Siderophore activity of myo-inositol hexakisphosphate in Pseudomonas aeruginosa

Myo-Inositol hexakisphosphate (InsP6), which is found in soil and most, if not all, plant and animal cells, has been estimated to have an affinity for Fe3+ in the range of 10(25) to 10(30) M-1. In this report, we demonstrate that the Fe-InsP6 complex has siderophore activity and is able to reverse the iron-restricted growth inhibition of Pseudomonas aeruginosa by ethylene diamine di(o-hydroxyphenyl)acetic acid. With 55Fe-InsP6 in transport studies, iron uptake is strongly iron regulated, being repressed after growth in iron-replete conditions and inhibited by treatment with potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone. The kinetics of iron transport revealed a Km of 100 nM. Self-displacement of binding of [3H]InsP6 to isolated membranes by InsP6 revealed a single class of binding sites (Kd = 143 +/- 6 nM; Hill coefficient, 1.1 +/- 0.1). The binding of [3H]InsP6 to membranes was not dependent on whether cells had been grown under conditions of high or low iron concentrations. We believe that this is the first report of inositol polyphosphate activity in prokaryotic cells.

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20 h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular alpha-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

From therapeutic to political education: the centrality of affective sensibility in critical pedagogy

While the need for humanising education is pressing in neoliberal societies, the conditions for its possibility in formal institutions have become particularly cramped. A constellation of factors – the strength of neoliberal ideologies, the corporatisation of universities, the conflation of human freedom with consumer satisfaction, and a wider crisis of hope in the possibility or desirability of social change – make it difficult to apply classical theories of subject-transformation to new work in critical pedagogy. In particular, the growth of interest in pedagogies of comfort (as illustrated in certain forms of ‘therapeutic’ education and concerns about student ‘satisfaction’) and resistance to critical pedagogies suggest that subjectivty has become a primary site of political struggle in education. However, it can no longer be assumed that educators can (or should) liberate students’ repressed desires for ‘humanisation’ by politicising curricula, pedagogy or institutions. Rather, we must work to understand the new meanings and affective conditions of critical subjectivity itself. Bringing critical theories of subject transformation together with new work on ‘pedagogies of discomfort’, I suggest we can create new ways of opening up possibilities for critical education that respond to neoliberal subjectivities without corresponding to or affirming them.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.