César Aguilar

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

BackgroundEscherichia 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.ResultsWhole 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.ConclusionsThe 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.

Acetate Metabolism in Escherichia coli Strains Lacking Phosphoenolpyruvate: Carbohydrate Phosphotransferase System; Evidence of Carbon Recycling Strategies and Futile Cycles

The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS–). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS–Glc+), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (μ) higher than PB12 (0.21 and 0.13 h–1, respectively) while JM101 had a μ of 0.28 h–1. To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS– strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS– derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12.

Hacia la identificación de relaciones de hiponimia/hiperonimia en Internet

Resumen es: En este trabajo se presenta un enfoque para la extraccion automatica de pares hiponimo-hiperonimo. En particular se propone un metodo de extraccion de in...

Rayleigh scattering to study acoustic waves in a turbulent jet flow

Rayleigh scattering is used as a visualization technique to study density fluctuations due to acoustical waves produced at the exit of a turbulent air jet flow. Scattering is produced by sending a 532‐nm laser beam through the flow. The scattered light is collected by a lens and projected to a screen to achieve an image. Pictures showing the crossover between expansion and compression waves in the jet have been obtained. The technique allows the mapping of the pattern of shock waves along the jet. These pictures can provide information of the exit velocity by analyzing the angle formed by the waves closest to the nozzle. Rayleigh scattering can also be used in an alternative technique that detects the scattered light by heterodyning. The current that comes out of the photodetector is proportional to the spatial Fourier transform (for a particular wave vector) of the density fluctuations as a function of time. [Work supported by UNAM through the DGAPA Project No. IN107599.]

Rayleigh scattering and heterodyne detection as a nonintrusive microphone

Heterodyne detection of a monochromatic laser beam scattered by molecules in a transparent gas gives a signal that is proportional to the spatial Fourier transform of density fluctuations for a wave vector determined by the optics. In a turbulent air jet, density fluctuations are due to either acoustic waves or to entropy fluctuations. This technique captures both. The method can then be used as a nonintrusive microphone to study the propagation of acoustic waves inside the jet. At each point in the flow the direction of propagation of the acoustic wave is determined, and the acoustic field can be described. This will eventually help to localize the sources of the waves in the flow and hopefully determine the hydrodynamic event that generates these waves.