Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.

Bacteria form communities known as biofilms, which disassemble over time. Here we found that prior to biofilm disassembly Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of D-leucine, D-methionine, D-tyrosine and D-tryptophan that could act at nanomolar concentrations. D-amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of D-amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. D-amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. D-amino acids are produced by many bacteria and thus may be a widespread signal for biofilm disassembly.

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin–antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.

mazEF is a toxin-antitoxin module located on many bacterial chromosomes, including those of pathogens. Here, we report that Escherichia coli mazEF-mediated cell death is a population phenomenon requiring a quorum-sensing molecule that we call the extracellular death factor (EDF). Structural analysis revealed that EDF is a linear pentapeptide, Asn-Asn-Trp-Asn-Asn. Each of the five amino acids of EDF is important for its activity.

Most bacteria form matrix-enclosed communities, or biofilms, when growing on surfaces. In clinical settings, biofilms are particularly problematic since they tend to form on indwelling devices and cause persistent infections and sepsis. Biofilmassociated bacteria are much less sensitive to antibiotics, making biofilm-related infections especially difficult to cure (7, 13). Often the only solution for a biofilm-infected catheter is complete replacement, a procedure that can range from uncomfortable and inconvenient to painful, expensive, and life threatening. Consequently, the development of methods to prevent biofilm formation may be just as important for treating hospital-acquired infections as the development of new antibiotics.Bacterial signaling molecules that trigger the dispersal of old biofilms hold promise as possible therapeutic agents. Recent work has demonstrated that D-amino acids may be an exemplary class of such compounds (10, 19). Certain D-amino acids isolated from the supernatants of disassembled Bacillus subtilis biofilms were shown to prevent biofilm formation in fresh cultures by disrupting the connection between an extracellular matrix protein and the cell. Similar inhibitory effects for Staphylococcus aureus and Pseudomonas aeruginosa biofilms suggested that D-amino acids might constitute a general strategy for inhibiting biofilm formation in opportunistic pathogens (10). Genes whose products are involved in biofilm formation are not orthologous across these species, however, and the mechanism of action of D-amino acids against biofilms formed by these dissimilar pathogens remains unknown.Here we describe investigations of the mechanism by which D-amino acids inhibit biofilm formation by S. aureus using fluorescence and confocal scanning laser microscopy. These techniques provide a more detailed picture of biofilm development on surfaces than visual inspection and bulk staining alone. Using dyes for specific components of the biofilm, such as cells, proteins, and polysaccharides, we have found that D-amino acids inhibit biofilm formation in S. aureus in much the same way as in B. subtilis: by preventing protein localization at the cell surface. Since S. aureus employs cell surface-associated proteins to connect neighboring cells in large aggregates (9), D-amino acids could prove effective at preventing mature biofilm development. MATERIALS AND METHODSBacterial growth. Staphylococcus aureus wild-type (WT) strain SC01 (2) was obtained from the Kolter lab collection. Tryptic soy broth (TSB) medium and D and L isomers of proline, tyrosine, phenylalanine, tryptophan, and leucine were obtained from Sigma-Aldrich (Atlanta). Cells were cultured in a shaking LB medium overnight and diluted 1:100 in TSB medium supplemented with NaCl (3%), glucose (0.5%), and the appropriate concentrations of L-or D-amino acids (or lack thereof). Cells were grown for the specified period of time in the bottom of 6-or 12-well polystyrene plates or in 6-well plates with submerged substrates without shaking at 37°C. Plan...

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The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli and Staphylococcus aureus.