Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli

The chlD gene in Escherichia coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. We constructed chlD-lac operon fusions and measured expression of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it had an identical affect on chlD expression. Expression of chlD-lac was increased in cells grown with nitrate. Strains with chlD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chlD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response to nitrate; mutations in chlB, narC, or narI resulted in partial constitutive expression of the chlD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chlA, chlE, or chlG which affect Mo cofactor metabolism, did not affect the expression of chlD-lac.     

Cistrome: an integrative platform for transcriptional regulation studies

The increasing volume of ChIP-chip and ChIP-seq data being generated creates a challenge for standard, integrative and reproducible bioinformatics data analysis platforms. We developed a web-based application called Cistrome, based on the Galaxy open source framework. In addition to the standard Galaxy functions, Cistrome has 29 ChIP-chip- and ChIP-seq-specific tools in three major categories, from preliminary peak calling and correlation analyses to downstream genome feature association, gene expression analyses, and motif discovery. Cistrome is available at http://cistrome.org/ap/.     

Envelope-associated folded chromosomes for Escherichia coli: variations under different physiological conditions.

The folded chromosome of Escherichia coli has been investigated under various lysis and physiological conditions. A new gradient system was devised that allows excellent separation between unlysed cells and envelope-associated and envelope-free chromosomes. Isotope incorporation experiments showed that the fraction often called "membrane-bound nucleoids" contains cell wall in addition to nucleic acids, membranes, and proteins. The amount of lysozyme added and the lysozyme digestion time were found to be important when comparing the rate of sedimentation of envelope-associated chromosomes obtained under various physiological conditions. Amino acid-starved cells were found to be much harder to lyse with lysozyme than exponentially grown cells, The difference in sedimentation coefficient of envelope-associated chromosomes described earlier (Ryder and Smith, 1974) was not detected when the latter two types of cells had been given equivalent, but not identical, lysozyme treatment such that detergent-mediated lysis proceeded at the same rate. Analysis of pulse- and uniformly labeled chromosomes from amino acid-starved cultures revealed no preferential labeling of either envelope-associated or -released nucleoids. Nor was there a difference in sedimentation coefficient between uniform and pulse-labeled envelope-associated nucleoids. These results are in disagreement with the models for chromosome replication of Worcel and Burgi (1974) and Ryder and Smith (1974), respectively. Growing cells on carbon sources poorer than glucose demonstrated that the replicating chromosomes sediment faster than the bulk of envelope-associated nucleoids. The slower the growth rate, the greater this difference became. An alternative hypothesis regarding chromosome replication and its association with the cell envelope is presented.     

Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor

Neb-LFamide or AYRKPPFNGSLFamide was originally purified from the grey flesh fly Neobellieria bullata as a myotropic neuropeptide. We studied the occurrence of this peptide and its isoforms in the central nervous system of different insect species by means of whole mount fluorescence immunohistochemistry, mass spectrometry, and data mining. We found that both sequence and immunoreactive distribution pattern are very conserved in the studied insects. In all species and stages we counted two pairs of immunoreactive cells in the pars intercerebralis. These cells projected axons throughout the ventral nerve cord. In the adult CNSs they formed a large number of immunoreactive varicosities as well. Mass spectrometry and data mining revealed that SIFamide exists in two isoforms: [G1]-SIFamide and [A1]-SIFamide. In addition, the SIFamide joining peptide is relatively well conserved throughout arthropod species. The conserved presence of two cysteine residues, separated by six amino acid residues, allows the formation of disulphide bridges.