The effect of streptomycin and hypotonicity on survival of Pseudomonas aeruginosa

Summary P. aeruginosa proliferates well in a water environment; however, when subjected to high doses of streptomycin or gentamicin, the residual viable bacteria are killed by moderate water dilution of their media. These results lead to the suggestion that the mechanism of lethal action of aminoglycosides may operate through interference with the water balance system of the P. aeruginosa.     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.     

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.     

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO₂ on growth, CO₂-fixation and N₂-fixation rates by strains of the unicellular marine N₂-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO₂-acclimated cultures (190 ppm) of WH0401, growth, CO₂-fixation and N₂-fixation rates were significantly lower than those in cultures acclimated to higher (present-day ～385 ppm, or future ～750 ppm) pCO₂ treatments. Growth rates were not significantly different, however, in low-pCO₂-acclimated cultures of WH0402 in comparison with higher pCO₂ treatments. Unlike previous reports for C. watsonii (strain WH8501), N₂-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both light and pCO₂ had a significant negative effect on gross : net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO₂, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO₂-fixation rates of C. watsonii may be positively affected by elevated light and pCO₂ within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.     

Osmotic stress induces gut microbiota community shift in fish

Alteration of the gut microbiota plays an important role in animal health and metabolic diseases. However, little is known with respect to the influence of environmental osmolality on the gut microbial community. The aim of the current study was to determine whether the reduction in salinity affects the gut microbiota and identify its potential role in salinity acclimation. Using Oryzias melastigma as a model organism to perform progressive hypotonic transfer experiments, we evaluated three conditions: seawater control (SW), SW to 50% sea water transfer (SFW) and SW to SFW to freshwater transfer (FW). Our results showed that the SFW and FW transfer groups contained higher operational taxonomic unit microbiota diversities. The dominant bacteria in all conditions constituted the phylum Proteobacteria, with the majority in the SW and SFW transfer gut comprising Vibrio at the genus level, whereas this population was replaced by Pseudomonas in the FW transfer gut. Furthermore, our data revealed that the FW transfer gut microbiota exhibited a reduced renin–angiotensin system, which is important in SW acclimation. In addition, induced detoxification and immune mechanisms were found in the FW transfer gut microbiota. The shift of the bacteria community in different osmolality environments indicated possible roles of bacteria in facilitating host acclimation.     

Alpine grassland plants grow earlier and faster but biomass remains unchanged over 35 years of climate change

Satellite data indicate significant advancement in alpine spring phenology over decades of climate warming, but corresponding field evidence is scarce. It is also unknown whether this advancement results from an earlier shift of phenological events, or enhancement of plant growth under unchanged phenological pattern. By analyzing a 35‐year dataset of seasonal biomass dynamics of a Tibetan alpine grassland, we show that climate change promoted both earlier phenology and faster growth, without changing annual biomass production. Biomass production increased in spring due to a warming‐induced earlier onset of plant growth, but decreased in autumn due mainly to increased water stress. Plants grew faster but the fast‐growing period shortened during the mid‐growing season. These findings provide the first in situ evidence of long‐term changes in growth patterns in alpine grassland plant communities, and suggest that earlier phenology and faster growth will jointly contribute to plant growth in a warming climate.