Robert W. Hutkins

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.

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

ABSTRACT Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16Lactobacillus strains and 7 of 8Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.

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.

Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus

Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter–operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract.

Barcoded Pyrosequencing Reveals That Consumption of Galactooligosaccharides Results in a Highly Specific Bifidogenic Response in Humans

Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS) on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram) of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by competitive interactions within the intestinal environment.

A dose dependent impact of prebiotic galactooligosaccharides on the intestinal microbiota of healthy adults.

The goal of this research was to determine the effect of different doses of galactooligosaccharide (GOS) on the fecal microbiota of healthy adults, with a focus on bifidobacteria. The study was designed as a single-blinded study, with eighteen subjects consuming GOS-containing chocolate chews at four increasing dosage levels; 0, 2.5, 5.0, and 10.0g. Subjects consumed each dose for 3 weeks, with a two-week baseline period preceding the study and a two-week washout period at the end. Fecal samples were collected weekly and analyzed by cultural and molecular methods. Cultural methods were used for bifidobacteria, Bacteroides, enterobacteria, enterococci, lactobacilli, and total anaerobes; culture-independent methods included denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qRT-PCR) using Bifidobacterium-specific primers. All three methods revealed an increase in bifidobacteria populations, as the GOS dosage increased to 5 or 10g. Enumeration of bifidobacteria by qRT-PCR showed a high inter-subject variation in bifidogenic effect and indicated a subset of 9 GOS responders among the eighteen subjects. There were no differences, however, in the initial levels of bifidobacteria between the responding individuals and the non-responding individuals. Collectively, this study showed that a high purity GOS, administered in a confection product at doses of 5g or higher, was bifidogenic, while a dose of 2.5g showed no significant effect. However, the results also showed that even when GOS was administered for many weeks and at high doses, there were still some individuals for which a bifidogenic response did not occur.

Role of sigma(B) in adaptation of Listeria monocytogenes to growth at low temperature.

The activity of sigma(B) in Listeria monocytogenes is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, sigma(B) contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.

Inhibition of Listeria monocytogenes by bacteriocin-producing Pediococcus during the manufacture of fermented semidry sausage.

A bacteriocin-producing Pediococcus species inhibitory to Listeria monocytogenes was used to manufacture fermented semidry sausage. Separate 13.6 kg batches of a commercial summer sausage formulation were inoculated to contain an initial level of 106 cells/g of Listeria monocytogenes Scott A. In each of two independent studies, an ca. 2 log10 CFU/g reduction of L. monocytogenes occurred over the fermentation period, as compared to a less than 1 log10 CFU/g reduction in sausage fermented with a non-inhibitory Pediococcus strain. Inactivation of L. monocytogenes was also observed in one study where adequate acid production did not occur (pH>5.5), indicating that bacteriocin production occurred independently of carbohydrate fermentation. Following heating to an internal temperature of 64.4°C and storage up to 2 weeks, 9 of 90 sausages sampled were positive for Listeria . Recovery was intermittent and did not indicate that the bacteriocin was effective in eliminating L. monocytogenes that had survived the heating process.

Intracellular pH and survival of Listeria monocytogenes Scott A in tryptic soy broth containing acetic, lactic, citric, and hydrochloric acids

To study the effect of citric, acetic, lactic, and hydrochloric acids on Listeria monocytogenes Scott A, growth, survival, and intracellular pH (pHin) values were determined during growth in a pH-controlled fermentation vessel. Under the experimental conditions, L. monocytogenes Scott A grown in tryptic soy (plus yeast extract) broth survived even when the pH was reduced to 3.5. For most acids, L. monocytogenes maintained a pH gradient (intracellular pH-external pH) of about 1.0 pH unit and a pHin near 5.0. When the citric and lactic acid-treated cells at pH values 3.5, 4.0, and 4.5 were incubated for a longer time (24 h), both the pH gradient and the pHin values decreased. Although citric and lactic acids were more effective in lowering the pHin, acetic acid had the greatest effect on cell survival. A greater than 4-log reduction in cell number occurred when L. monocytogenes was held in acetic acid-treated broth for 24 h at pH 3.5 even though the pHin was 5.0. The results suggest that inhibition of L. monocytogenes by acids is caused not by a decrease in the intracellular pH, per se, but rather by specific effects of undissociated acid species on metabolic or other physiological activities.

Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195

ABSTRACT Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [3H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) α-1,2 linked to two, three, or four fructose (F) units (GF2, GF3, and GF4, respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF2 and GF3 was rapid, whereas little GF4 uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.