on alternative carbon sources, and 2) whether or not the L. monocytogenes Lin0142 homolog, Lmo0095, is involved in this process. Growth in media lacking glucose is known to cause a reduction in mpt transcription and decreases the susceptibility of the bacterium to class IIa bacteriocins. Here, we will determine if this is achieved via modulation of the levels of transcription of the mpt regulatory genes, lmo0095, manR, or rpoN, and/or by alteration of the phosphorylation state of the ManR protein. Studies will be conducted in a lmo0095 deletion mutant to see if this gene modulates ManR phosphorylation state.
Non-Technical Summary: Foodborne illness is a major public health concern in the USA. It is estimated that about 75 million cases of foodborne disease resulting in 325,000 hospitalizations, 5,000 deaths, and $5 to 6 billion in costs occur each year. One of the most important causative agents of foodborne illness in the USA is the pathogenic bacterium, Listeria monocytogenes. This bacterium is responsible for ~2,500 cases of the severe disease known as listeriosis and 500 deaths each year. Research has shown that growth of L. monocytogenes in foods can be controlled by antimicrobial peptides known as class IIa bacteriocins produced by beneficial lactic acid bacterial species used in food production. However, the susceptibility of L. monocytogenes to bacteriocins may be influenced by the type of sugar present in the food. The proposed research will investigate the mechanism by which different sugars regulate class IIa bacteriocin susceptibility. It therefore will lead to better methods for prevention of food contamination and disease. <P> Approach: Objective 1. Genes encoding the Lin0142 homologs that are linked to the fructose (lmo2131 & 2132), B-glucoside (lmo0740), and trehalose (lmo1251) PTS operons will be knocked out by in-frame deletion methods in the sequenced L. monocytogenes EGD-e strain. The procedure involves PCR amplification of the proximal and distal coding regions of each targeted gene and fusing them together in-frame by a second round of PCR called splice-by-overlap-extension PCR. The gene fragment, in which the central part of the coding region is missing, is cloned into the vector, pKSV7, and the vector is introduced into the host by electroporation. pKSV7 contains a chloramphenicol resistance determinant (cm) and a temperature-sensitive origin of replication. Thus, recombinants in which the internally deleted gene has inserted into the chromosome are selected by plating cells on Cm agar at 41 C. Subsequently, the vector and wild-type gene are cured from the strain by growing cells for several passages at 41 C on agar lacking the antibiotic. The presence of the deletion is confirmed by PCR analysis of genomic DNA. Objective 2. The effects of lin0142 homolog disruption on carbohydrate utilization will be determined by growing strains on minimal media supplemented with the relevant carbohydrates. As a positive control for growth, strains will be grown on minimal media supplemented with glucose. The function of the B-glucoside PTS will be tested by growing strains on the B-glucosides, salicin and cellobiose. The effects of gene disruption on transcription of the operons will be analyzed by real-time reverse transcriptase (RT)-PCR. In brief, this technique involves isolation of total RNA, RT synthesis of cDNA from RNA using random hexamer primers, and real-time RT-PCR amplification and measurement of product levels. Results from three RNA preparations per strain and triplicate real-time amplification runs for each prep will be pooled and the data analyzed to evaluate statistical significance. Objective 3. To determine how alternative sugars down regulate mpt expression, the levels of mRNAs for the mpt regulators lmo0095, manR, and rpoN will be measured as discussed above in strains grown in glucose, fructose, B-glucoside, and trehalose media. To assess the ManR phosphorylation state, a C-terminal-His6-tagged manR gene will be constructed and cloned under its own promoter in pAM401. The plasmid will be introduced into both wild-type and lmo0095 knockout strains, and cells will be grown in a synthetic minimal media where [32P] H2PO4 is the phosphate source, and glucose or the other sugars are carbon sources. The His6-tagged ManR protein will be isolated by nickel affinity chromatography and its activity determined as a function of lmo0095 expression and the type of carbohydrate in the media. The location and number of phosphorylation sites will be determined by fragmenting the protein with trypsin and analyzing phosphopeptides by mass spectrometry.