Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.     

Selecting appropriate methods of knowledge synthesis to inform biodiversity policy

Responding to different questions generated by biodiversity and ecosystem services policy or management requires different forms of knowledge (e.g. scientific, experiential) and knowledge synthesis. Additionally, synthesis methods need to be appropriate to policy context (e.g. question types, budget, timeframe, output type, required scientific rigour). In this paper we present a range of different methods that could potentially be used to conduct a knowledge synthesis in response to questions arising from knowledge needs of decision makers on biodiversity and ecosystem services policy and management. Through a series of workshops attended by natural and social scientists and decision makers we compiled a range of question types, different policy contexts and potential methodological approaches to knowledge synthesis. Methods are derived from both natural and social sciences fields and reflect the range of question and study types that may be relevant for syntheses. Knowledge can be available either in qualitative or quantitative form and in some cases also mixed. All methods have their strengths and weaknesses and we discuss a sample of these to illustrate the need for diversity and importance of appropriate selection. To summarize this collection, we present a table that identifies potential methods matched to different combinations of question types and policy contexts, aimed at assisting teams undertaking knowledge syntheses to select appropriate methods.     

Modeling denitrification in aquatic sediments

Sediment denitrification is a major pathway of fixed nitrogen loss from aquatic systems. Due to technical difficulties in measuring this process and its spatial and temporal variability, estimates of local, regional and global denitrification have to rely on a combination of measurements and models. Here we review approaches to describing denitrification in aquatic sediments, ranging from mechanistic diagenetic models to empirical parameterizations of nitrogen fluxes across the sediment-water interface. We also present a compilation of denitrification measurements and ancillary data for different aquatic systems, ranging from freshwater to marine. Based on this data compilation we reevaluate published parameterizations of denitrification. We recommend that future models of denitrification use (1) a combination of mechanistic diagenetic models and measurements where bottom-waters are temporally hypoxic or anoxic, and (2) the much simpler correlations between denitrification and sediment oxygen consumption for oxic bottom waters. For our data set, inclusion of bottom water oxygen and nitrate concentrations in a multivariate regression did not improve the statistical fit.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.     

Investigating the long‐term legacy of drought and warming on the soil microbial community across five European shrubland ecosystems

We investigated how the legacy of warming and summer drought affected microbial communities in five different replicated long‐term (>10 years) field experiments across Europe (EU‐FP7 INCREASE infrastructure). To focus explicitly on legacy effects (i.e., indirect rather than direct effects of the environmental factors), we measured microbial variables under the same moisture and temperature in a brief screening, and following a pre‐incubation at stable conditions. Specifically, we investigated the size and composition of the soil microbial community (PLFA) alongside measurements of bacterial (leucine incorporation) and fungal (acetate in ergosterol incorporation) growth rates, previously shown to be highly responsive to changes in environmental factors, and microbial respiration. We found no legacy effects on the microbial community size, composition, growth rates, or basal respiration rates at the effect sizes used in our experimental setup (0.6 °C, about 30% precipitation reduction). Our findings support previous reports from single short‐term ecosystem studies thereby providing a clear evidence base to allow long‐term, broad‐scale generalizations to be made. The implication of our study is that warming and summer drought will not result in legacy effects on the microbial community and their processes within the effect sizes here studied. While legacy effects on microbial processes during perturbation cycles, such as drying–rewetting, and on tolerance to drought and warming remain to be studied, our results suggest that any effects on overall ecosystem processes will be rather limited. Thus, the legacies of warming and drought should not be prioritized factors to consider when modeling contemporary rates of biogeochemical processes in soil.     

Ascorbate biosynthesis in Arabidopsis cell suspension culture.

The biosynthesis of L-ascorbic acid (L-AA) in an Arabidopsis (L.) Heynh. cell suspension culture was studied by quantifying the effects of incubation with a range of potential biosynthetic precursors, analogs, and inhibitors on the intracellular levels of reduced and oxidized forms of L-AA. Our results support the recently published biosynthetic pathway of L-AA from L-galactose (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365-369), but suggest that Arabidopsis cell suspension culture simultaneously contains two other routes leading to L-AA. The possible physiological significance of these alternate routes is discussed.