Effects of genetic, processing, or product formulation changes on efficacy and safety of probiotics.

Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.

H(2)O(2) production in species of the Lactobacillus acidophilus group: a central role for a novel NADH-dependent flavin reductase.

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H(2)O(2), inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H(2)O(2) production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 µM, in accordance with its proposed in vivo role in H(2)O(2) production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H(2)O(2) production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H(2)O(2) production in L. johnsonii.

Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533.

Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO2 during growth. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas phase. Switching off the CO2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO2 dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO2 requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO2 and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO2 dependency.

Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics.

A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.

Construction of a reporter vector for the analysis of Bifidobacterium longum promoters.

In order to initiate studies on promoter activities in Bifidobacterium longum and to independently confirm transcriptional data generated by microarray experiments, we have constructed a versatile reporter plasmid based on a B. longum cryptic plasmid and the Escherichia coli gusA gene. The resulting plasmid, pMDY23, has been tested using three B. longum promoters.

The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis.

Two non-inducible, but apparently complete prophages were identified in the genome of the sequenced Lactobacillus johnsonii strain NCC 533. The 38- and 40-kb-long prophages Lj928 and Lj965 represent distinct lineages of Sfi11-like pac-site Siphoviridae unrelated at the DNA sequence level. The deduced structural proteins from Lj928 demonstrated aa sequence identity with Lactococcus lactis phage TP901-1, while Lj965 shared sequence links with Streptococcus thermophilus phage O1205. With the exception of tRNA genes, inserted between DNA replication and DNA packaging genes, the transcription of the prophage was restricted to the genome segments near both attachment sites. Transcribed genes unrelated to phage functions were inserted between the phage repressor and integrase genes; one group of genes shared sequence relatedness with a mobile DNA element in Staphylococcus aureus. A short, but highly transcribed region was located between the phage lysin and right attachment site; it lacked a protein-encoding function in one prophage.

The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533.

Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their "probiotic" activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L. johnsonii NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected. In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.

Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter.

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri.

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.

The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract.

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of Bifidobacterium longum, and identified 1,730 possible coding sequences organized in a 60%-GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on "nondigestible" plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.

DNA sequence and functional analysis of Lactobacillus delbrueckii subsp. lactis plasmids pN42 and pJBL2.

The plasmids pN42 and pJBL2 were isolated from the Lactobacillus delbrueckii subsp. lactis strains NCC88 and JCL414. DNA sequence determination and bioinformatic analysis revealed a strikingly conserved genetic organization containing five major, highly conserved open reading frames (ORFs). Transformation studies indicated that ORF2 (consisting of a primase fused to a replicative DNA helicase), ori, and ORF3 constitute the minimal requirements for replication of pN42 in the heterologous host Lactococcus lactis. The ORF1's are predicted to encode type I restriction-modification (R-M) system HsdS subunits with different specificities on either plasmid, suggesting that these plasmids may be involved in host defense by expanding their host R-M system repertoire. These plasmids constitute the basis for the construction of novel L. delbrueckii vectors.

A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization.

Changes in iron supply to oceanic plankton are thought to have a significant effect on concentrations of atmospheric carbon dioxide by altering rates of carbon sequestration, a theory known as the 'iron hypothesis'. For this reason, it is important to understand the response of pelagic biota to increased iron supply. Here we report the results of a mesoscale iron fertilization experiment in the polar Southern Ocean, where the potential to sequester iron-elevated algal carbon is probably greatest. Increased iron supply led to elevated phytoplankton biomass and rates of photosynthesis in surface waters, causing a large drawdown of carbon dioxide and macronutrients, and elevated dimethyl sulphide levels after 13 days. This drawdown was mostly due to the proliferation of diatom stocks. But downward export of biogenic carbon was not increased. Moreover, satellite observations of this massive bloom 30 days later, suggest that a sufficient proportion of the added iron was retained in surface waters. Our findings demonstrate that iron supply controls phytoplankton growth and community composition during summer in these polar Southern Ocean waters, but the fate of algal carbon remains unknown and depends on the interplay between the processes controlling export, remineralisation and timescales of water mass subduction.

