Genetic changes during a laboratory adaptive evolution process that allowed fast growth in glucose to an Escherichia coli strain lacking the major glucose transport system

ABSTRACT: BACKGROUND: Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400 % increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways. RESULTS: Whole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna) and in other putative regulatory loci (yjjU, rssA and ypdA). In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented. CONCLUSIONS: The deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased its growth rate, very likely by increasing glycolytic mRNA genes stability. Furthermore, galR inactivation allowed glucose transport by GalP into the cell. The deletion of mutH in an already stressed strain that lacks PTS is apparently responsible for the very high mutation rate observed.     

Cell‐Cycle‐Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches

It is a long‐standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell‐cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell‐cycle‐dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell‐cycle‐dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell‐cycle‐dependent manner. Novel techniques allowing a genome‐wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell‐cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.     

Perspectives on facilitating dynamic ecology courses online using active learning

As education methodology has grown to incorporate online learning, disciplines with a field component, like ecology, may find themselves sidelined in this transition. In response to challenges posed by moving classes online, previous studies have assessed whether an online environment can be effective for student learning. This work has found that active learning structures, which maximize information processing and require critical thinking, best support student learning. All too commonly, online and active learning are perceived as mutually exclusive. We argue the success of online learning requires facilitating active learning in online spaces. To highlight this intersection in practice, we use a case study of an online, active, and synchronous ecology and conservation biology course from the College of Natural Sciences at Minerva Schools at KGI. We use our perspectives as curriculum designers, instructors, and students of this course to offer recommendations for creating active online ecology courses. Key components to effective course design and implementation are as follows: facilitating critical “thinking like a scientist”, integrating open‐ended assignments into class discussion, and creating active in‐class dialogues by minimizing lecturing. Based on our experience, we suggest that by employing active learning strategies, the future of ecology in higher education is not inhibited, but in fact supported, by opportunities for learning online.     

The effect of alpha-melanophor-stimulating hormone on liver regeneration and incorporation of amino acid in rats' liver protein.

Following 25 mug/day synthetic alpha-MSH administration, the liver regeneration of partially hepatectomized rats proved to be increased. The hormone treatment resulted in an enhanced alanine incorporation of the liver proteins, but this effect was uncertain on partially hepatectomized rats. Due to the hormone treatment the low liver protein content of the operated rats became normal. The pseudocholinesterase activity of the liver homogenate of alpha-MSH treated rats was also elevated. On the basis of these experiments authors are supposing some protein synthesis increasing effect of synthetic alpha-MSH.