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Department of Molecular Biology|College of Agriculture and Natural Resources

Dr. Kurt W. Miller

Department of Molecular Biology
University of Wyoming
Laramie, WY 82071
email: kwmiller@uwyo.edu

Education

B.S., Biochemistry, The Pennsylvania State University, 1977
Ph.D., Biochemistry, Boston University, 1982

Background

Listeria monocytogenes--L. monocytogenes is a foodborne pathogen that can be isolated from many types of foods (www.niaid.nih.gov/factsheets/foodbornedis.htm). This pathogen is a remarkably well adapted bacterium which is able to grow in vegetable matter, yet invade human cells and propagate intracellularly. L. monocytogenes also is able to grow at low temperature. If introduced post cooking into foods, it can grow during refrigeration.

Listeriosis--About 75 million cases of foodborne illness resulting in 325,000 hospitalizations and 5,000 deaths occur annually in the USA (www.niaid.nih.gov/factsheets/foodbornedis.htm). Although only ~2,500 cases of listeriosis caused by L. monocytogenes occur in a typical year, it is the most deadly of the common foodborne diseases. About 90% of cases require hospitalization, 20% (500) result in death, and of those surviving the disease, 10 to 30% experience residual neurological problems (http://www.foodsafety.gov/poisoning/causes/bacteriaviruses/listeria/). Due to the severity of the disease, bacteriocins such as pediocin AcH that are produced by lactic acid bacteria have been promoted to help control the growth of L. monocytogenes in foods.

Pediocin AcH--Pediocin AcH is produced by certain strains of Pediococcus, which is one of the core genera of the lactic acid bacteria. Foodgrade lactic acid bacterial strains are used in hundreds of food production applications worldwide. Bacteriocins naturally serve as germ warfare agents that help the producer strain compete for resources in the growth environment (Fig. 1). They have the added benefit of limiting the growth of pathogenic bacteria such as L. monocytogenes and Clostridium botulinum in foods. The lactic acid bacterium, Pediococcus acidilactici strain H, produces the 44-amino-acid, class IIa bacteriocin known as pediocin AcH. This peptide has the highest activity against L. monocytogenes of the class IIa group.

Colony overlay analysis of pediocin AcH production

Fig. 1. Colony overlay analysis of pediocin AcH production. Colonies of the pediocin AcH production strain, P. acidilactici H, were grown in soft agar, and overlayed with soft agar containing the susceptible indicator strain Lactobacillus plantarum NCDO955. Zones of inhibition appear in the upper agar layer due to inhibition of L. plantarum growth by pediocin AcH.

The structure and mechanism of action of class IIa bacteriocins have been studied by ourselves and others. These cationic peptides are unstructured in aqueous solution but adopt a ßa fold when bound to membranes containing high percentages of anionic phospholipids. After they bind to the cytoplasmic membrane, they are thought to aggregate and form ion conductance pores (Fig. 2). Pore formation leads to cell death via dissipation of the proton gradient. Class IIa peptides appear to bind to the EIItMan PTS transporter prior to forming pores in the cytoplasmic membrane. Phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS systems) are high affinity sugar transporters that are prevalent in bacteria.

Mechanism of action of pediocin AcH against Listeria cells.
Fig. 2. Mechanism of action of pediocin AcH against Listeria cells. Pediocin AcH (Ped) interacts with lipoteichoic (LTA) and teichoic (TA) acids, and the EIItMan transporter prior to forming ion conductance pores in the cell membrane. CM-cytoplasmic membrane; CW-cell wall.

Recent and ongoing research.

The research in my lab is primarily aimed at 1) isolating Listeria genes that are involved in pediocin AcH-cell interactions and resistance, and 2) studying the structure of pediocin AcH.

1. Pediocin AcH-Listeria cell interactions and resistance--As with all antimicrobial agents, strains resistant to pediocin AcH and other class IIa bacteriocins can be isolated from the natural environment and in the laboratory. Analysis of such mutants provides important information about cellular components needed for peptide recognition, and the mechanisms that cells can use to defend themselves against attack. We have applied the technique of transposon Tn917 mutagenesis to isolate Listeria mutants that are resistant to pediocin AcH. The properties of one of these mutants are summarized here.

  1. A L. innocua mutant (G7) was isolated that is >1,000-fold resistant to pediocin AcH (Xue et al., 2005). The Tn917 insertion in G7 occurs in the promoter region of the lin0142 gene, which encodes a putative transcription factor in the Crp/Fnr family.

  2. The transposon insertion in G7 inactivates transcription of the lin0142 gene. It further inactivates transcription of the mpt operon (encodes the EIItMan PTS transporter), which is located immediately downstream.

  3. Introduction of the lin0142 gene into the G7 mutant on a complementation vector restores pediocin AcH resistance and expression of the mpt operon (Fig. 3). This indicates that the Tn917 insertion does not inactivate mpt expression simply due to a polar effect on mpt transcription.

  4. The Lin0142 protein does not control the transcription of the mpt regulatory factors, s54 and ManR. The mechanism by which Lin0142 activates mpt transcription currently is unknown.

The lin0142 gene complements the pediocin AcH resistant phenotype of the G7 mutant.
Fig. 3. The lin0142 gene complements the pediocin AcH resistant phenotype of the G7 mutant. The pediocin AcH susceptibility of the wild-type strain (Lin11) and other controls were compared to the G7 mutant, and the G7 mutant expressing the lin0142 gene from the multicopy-number plasmid, pJX0142. Non-mutant control strains are inhibited by 0.2 mg/ml pediocin AcH, whereas the G7 mutant grows at 20 mg/ml pediocin AcH. Introduction of the pJX0142 plasmid into the G7 mutant reverts it to the susceptible phenotype.