Comparative genomics of the late gene cluster from Lactobacillus phages.

Three prophage sequences were identified in the Lactobacillus johnsoni strain NCC533. Prophage Lj965 predicted a gene map very similar to those of pac-site Streptococcus thermophilus phages over its DNA packaging and head and tail morphogenesis modules. Sequence similarity linked the putative DNA packaging and head morphogenesis genes at the protein level. Prophage Lj965/S. thermophilus phage Sfi11/Lactococcus lactis phage TP901-1 on one hand and Lactobacillus delbrueckii phage LL-H/Lactobacillus plantarum phage phig1e/Listeria monocytogenes phage A118 on the other hand defined two sublines of structural gene clusters in pac-site Siphoviridae from low-GC Gram-positive bacteria. Bacillus subtilis phage SPP1 linked both sublines. The putative major head and tail proteins from Lj965 shared weak sequence similarity with phages from Gram-negative bacteria. A clearly independent line of structural genes in Siphoviridae from low-GC Gram-positive bacteria is defined by temperate cos-site phages including Lactobacillus gasseri phage adh, which also shared sequence similarity with phage D3 infecting a Gram-negative bacterium. A phylogenetic tree analysis demonstrated that the ClpP-like protein identified in four cos-site Siphoviridae from Lactobacillus, Lactococcus, Streptococcus, and Pseudomonas showed graded sequence relationships. The tree suggested that the ClpP-like proteins from the phages were not acquired by horizontal gene transfer from their corresponding bacterial hosts.

Genomics, molecular genetics and the food industry.

The production of foods for an increasingly informed and selective consumer requires the coordinated activities of the various branches of the food chain in order to provide convenient, wholesome, tasty, safe and affordable foods. Also, the size and complexity of the food sector ensures that no single player can control a single process from seed production, through farming and processing to a final product marketed in a retail outlet. Furthermore, the scientific advances in genome research and their exploitation via biotechnology is leading to a technology driven revolution that will have advantages for the consumer and food industry alike. The segment of food processing aids, namely industrial enzymes which have been enhanced by the use of biotechnology, has proven invaluable in the production of enzymes with greater purity and flexibility while ensuring a sustainable and cheap supply. Such enzymes produced in safe GRAS microorganisms are available today and are being used in the production of foods. A second rapidly evolving segment that is already having an impact on our foods may be found in the new genetically modified crops. While the most notorious examples today were developed by the seed companies for the agro-industry directed at the farming sector for cost saving production of the main agronomical products like soya and maize, its benefits are also being seen in the reduced use of herbicides and pesticides which will have long term benefits for the environment. Technology-driven advances for the food processing industry and the consumer are being developed and may be divided into two separate sectors that will be presented in greater detail: 1. The application of genome research and biotechnology to the breeding and development of improved plants. This may be as an aid for the cataloging of industrially important plant varieties, the rapid identification of key quality traits for enhanced classical breeding programs, or the genetic modification of important plants for improved processing properties or health characteristics. 2. The development of advanced microorganisms for food fermentations with improved flavor production, health or technological characteristics. Both yeasts and bacteria have been developed that fulfill these requirements, but are as yet not used in the production of foods.

Complete sequence of plasmid pLH1 from lactobacillus helveticus ATCC15009: analysis reveals the presence of regions homologous to other native plasmids from the host strain.

The complete sequence for plasmid pLH1 from Lactobacillus helveticus ATCC15009 has been determined. Analysis of the 19,360-bp primary sequence revealed a putative replication origin and initiation protein, information that could provide the basis for the construction of cloning vectors for L. helveticus. Evidence that pLH1 is theta-replicating could be deduced from the plasmid size, from the homology to the replication protein of the Bacillus natto theta-replicating plasmid pLS32, and from the identification of a putative resolvase gene (orf-195). Although 14 open reading frames capable of encoding polypeptides longer than 100 amino acids were identified, none, on the basis of homology with known sequences, appeared to encode a well-characterized trait relevant to milk fermentation. Plasmid pLH1 revealed regions of identity with the smaller cryptic plasmids (pLH2 and pLH3) from the same strain and with other tracts of DNA, including insertion sequence elements, from a variety of other lactic acid bacteria. The presence of such regions provides a basis for developing an explanation of the phenotypic variability observed in these bacteria. The plasmid also appears to possess a number of genetic elements present in other lactic acid bacterial plasmids, conservation of which would be consistent with an important functional or evolutionary role. It could be argued that the plasmid complement of L. helveticus ATCC15009 consists of parasitic entities concerned only with their own replication and survival.

