Todd R. Klaenhammer

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.

Comparative genomics of the lactic acid bacteria

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

New scientific paradigms for probiotics and prebiotics.

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics.

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFα, and IL-1β were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA–DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM

ABSTRACT Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organisms ability to attach to intestinal cells in vitro.

Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes

Fourteen bacteriocin-producing strains from the genera Lactobacillus , Leuconostoc , Pediococcus , and Lactococcus were evaluated for their ability to inhibit the growth of eight strains of Listeria monocytogenes . Seven strains of lactic acid bacteria were antagonistic toward L. monocytogenes by deferred antagonism testing on agar. Cell-free supernatants from cultures of three of the seven bacteriocin-producing strains which inhibited growth of L. monocytogenes in deferred antagonism testing also inhibited growth in well diffusion assays. The eight strains of L. monocytogenes were identical in their sensitivity or resistance to bacteriocins. The action of the bacteriocins was eliminated by proteolytic enzymes.

Selection and design of probiotics

Over the past 5 years the probiotic field has exploded with a number of new cultures, each purported to elicit a variety of benefits. Lists of functional characteristics and benefits, in vivo, are now commonplace to any presentation on probiotics. Scientifically established health claims remain among the highest priorities to companies who seek to establish solid health benefits that will promote their particular probiotic. The scientific community faces a greater challenge and must objectively seek cause and effect relationships for many potential and currently investigated probiotic species and strain combinations. Rational selection and design of probiotics remains an important challenge and will require a platform of basic information about the physiology and genetics of candidate strains relevant to their intestinal roles, functional activities, and interactions with other resident microflora. In this context, genetic characterization of probiotic cultures is essential to unequivocally define their contributions to the intestinal microbiota and ultimately identify the genotypes that control any unique and beneficial properties. Strain selection and differentiation, based on the genetic complement and programming of a candidate probiotic, then becomes feasible. Looking ahead, it will be vital to the development of this exploding field to correlate important characteristics in probiotics with known genotypes and regulatory controls that are likely to affect functionality and beneficial outcomes, in vivo.

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.

Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, LactobacHlus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent‐cell or protoplast transformation. Plasmid‐containing transformants were recovered in these recipients at frequencies ranging from 101 105 transformants μg−1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 andpVA797. The ability to transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram‐positive bacteria for cloning and analysis of significant genes.