Research and Applications in Bacteriocins Chapter Abstracts

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Chapter 2
Bacteriocin Biosynthesis, Structure and Function
Des Field, Paul Cotter, Colin Hill, and R. Paul Ross

Gene-encoded antimicrobial peptides have been observed in virtually every living organism, and those produced by bacteria, termed bacteriocins, have attracted widespread scientific attention in recent years as potential chemotherapeutic agents. As a consequence, significant advances have been made in our understanding of the genetic determinants encoding bacteriocin production, their organization, and the molecular mechanisms through which they mediate antibiosis. These advances have led to the development of a number of novel expression systems for the production and bioengineering of bacteriocins that not only facilitates their further structural characterization but could also lead to their large scale industrial production. This review will focus primarily on recent developments in the biosynthesis, structure-function relationships and biotechnological production of bacteriocins from the lactic acid bacteria. In addition, potential applications for the enzymes involved in the post translational modification of lantibiotics, a class of bacteriocins that contain unusual amino acids, will be discussed.

Chapter 3
Genetically Modified Bacteriocins
Gunnar Fimland and Jon Nissen-Meyer

Genetic modification of bacteriocins has revealed structural features that are important for their biosynthesis, mode of action, and potency, and some modifications have interesting properties from an applied point of view. Construction of chimeric colicins and pyocins resulted in custom designed bacteriocins with optimised killing spectra. Some genetically modified lantibiotics have increased antimicrobial activity, solubility, and/or temperature-stability, and some have increased resistance to proteolytic and acid/base-catalyzed chemical degradation. Some modified lantibiotics have also been rendered active against several gram-negative strains. Some genetically modified pediocin-like bacteriocins with increased positive charge, have somewhat improved potencies, and replacement of their methionine residues protects against oxidative inactivation without decreasing antimicrobial activity. Moreover, introducing a structure-stabilizing C-terminal disulfide bridge resulted in broadened target cell specificity and increased their thermostablity. Replacement of non-standard residues in lantibiotics revealed that these residues generally stabilize conformations that are essential for activity and protection against proteases, and that the residues are often required for optimal bacteriocin-production. Genetic modification of lantibiotics has also revealed that the lantibiotic-modifying enzyme is rather permissive with respect to substrate specificity, but the presence of a correct leader sequence is a requirement for substrate-recognition as well as for bacteriocin-export. Genetically modified constructs of lantibiotics and pediocin-like bacteriocins have especially been useful for elucidating their structure and interaction with the wall and membrane of target-cells.

Chapter 4
Potential Application of Bacteriocins as Antimicrobials
Osnat Gillo, L.M. Nigro and Margaret A. Riley

The discovery of penicillin by Fleming in 1928 was an historical milestone in the fight against infectious disease. Over the following fifty years, pharmaceutical companies discovered and developed over 100 antibiotics effective against a wide range of human pathogens. More recently, the dramatic rise in antibiotic-resistant pathogens has stimulated renewed efforts to identify, develop or redesign antibiotics active against these multi-resistant bacteria. This chapter focuses on such efforts directed at one large and highly diverse family of toxins, the bacteriocins, which hold great promise as the next generation of antimicrobials. The majority of bacteriocins differ from traditional antibiotics in one critical way: they have a relatively narrow killing spectrum and are, therefore, toxic only to bacteria closely related to the producing strain. Accordingly, they can be considered "designers drugs" that target specific bacterial pathogens. In this chapter we will illustrate the potential of using bacteriocins and genetically-modified bacteriocins to solve some of the most challenging problems in infectious disease control.

Chapter 5
Cytotoxic Activity of Bacteriocins Against Eukaryotic Cells
Rosalba Lagos

Bacteriocins have been defined by their bactericidal or bacteriostatic action on strains closely related to the producer bacteria, while bacterial toxins (exotoxins) by their toxic effect on an animal host through their specific cytotoxic action on specialized cells. In both cases the specificity of action is given by the interaction between the toxin and a receptor. Therefore, it might seem unusual to find bacteriocins that behave as toxins and vice a versa. Nonetheless, there are some examples in which bacteriocins have been shown to have cytotoxic effect on animal cells. These bacteriocin/toxin molecules are mostly pore formers, and their broad range of action provides an excellent opportunity for studying the molecular basis of their specificity. These bacteriocins exert their toxic action on eukaryotic cells through mechanisms that can lead the induction of necrosis or apoptosis. In the latter case, individual animal cells are eliminated without the induction of an inflammatory response. Thus, modulation of apoptosis has an immense potential in the treatment of some diseases, particularly cancer. The properties and potential applications of this group of bacteriocins are discussed in this chapter.