Bezold-Brucke hue-shift as functions of luminance level, luminance ratio, interstimulus interval and adapting white for aperture and object colors.

UNLABELLED: For spectral and nonspectral aperture color 3 s stimuli, Bezold-Brucke (B-B) hue-shift was measured for one to six subjects for four functions: (1) luminance (L) level from 0.1:1.0 through 100:1000 cd/m2; (2) L ratio from 1:2 through 1:1000; (3) illuminant color temperature (for seven illuminants including A and D65) by two adaptation methods; and (4) interstimulus interval from 0 through 40 s; the latter removes contrast effects and inverts hue-shift, so B-B 'invariants' become hue-shift maxima. SUMMARY: four wavelengths are 'invariant' for a given L level or ratio. With higher L level, invariants shift as opposed pairs: to longer wavelength for b and y, shorter for g and r. With higher L ratio, b and y invariants shift some 10 nm longer but g and r remain about 508 nm and 493.5 c. With higher illuminant color temperature, invariants (1:10 cd/m2) shift shorter for b and y, r slightly longer (spectral to nonspectral 494 c) but g seems constant. Hue-shift data for real object color are very similar to aperture color. The full hue cycle is graphed to the recent extended wavelength scale for CIE dominant wavelength. Many effects are newly reported.

Unique and binary hues as functions of luminance and illuminant color temperature, and relations with invariant hues.

For aperture color 3 s stimuli, the four unique hues, and at times the four (equally-balanced) binary hues, were measured for: (1) adapting white color temperatures 2850, 3400, 5500, 6500 K; and (2) luminance (L) for 5 and 6 log L ranges (about 0.01-3200 cd/m2) in 3.2:1 L steps (viewed singly) for 1 subject per white, 3 log L in 10:1 steps for 6 subjects for white 6500 K, and 1 log L for 2-11 subjects per white. The full hue cycle is graphed to the extended wavelength scale. With higher L, unique hues *b and *y shift longer, *g and *r shorter, and spectral binaries and 460 nm are invariant wavelengths, for Ls viewed singly. But for Ls viewed in successive contrast (Bezold-Brucke effect), unique hues are practically invariants. As interstimuli interval increases from 0-40 s (from successive L contrast to no-contrast), invariants shift away from uniques (which become hue-shift maxima) and coincide with spectral binaries (but not r/b, 565 c) at 495, 546, 600 nm. Successive L contrast switches spectral uniques' hue-shift off (as invariants), and spectral binaries on (as hue-shift maxima); and no-contrast switches the reverse. With higher illuminant color temperature 2850-6500 K, wavelength of 10 constant hues shortens 5-10 nm for *b, *y, g/y, y/r, but others are constant +/- 2 nm.

Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO2054.

The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the beta-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is galK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, and lacA encodes a galactoside acetyltransferase. The galT and galE genes of L. lactis LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L. lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054. Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless beta-glucuronidase gene (gusA) from E. coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon. The greater similarity of the lacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L. lactis NCDO2054 have been recently acquired. Thus, the lacA-lacZ genes appear to have engaged the promoters of the gal operon in order to direct and control their expression.

Patchy distributions: Optimising sample size.

A method for estimating sample size which does not require an a priori definition of desired precision, or the assumption that the population is normally distributed with constant variance, has recently been proposed. This paper discusses this method and presents five modifications which make the method easier to use and reduce the probability of estimating a larger sample size than is actually required. The method is extended and used to estimate the mean abundance of patchily distributed benthic organisms. The technique can be used to guide the design of any environmental sampling programme, be it physical, chemical or biological, where comparisons between times and/or locations are required. Trade-offs between numbers of replicates and numbers of levels/sites are discussed.