An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Enterococcus faecium SF68 as a model for efficacy and safety evaluation of pharmaceutical probiotics.

As normal inhabitants of diverse ecosystems, including the human gastrointestinal tract, the enterococci, and especially the two species Enterococcus faecalis and Enterococcus faecium, can be considered ubiquitous with regard to our natural environment. E. faecium has gained special importance thanks to beneficial strains marketed as probiotics, and because of its beneficial role in traditional fermented foods such as artisanal cheeses in some Southern European countries. Yet, following reports on the increasing association of some enterococcal strains with nosocomial infections such as endocarditis and bacteraemia, it became evident that strains from clinical origin are frequently highly resistant to 'last-defence-line' antibiotics such as the glycopeptide derivatives. For this reason enterococci have been classified in risk group 2 in the European Directive 93/88. With this paper it is intended to clarify the uncertain situation around the safety of the species E. faecium, also with referring to intra-species heterogeneity. In fact, well established scientific and surveillance data support the safety of some probiotic E. faecium strains for both human and animal applications. As a model, summarising yet extensive information is provided on the efficacy and safety of E. faecium SF68®, a pharmaceutical probiotic with a long history of safe use. We propose the approach presented in this review as a model for the evaluation of safety of probiotic strains of this species.

The translation initiation factor eIF4A from Schizosaccharomyces pombe is closely related to its mammalian counterpart.

We have isolated a cDNA clone encoding eIF4A from Schizosaccharomyces pombe. The deduced protein sequence is similar in length and sequence to other eIF4A proteins and exhibits highest similarity with the mammalian eIF4A protein. Hybridization with genomic DNA reveals two eIF4A genes located on two different chromosomes.

A new yeast translation initiation factor suppresses a mutation in the eIF-4A RNA helicase.

We have isolated a gene, STM1, which encodes a new translation initiation factor from Saccharomyces cerevisiae. The gene acts, if present on a multicopy plasmid, as a suppressor of a temperature-sensitive mutation in eIF-4A. The single copy STM1 gene is not essential, but disruption causes a slow growth phenotype. Analysis of polysomes from a strain carrying a disrupted stm1 allele shows a clear defect in translation initiation as shown by a strong reduction in polysomes and an increase in the monosomes. Sequence analysis revealed interesting features of the putative Stm1 protein. Comparison of the entire protein sequence with databanks showed some similarity with the human eIF-4B protein. The Stm1 protein has potential RNP1 and RNP2 motifs characteristic for RNA-binding proteins. The protein also contains six highly conserved direct repeats of 21-26 amino acids and one partial repeat.

Deletion of spoIIAB blocks endospore formation in Bacillus subtilis at an early stage.

During an early stage of endospore formation in Bacillus subtilis, the cell divides asymmetrically into two compartments that follow different developmental paths. The differential expression of genes in these two compartments is controlled in part by the production of compartment-specific transcription factors, sigma G and sigma K. It is not known how sigma G accumulation is restricted to one of the two compartments, the forespore. However, the observations that sigma F directs transcription of the structural gene for sigma G and that sigma F activity can be modified by the product of a gene, spoIIAB, has led us to investigate the role of spoIIAB during sporulation. We have isolated mutants that carry deletion alleles of spoIIAB. Electron microscopic examination of these mutants revealed that these mutations blocked endospore formation at an early stage before septation and caused extensive cell lysis. The spoIIAB deletion alleles caused hyperexpression of genes that are normally expressed exclusively in the forespore compartments of sporulating wild-type cells, whereas these alleles reduced expression of other genes, including spoIIE, which is expressed before septation in wild-type cells. These observations confirm that spoIIAB is essential for sporulation and are consistent with models in which the product of spoIIAB plays a role in regulating the timing and/or compartment specificity of sigma F- and sigma G-directed transcription.

Control of developmental transcription factor sigma F by sporulation regulatory proteins SpoIIAA and SpoIIAB in Bacillus subtilis.

The sporulation operon spoIIA of Bacillus subtilis consists of three cistrons called spoIIAA, spoIIAB, and spoIIAC. Little is known about the function of spoIIAA and spoIIAB, but spoIIAC encodes a sigma factor called sigma F, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spoIIA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of SpoIIAB) and SpoIIAB is, in turn, an antagonist of SpoIIAC (sigma F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits sigma F-directed gene expression, (ii) a mutation in spoIIAB stimulates sigma F-directed gene expression, (iii) a mutation in spoIIAA blocks sigma F-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in sigma F-directed gene expression caused by a mutation in spoIIAA. The SpoIIAA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of sigma F-directed gene expression and/or could be responsible for restricting sigma F-directed gene expression to the forespore chamber of the sporangium.

Negative regulator of sigma G-controlled gene expression in stationary-phase Bacillus subtilis.

In some media, Bacillus subtilis can maintain a prolonged stationary growth phase; however, in other media, nutrient depletion triggers a complex differentiation that culminates in production of a dormant endospore. This differentiation requires the expression of many genes. We found that during the stationary phase in media in which the cells do not form endospores and do not normally express these sporulation-essential genes, a recessive mutation in spoIIAB caused increased transcription of a set of genes essential for sporulation. Evidently, the wild-type product of spoIIAB acts during the stationary phase to prevent expression of additional sporulation-specific genes.

Competence proteins in Bacillus subtilis com mutants.

The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.