Chapter 6
Lantibiotic Production by Streptococcus mutans: Their Uses in Replacement Therapy for the Prevention of Dental Caries and as Antibiotics for the Treatment of Various Infectious Diseases
James L. Smith, Ravi S. Orugunty and J.D. Hillman

Five billion people worldwide suffer from dental caries, making it the most common, chronic infectious disease of humankind. Mutans streptococci (MS) are the primary etiological agent of dental caries. Streptococcus mutans strain JH1000 was originally isolated based on its superior ability to colonize teeth, which was due to its production of a bacteriocin capable of killing all other strains of S. mutans. Its cariogenic potential was essentially eliminated by genetically modifying its ability to produce lactic acid, which is directly responsible for eroding tooth mineral. The resulting strain is intended for use in a novel method for prevention of caries called "replacement therapy". A single application of the replacement strain is expected to provide lifelong protection from most human decay by displacing disease-causing strains that are naturally present on tooth surfaces. Once established in the MS niche, it will also prevent colonization by disease-causing strains whenever the host comes in contact with them. The bacteriocin produced by the replacement strain belongs to the lantibiotic family. These are an important group of antimicrobials compounds. In addition to discussing our progress with replacement therapy, we detail the current state of the art regarding the potential use of these antibiotics as therapeutic agents.

Chapter 7
Use of Bacteriocins in Livestock
Francisco Diez-Gonzalez

The utilization of bacteriocin-producing bacteria (BPB) in livestock has been evaluated as a potential alternative to antibiotic usage. Studies that have administered BPB to farm animals have been conducted to increase productivity, improve animal health, and prevent the spread of human pathogens. Colicinogenic bacteria appear to be promising in controlling enterohemorrhagic E. coli in cattle and reducing the incidence of neonatal diarrhea caused by enterotoxigenic E. coli in calves. Microcin-producing E. coli have been used to reduce Salmonella carriage in poultry. Lantibiotic-producing lactic acid bacteria (LLAB) have been effective to reduce the prevalence of E. coli O157:H7 in feedlot cattle. The manipulation of the rumen fermentation to increase productivity has been investigated using a variety of LLAB including Streptococcus bovis (bovicin) and Butyrivibrio fibrisolvens (butyrivibriocin). Lacticin produced by Lactococcus lactis has been reported to be effective to reduce cow's mastitis caused by Staphylococcus aureus and Strepococcus dysgalactia. Despite that, there are relatively few commercial bacteriocins or BPB. These findings indicate that their utilization will be a feasible antimicrobial intervention in livestock for the future.

Chapter 8
Bacteriocins of Plant-Associated Bacteria and Their Potential as Biocontrol Agents Against Phytopathogens
René De Mot

Bacteria occupying plant niches exhibit diverse lifestyles ranging from epiphytic colonization of phyllospheres or rhizospheres to infection of plant tissues as endophytes, symbionts, or pathogens. A number of studies indicate that bacteriocin production plays a role in the competitive colonization of the plant environment by phytobacteria. A large bacteriocinogenic potential is present in these bacteria, but only a limited number of these allelopathic agents has been characterized biochemically and/or genetically. Genomics and metagenomics provide alternative ways for exploration of phytobacterial bacteriocin diversity. In a number of cases, proof-of-principle for control of specific bacterial plant diseases, using bacteriocin producers or bacteriocin preparations, has been provided but no commercial bacteriocin-based formulations are as yet used in plant protection. In this area, new opportunities emerge through the engineering of bacteriocins and bacteriocin producers, incited by the molecular characterization of novel types of bacteriocins.

Chapter 9
Application of Bacteriocins in the Food Industry
E.B. O'Connor, R. Paul Ross and Colin Hill

Bacteriocins are ribosomally-synthesised antimicrobial peptides produced by some bacteria that are inhibitory to other bacteria, either within the same species (narrow spectrum), or across genera (medium to broad spectrum). Many lactic acid bacteria (LAB) used in the food industry produce bacteriocins and thus are a rich source of these natural inhibitors. These bacteriocins have widespread potential applications in preservation for improving the safety and quality of foods. Indeed, bacteriocins may be viewed as an innate immunity system which can be inbuilt into food systems to self-protect them against contamination and/or outgrowth with undesirable flora. This review will focus on the potential applications of a wide range of bacteriocins produced by LAB for food improvement.

Chapter 10
Nisin: Past, Present and Future
Timo M. Takala and Per E. J. Saris

Nisin is the star of bacteriocins. It is the only bacteriocin allowed as a food additive (E234, 1983), recognized as safe (FAO/WHO, 1968), and accepted by the American Food and Drug Administration (1988). Its structure has been solved, revealing modified amino acids and lanthionine rings, and its chemical and enzymatic syntheses have been successful. Killing activities of nisin include formation of membrane pores, blockage of cell wall biosynthesis, release of cell wall hydrolases, and prevention of spore outgrowth. The structural gene (nisA, nisZ, nisQ, and nsuA), and the genes (nisBTCIPRKFEG) required for its synthesis, regulation, and self-protection have been cloned, sequenced, and functionally characterized. The regulation includes autoinduction by nisin. The nisin inducible promoters have been used for protein production and for bioassays to quantify nisin in food. The immunity system has been used to construct food-grade cloning vectors enabling usage of nisin as a selective agent. The modification machinery has been exploited to construct novel peptides containing dehydrated residues and lanthionines. Properties of nisin have been improved by genetic engineering. Future research is still needed to solve open questions, e.g. Is heterologous production of nisin possible? What are the molecular mechanisms of nisin self-immunity?

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