Future Research

We plan to determine the mechanism by which the Lin0142 protein activates transcription of the mpt promoter. We also will study the mechanism of action of other genes we have isolated that cause pediocin AcH resistance when mutated.


2. Structure of pediocin AcH--We have determined the secondary structure of pediocin AcH in membrane vesicles and have identified amino acids important for attack on Listeria membranes. Some of our main findings are summarized here:

  1.  
    1. Pediocin AcH is inactivated by substitutions at many of its 44 amino acids (Miller et al., 1998) (Fig. 4). A number of inactivating mutations alter the sequence of key secondary structure elements such as ß turns that are needed for folding. Other mutations reduce the hydrophobicity of the amphipathic a-helical region in the C-terminal half of the peptide. On the other hand, we have determined that variants with increased specific activity (e.g., K11E) can be isolated. This suggests that it may be possible to engineer the peptide to more effectively attack wild-type and perhaps mutant cells.

    2. Pediocin AcH is relatively unstructured in water, but adopts a ß/a secondary structure when bound to Listeria phospholipid vesicles (Watson et al., 2001) (Fig. 4). The secondary structure content depends on the percentage of anionic lipids in target membranes.

Specific activities (relative to wild-type) of pediocin AcH mutants.
Fig. 4. Specific activities (relative to wild-type) of pediocin AcH mutants.

Future Research

In the future, we plan to further study the structure of pediocin AcH, and will attempt to genetically engineer it so that it becomes more active against native and resistant cells.

Selected recent publications

  • Vu-Khac, H., and Miller, K. W. 2009. Appl. Environ. Microbiol. 75, 6671-6678. "Regulation of Mannose Phosphotransferase System Permease and Virulence Gene Expression in Listeria monocytogenes by the EIItMan Transporter.

  • Xue, J., C. M. Murrieta, D. C. Rule, and K. W. Miller. 2008. Exogenous or L-rhamnose-derived 1,2-propanediol is metabolized via a pduD-dependent pathway in Listeria innocua. Appl. Environ. Microbiol. 74:7073-7079.

  • Xue, J., and Miller, K. W. 2007. Appl. Environ. Microbiol. 73, issue 17 in press. "Regulation of the mpt Operon in Listeria innocua by the ManR Protein."

  • Miller, K. W., Ray, P., Steinmetz, T., Hanekamp, T., and Ray, B. 2005. Lett. Appl. Microbiol. 40, 56-62. "Gene Organization and Sequences  of Pediocin AcH/PA-1 Production Operons in Pediococcus and Lactobacillus Plasmids."

  • Xue, J., Hunter, I., Steinmetz, T., Peters, A., Ray, B., and Miller, K. W. 2005. Appl. Environ. Microbiol. 71, 1283-1290. "Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance."

  • Ray, B., and Miller, K. W. 2003. In "Natural Antimicrobials for the Minimal Processing  of Foods," S. Roller, Ed. Woodhead Publishing Limited, Cambridge , UK . "Bacteriocins Other Than Nisin: the Pediocin-like Cystibiotics of Lactic Acid Bacteria." pp. 64-81

  • Lewis, G. S., Jewell, J. E., Phang, T., and Miller, K. W. 2003. Biochem. Biophys. Res. Comm. 305, 1067-1072. "Mutational and Sequence Analysis of Transmembrane Segment 6 Orientation in TetA Proteins."

  • Lewis, G. S., Jewell, J. E., Phang, T., and Miller, K. W. 2002. Arch. Biochem. Biophys. 404, 317-325. "Mutational Analysis of Tetracycline Resistance Protein Transmembrane Segment Insertion."

  • Watson, R. M., Woody, R. W., Lewis, R. V., Bohle, D. S., Andreotti, A. H., Ray, B., and Miller, K. W. 2001. Biochemistry  40, 14037-14046. "Conformational Changes in Pediocin AcH upon Vesicle Binding and Approximation of the Membrane-bound Structure in Detergent Micelles."

  • Ray, B., and Miller, K. W. 2000. In "Natural Food Antimicrobial Systems," A. S. Naidu, Ed. CRC Press, Boca Raton, FL. "Pediocins of Pediococcus Species." pp. 525-566.

  • Ray, B., Schamber, R., and Miller, K. W. 1999. Appl. Environ. Microbiol. 65, 2281-2286. "The Pediocin AcH Precursor Is Biologically Active."

  • Jewell, J. E., Orwick, J., Liu, J., and Miller, K. W. 1999. J. Bacteriol. 181, 1689-1693. "Functional Importance and Local Environments of the Cysteines in the Tetracycline Resistance Protein Encoded by Plasmid pBR322."

  • Miller, K. W., Schamber, R., Osmanagaoglu, O., and Ray, B. 1998. Appl. Environ. Microbiol. 64, 1997-2005. "Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity."

  • Miller, K. W., Schamber, R., Chen, Y., and Ray, B. 1998. Appl. Environ. Microbiol.  64, 14-20. “Production of Active Chimeric Pediocin AcH in Escherichia coli  in the Absence of Processing and Secretion Genes from the Pediococcus pap Operon.”

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