Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells

By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH. During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP. Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed [Rosen, B. P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.     

Plastid division: across time and space

Plastid division is executed by the coordinated action of at least two molecular machineries--an internal machinery situated on the stromal side of the inner envelope membrane that was contributed by the cyanobacterial endosymbiont from which plastids evolved, and an external machinery situated on the cytosolic side of the outer envelope membrane that was contributed by the host. Here we review progress in defining the components of the plastid division complex and understanding the mechanisms of envelope constriction and division-site placement in plants. We also highlight recent work identifying the first molecular linkage between the internal and external division machineries, shedding light on how their mid-plastid positioning is coordinated across the envelope membranes. Little is known about the mechanisms that regulate plastid division in plant cells, but recent studies have begun to hint at potential mechanisms.     

Purification and Characterization of the Pasteurella Haemolytica 35 Kilodalton Periplasmic Iron-Regulated Protein

ABSTRACT

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrahgic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 μM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA.     

Herbicidal 4-hydroxyphenylpyruvate dioxygenase inhibitors—A review of the triketone chemistry story from a Syngenta perspective

A review, outlining the origins and subsequent development of the triketone class of herbicidal 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.     

PhyTB: Phylogenetic tree visualisation and sample positioning for M. tuberculosis

Background

Phylogenetic-based classification of M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.

Results

We have developed a web-based tool called PhyTB (http://pathogenseq.lshtm.ac.uk/phytblive/index.php) to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.

Conclusion

PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (http://sourceforge.net/projects/phylotrack).