Widespread fungal-bacterial competition for magnesium lowers bacterial susceptibility to polymyxin antibiotics.

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal-bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.

Stickiness of extracellular polymeric substances on different surfaces via magnetic tweezers.

Organic particle dynamics in the surface ocean plays a critical part in the marine carbon cycle. Aggregation of marine organic particles drives their downward transport to support various marine organisms on their transit to the sediments. Extracellular polymeric substances (EPS) from various microbes are a major contributor to the oceanic organic particle pool. The stickiness of EPS is expected to play a determining role in the aggregation process of particles; however, stickiness parameters are usually indirectly estimated through data fitting without direct assessment. Here a magnetic tweezer method was developed to quantitatively assess the stickiness of three model EPS produced by: Amphora sp., (diatom), Emiliania huxleyi (coccolithophore), and Sagittula stellata (bacteria), under different in vitro environmental conditions (salinity or EDTA complexed cations) and surface matrices (EPS-EPS and bare glass). Our results showed the stickiness of three microbial EPS decreasing for S. stellata > E. huxleyi > Amphora sp., in line with their decreasing protein-to-carbohydrate (P/C) ratios (related to their relative hydrophobicity). The data not only emphasize the importance of hydrophobicity on EPS stickiness, but also demonstrates that salinity and the nature of the substrate surface can influence the stickiness. Furthermore, we investigated stickiness between various types of EPS, and the observed selective stickiness of EPS between species may shed light on the interactions among heterogeneous marine microorganisms. Overall, this newly developed system provides a platform to assess the EPS stickiness to advance our understanding of the aggregation and sedimentation process of organic particles that are critical for the fate of organic carbon as well as for biofilm formation and microbial colonization of surfaces in the ocean.

Klotho Exerts an Emerging Role in Cytokinesis.

The Klotho gene functions as an anti-aging gene. A previous klotho-knockout mice study indicated that neither male nor female gametocytes could accomplish the first meiotic division. It suggested that Klotho might regulate cell division. In this study, we determined the roles of Klotho in cytokinesis in cultural human cells (HEK293 and HeLa) and in zebrafish embryos. Immunoprecipitation, mass spectrometry analysis, and a zebrafish model were used in this study. The results showed that Klotho is located in the midbody, which correlated with cytokinesis related kinases, Aurora kinase B and citron kinases, in the late stage of cytokinesis. There was a spatial correlation between the abscission site and the location of Klotho in the cytokinesis bridge. A three-dimensional structural reconstruction study demonstrated there was a spatial correlation among Klotho, Aurora kinase B, and citron kinases in the midbody. In addition, Klotho depletion inactivated Aurora kinases; it was also indicated that Klotho depletion caused aberrant cell cycle and delayed cytokinesis in a cell model. The study with zebrafish embryos suggested that klotho knockdown caused early embryo development abnormality due to dysregulated cytokinesis. In conclusion, Klotho might have a critical role in cytokinesis regulation by interacting with the cytokinesis related kinases.

Evolutionary Repair Experiments as a Window to the Molecular Diversity of Life.

Comparative genomics reveals an unexpected diversity in the molecular mechanisms underlying conserved cellular functions, such as DNA replication and cytokinesis. However, the genetic bases and evolutionary processes underlying this 'molecular diversity' remain to be explained. Here, we review a tool to generate alternative mechanisms for conserved cellular functions and test hypotheses concerning the generation of molecular diversity - evolutionary repair experiments, in which laboratory microbial populations adapt in response to a genetic perturbation. We summarize the insights gained from evolutionary repair experiments, the spectrum and dynamics of compensatory mutations, and the alternative molecular mechanisms used to repair perturbed cellular functions. We relate these experiments to the modifications of conserved functions that have occurred outside the laboratory. We end by proposing strategies to improve evolutionary repair experiments as a tool to explore the molecular diversity of life.

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis.

The role of proteins often changes during evolution, but we do not know how cells adapt when a protein is asked to participate in a different biological function. We forced the budding yeast, Saccharomyces cerevisiae, to use the meiosis-specific kleisin, recombination 8 (Rec8), during the mitotic cell cycle, instead of its paralog, Scc1. This perturbation impairs sister chromosome linkage, advances the timing of genome replication, and reduces reproductive fitness by 45%. We evolved 15 parallel populations for 1,750 generations, substantially increasing their fitness, and analyzed the genotypes and phenotypes of the evolved cells. Only one population contained a mutation in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and delay genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow cells to use an existing protein to participate in new biological functions.

Hsp90 regulates nongenetic variation in response to environmental stress.

Nongenetic cell-to-cell variability often plays an important role for the survival of a clonal population in the face of fluctuating environments. However, the underlying mechanisms regulating such nongenetic heterogeneity remain elusive in most organisms. We report here that a clonal yeast population exhibits morphological heterogeneity when the level of Hsp90, a molecular chaperone, is reduced. The morphological heterogeneity is driven by the dosage of Cdc28 and Cla4, a key regulator of septin formation. Low Hsp90 levels reduce Cla4 protein stability and cause a subpopulation of cells to switch to a filamentous form that has been previously suggested to be beneficial under certain hostile environments. Moreover, Hsp90-dependent morphological heterogeneity can be induced by environmental stress and is conserved across diverse yeast species. Our results suggest that Hsp90 provides an evolutionarily conserved mechanism that links environmental stress to the induction of morphological diversity.

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae.

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ∼6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.

Molecular packing and lateral interactions of distearoylphosphatidylcholine with dihexadecyldimethylammonium bromide in Langmuir monolayers and vesicles.

The behavior of distearoylphosphatidylcholine (DSPC) mixed with dihexadecyldimethylammonium bromide (DHDAB) in the monolayer was investigated by means of Langmuir trough, interfacial thermodynamic analysis, and Brewster angle microscopy. It was found that the cationic surfactant, DHDAB, was miscible with DSPC and a condensing effect, indicating stronger contraction of area per molecule and stronger ordering molecular packing, appeared in the mixed DSPC/DHDAB monolayers. Condensed structures of the mixed monolayers were visible as the molar fraction of DHDAB (X(DHDAB)) ≤ 0.7. The negative deviations of excess area and mixing Gibbs free energy were obtained, and their minimum values occurred at X(DHDAB) = 0.3, suggesting that a DSPC/DHDAB monolayer with this composition exhibited the most pronounced intermolecular interactions with a compact molecular arrangement than the monolayers with separation between individual components. Furthermore, characteristics of mixed DSPC/DHDAB vesicles dispersed in water were studied by dynamic light scattering, transmission electron microscopy, and fluorescence polarization. The DSPC vesicles added with DHDAB showed zeta potentials of about +50 mV and narrower size distributions than those of pure DSPC vesicles. DSPC formed more rigid membranes than DHDAB, and the minimum disordering effect on membrane packing of vesicles was found at X(DHDAB) = 0.5, which was the most stable vesicle composition against aggregation. By contrast, the condensing effect and the increase of intermolecular attraction in mixed DSPC/DHDAB monolayers may be related to the stability enhancement of mixed vesicles as compared with the stability of pure component vesicles.