Hopanoid-free Methylobacterium extorquens DM4 overproduces carotenoids and has widespread growth impairment

Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of Methylobacterium extorquens DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids–the other major isoprenoid product of this strain–and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in M. extorquens DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for M. extorquens DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane

Search for the Xb and other hidden-beauty states in the π+π−ϒ(1S) channel at ATLAS

This Letter presents a search for a hidden-beauty counterpart of the X(3872) in the mass ranges of 10.05–10.31 GeV and 10.40–11.00 GeV, in the channel Xb→π+π−ϒ(1S)(→μ+μ−), using 16.2 fb−1 of pp   collision data collected by the ATLAS detector at the LHC. No evidence for new narrow states is found, and upper limits are set on the product of the Xb cross section and branching fraction, relative to those of the ϒ(2S), at the 95% confidence level using the CLS approach. These limits range from 0.8% to 4.0%, depending on mass. For masses above 10.1 GeV, the expected upper limits from this analysis are the most restrictive to date. Searches for production of the ϒ(13DJ), , and states also reveal no significant signals

Exceptionally high-affinity Ras binders that remodel its effector domain

The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule–binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins

Growth rates of AM1Δ<i>cel</i> and PA1Δ<i>cel</i> on different media formulations and carbon sources.

<p>Measurements were taken every 50 minutes. The data for the Choi medium is not shown because the high concentrations of metals altered the OD readings (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062957#pone.0062957.s003" target="_blank">Fig. S3</a>) such that meaningful growth rate comparisons could not be made.</p

Summary of experimental questions and findings.

<p>Summary of experimental questions and findings.</p

Comparison of the main components of the different mediums.

<p>Comparison of the main components of the different mediums.</p

A comparison of the growth characteristics of <i>M.extorquens</i> with and without the cellulose genes removed.

<p>The top row shows pictures of individual wells after growth with (A) or without (B) the cellulose genes. A large clump of the wild-type strain is indicated by the white arrow. Such clumps were not found for AM1Δ<i>cel</i>, but occasionally small strands such as those shown in B can appear. The bottom row (C and D) shows example growth curves obtained for 12 replicates with or without the cellulose locus. The OD readings of wild-type were noisy and irregular (C), whereas the AM1Δ<i>cel</i> growth dynamics were more consistent (D).</p