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.

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.

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.

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.