SignificanceMultidrug-resistant enterococci are leading causes of hospital infection. The antibiotic-perturbed patient gut serves as a staging ground—small numbers of resistant hospital strains colonize and then, greatly amplify in the colon. Little is known of the colonization principles involved—whether hospital strains are competitive or noncompetitive with commensal enterococci or whether mobile elements comprising over 25% of the genome of the former impose significant fitness costs. We unexpectedly found that the prototype vancomycin-resistant Enterococcus faecalis strain V583 was actively killed by fecal organisms, and we traced that to pheromone production by commensal enterococci that trigger lethal mobile element cross-talk. This work highlights the importance of maintaining commensal enterococci in the gut of the hospitalized patient.
The effect of water on the flow behavior of glass microspheres dispersed in mineral oil was investigated for various levels of water, particle volume fractions, and particle sizes. The addition of small amounts of water leads to large increases in viscosity due to the formation of water bridges between particles that give rise to capillary forces between the particles. The capillary forces between the particles also make the flow profile highly shear-thinning across the range of particle volume fractions that were studied (0.10 to 0.25). The presence of water leads to a significant effect of particle size, and the viscosity of dispersion goes through a maximum as the amount of water is increased. Two hydrophobic surfactants, Span 80 and Arquad 2HT, were found to reduce the viscosity of the dispersions with added water, but the mechanism and extent of viscosity reduction differed.
Nutrient limitation causes Bacillus subtilis to develop into two different cell types, a mother cell and a spore. SpoIIID is a key regulator of transcription in the mother cell and positively or negatively regulates more than 100 genes, in many cases by binding to the promoter region. SpoIIID was predicted to have a helix-turn-helix motif for sequence-specific DNA binding, and a 10-bp consensus sequence was recognized in binding sites, but some strong binding sites were observed to contain more than one match to the consensus sequence, suggesting that SpoIIID might bind as a dimer or cooperatively as monomers. Here we show that SpoIIID binds with high affinity as a monomer to a single copy of its recognition sequence. Using charge reversal substitutions of residues likely to be exposed on the surface of SpoIIID and assays for transcriptional activation in vivo and for DNA binding in vitro, we identify two regions essential for DNA binding, the putative recognition helix of the predicted helix-turn-helix motif and a basic region near the C terminus. SpoIIID is unusual among prokaryotic DNA-binding proteins with a single helix-turn-helix motif in its ability to bind DNA monomerically with high affinity. We propose that the C-terminal basic region of SpoIIID makes additional contacts with DNA, analogous to the N-terminal arm of eukaryotic homeodomain proteins and the "wings" of winged-helix proteins, but structurally distinct. SpoIIID is highly conserved only among bacteria that form endospores, including several important human pathogens. The need to conserve biosynthetic capacity during endospore formation might have favored the evolution of a small transcription factor capable of high-affinity binding to DNA as a monomer, and this unusual mode of DNA binding could provide a target for drug design.
SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.
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