Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.The bacterium and model organism Escherichia coli K-12 has three basic metabolic modes: aerobic respiration, anaerobic respiration, and fermentation (1, 2). There is a hierarchy in which aerobic respiration is preferred to anaerobic respiration, which in turn is preferred to fermentation (1). This hierarchy reflects the relative amounts of energy that can be conserved by these metabolic modes, and oxygen availability is the major signal that governs which metabolic mode is adopted.Many environments, both natural (host intestinal tract) and man-made (bioreactors), are characterized by the presence of oxygen gradients and/or regions of variable oxygen availability (3, 4). Thus, how patterns of gene expression adapt across the range of physiologically relevant oxygen availabilities is important for the efficiency of biotechnological processes that use E. coli as a cell factory and for competitiveness in natural environments (4). However, obtaining reproducible data from E. coli cultures at low oxygen tensions is technically demanding, and the overwhelming majority of the relevant literature reports the results of experiments with fully aerobic or anaerobic cultures. Furthermore, as indicated by Alexeeva et al. (5), in the relatively few attempts to study E. coli grown at intermediate oxygen tensions, it was apparent that neither dissolved oxygen tension nor the gas input to a chemostat accurately describes the responses of the culture to changes in ...
N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine.
CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix ␣R from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H 2 O-D 2 O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.Life depends on multiple metals (1). Iron-, copper-, and zincresponsive transcriptional regulators are known in yeast (AFT1, MAC1, ACE1, and ZAP1) (2) and higher eukaryotes (MTF1) (3, 4). In bacteria, multiple families of metal-responsive transcriptional regulators have been described, including ArsR/ SmtB-like DNA-binding repressors (5-8). These are a subgroup of winged helix repressors. We previously discovered the cellular zinc sensor SmtB (5). In elevated zinc, SmtB repression of the smtA gene is alleviated to allow transcription of a metallothionein gene encoding a protein, which sequesters the surplus metal atoms (9, 10). In contrast, the related ArsR sensor responds to arsenite to regulate production of an arsenicaltranslocating ATPase (6). The location of the metal-sensing site, the complement of ligands and site geometry, vary in different members of this family of regulators (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). There is considerable interest in understanding how metal binding to metal sensors is transduced into altered levels of transcription of their target genes.Mycobacterium tuberculosis contains ten genes encoding ArsR/SmtB sensors making it a useful model organism for studies of these regulators. It is plausible that fluctuations in metal concentrations within macrophages have selected for multiple genes encoding such regulators in this pathogen. One of the M. tuberculosis sensors, CmtR, was chosen for structural and functional studies of a sensory mechanism. We previously established that CmtR responds in vivo to cadmium and lead to modulate production of a toxic metal-exporting P 1 -type ATPase (16). Residues Cys-57 and Cys-61 plus Cys-102 have been implicated ...
We report a cadmium-and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P 1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co(II)-, and Ni(II)-substituted CmtR revealed S؊ to metal-charge-transfer which was absent in NmtR, providing diagnostic metaldifference spectra that discriminated between metalbinding to these two proteins. Ni(II)-binding isothermal titrations of CmtR are complex, with K app ؍ 1.8 ؋ 10 4 M ؊1 for site 1 , three orders of magnitude weaker than K Ni for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd(II) gave Cd(II)-dependent difference spectra almost identical to Cd(II) 0.9 -CmtR. Thus, Cd(II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni(II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co(II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal ␣5 helices or helix ␣3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR.
Escherichia coli possesses two major systems for inorganic phosphate (P(i)) uptake. The Pst system (pstSCAB) is inducible by low phosphate concentrations whereas the low-affinity transporter (pitA) has been described as constitutively expressed. PitA catalyses transport of metal [Mg(II), Ca(II)]-phosphate complexes, and mutations in pitA confer Zn(II) resistance. Here we report that pitA transcription is not constitutive; activity of a single-copy pitA-lacZ transcriptional fusion (monolysogen) was maximal at high extracellular Zn(II) (150 microM), in the absence of added P(i), and in a well-defined pitA mutant strain. Intracellular zinc levels were unaffected by adding Zn(II) to the medium for both the wild-type and mutant strains. However, in the wild-type strain, Mg levels (per gram of dry biomass) fell by eightfold in cells grown with added Zn(II) and by 20-fold when Zn(II) and P(i) were added to cultures. Mutation of pitA reduced the effects of external Zn(II) and phosphate levels on Mg pools, consistent with competition or inhibition by Zn(II) of PitA. The mechanism of pitA regulation by extracellular Zn(II) and P(i) is unknown but appears not to involve Fur or other well-characterized regulators.
Many of the complex systems found in biology are comprised of numerous components, where interactions between individual agents result in the emergence of structures and function, typically in a highly dynamic manner. Often these entities have limited lifetimes but their interactions both with each other and their environment can have profound biological consequences. We will demonstrate how modelling these entities, and their interactions, can lead to a new approach to experimental biology bringing new insights and a deeper understanding of biological systems.
Research in higher education has revealed that only a small proportion of students complete assigned reading during semester. The current studies examined students' reading practices, and sought to explore potential factors underlying these practices. Three studies were conducted. Study 1 utilised a questionnaire to examine how long students spend reading academic material. Students reported spending an average of 14.1 hours per week reading a range of sources, including textbooks and journal articles for both guided and independent reading. The number of hours spent reading was lower than university expectations. Study 2 involved conducting focus groups to explore potential factors underlying students' reading practices. Six main themes emerged in the data; Expectations, Perceived benefits, Course structure, Lack of time, Practicalities, and Confidence. Study 3 further examined students' perceptions of a lack of time and a lack of confidence with reading using a diary exercise and a further questionnaire. The diary exercise revealed that students spent an average of 6.5 hours per day engaged in academic activities. In addition, students were generally confident with reading, although more so with reading textbooks than journal articles. The findings are discussed in terms of implications for staff teaching in higher education.
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