Biomedical subjects

Colin Scott

Publications and source records attributed to Colin Scott.

7 recordsLinked to original sources

PubMed2006

A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins.

Sulfur is essential for life on Earth, but its availability is limited in many environments. Here the sulfur-starvation response of the model soil bacterium Pseudomonas putida KT2440 is shown to be associated with an approximately fivefold reduction in the total soluble thiol content of the cell. A bioinformatic survey of the P. putida KT2440 genome identified 646 genes encoding proteins with a significantly lower than average sulfur content (low sulfur-content proteins, LSPs), the expression of which may have a role in the global reduction of cellular thiol content during sulfur starvation. Analysis of the genetic organization of the LSP-encoding genes showed that 31% were potentially transcriptionally associated with at least one other gene encoding a protein defined as an LSP. In particular, 55 LSP genes were located in three large clusters, termed low-sulfur islands (LSIs) here. The predicted identities of the proteins encoded by the LSIs strongly suggest that the LSIs have a role in acquiring sulfur from organic sulfur sources during sulfur starvation. This hypothesis was supported by transcription fusion studies on a limited number of LSP promoters under low-sulfur conditions. In a wider survey of bacterial species, LSIs were found to be more prevalent in free-living, Gram-negative bacteria than in Gram-positive or obligately intracellular bacteria.

Bacterial Proteins↗

PubMed2006

Escherichia coli transcriptome dynamics during the transition from anaerobic to aerobic conditions.

Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Several previous transcript-profiling experiments have compared separate anaerobic and aerobic cultures. Here the process of adaptation was investigated by determining changes in transcript profiles when anaerobic steady-state cultures were perturbed by the introduction of air. Within 5 min of culture aeration the abundances of transcripts associated with anaerobic metabolism were decreased, whereas transcripts associated with aerobic metabolism were increased. In addition to the rapid switch to aerobic central metabolism, transcript profiling, supported by experiments with relevant mutants, revealed transient changes suggesting that the peroxide stress response, methionine biosynthesis, and degradation of putrescine play important roles during the adaptation to aerobic conditions.

Adaptation, Physiological↗

PubMed2005

A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor.

Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

Aconitate Hydratase↗

PubMed2003

DNA target sequence and FNR-dependent gene expression.

FNR proteins are global transcription regulators that respond to fluctuations in environmental oxygen. They recognise a DNA target consisting of an inverted repeat, TTGATN(1)N(2)N(3)N(4)ATCAA (where N(1-4) represents a non-conserved tetrad, NCT). Analysis of 68 known and predicted FNR sites from the Escherichia coli K12 genome revealed a bias toward A or T at positions N(2) and N(3) of the NCT. The effect of the NCT sequence on FNR-dependent transcription in vivo was assessed using a series of class II and class I model promoters with different NCT sequences. Changing the NCT sequence did not affect basal activity but altered anaerobic induction by as much as an order of magnitude. Thus, the NCT sequence is a fundamental component in setting the dynamic range of the FNR switch.

Base Sequence↗

PubMed2003

Maldevelopment of convergence eye movements in macaque monkeys with small- and large-angle infantile esotropia.

PURPOSE: To describe symmetric convergence eye movements evoked by disparity and/or accommodative cues in esotropic macaque monkeys, with the goal of determining whether these animals have the vergence deficits found in humans with esotropia. METHODS: Physical far and near targets were used to evoke large (approximately 8 degrees) symmetric convergence eye movements in four adult macaque monkeys (two with strabismus, two normal), using positive-feedback rewards. One strabismic monkey had infantile-onset, small-angle esotropia (small-eso approximately 2 degrees) induced by alternating occlusion from birth to age 9 months. The other strabismic monkey had naturally occurring, large-angle (approximately 25 degrees) infantile-onset esotropia (large-eso). Visual acuity was normal in each eye as measured by spatial sweep visually evoked potentials (VEPs). Eye movements were recorded using magnetic search coils. RESULTS: When viewing binocularly, both normal monkeys exhibited accurate, stereotyped symmetric convergence movements that achieved 87% to 96% of the required change in vergence angle by the end of the initial movement. In contrast, the small-eso monkey's convergence response when viewing binocularly was variable, strikingly asymmetric, usually accompanied by a disjunctive saccade, and subnormal, achieving only 56% of required vergence. The convergence response of the large-eso monkey was also asymmetric and weak, achieving 18% of the required vergence and employing conjugate saccades to refixate the near target. Monocular viewing (i.e., accommodative vergence) caused substantial reductions in both convergence amplitudes and velocities in the normal monkeys, but had a minor effect on the vergence behavior of the strabismic animals. CONCLUSIONS: Monkeys with small- and large-angle infantile esotropia have striking maldevelopments of binocular (disparity-driven) convergence and use accommodative vergence and saccades to refixate near targets. Their vergence behavior resembles that in esotropic humans. The maldevelopment may be explained in large part by the paucity of binocular connections recently described in the visual cortex of esotropic macaques.

Accommodation, Ocular↗

PubMed2002

NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp.

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

Anaerobiosis↗

PubMed2001

Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation.

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Bacterial Proteins↗