| Popis: |
It has been thought for some time that the 76 minute region of the Escherichia coll chromosome may contain a large cluster of essential genes. The finding that the ftsYEX operon and rpoH are contiguous has given encouragement to this speculation. The extended homology of the FtsE protein with the superfamily of ATP binding cassette (ABC) proteins has created extra interest in this gene product in particular and the possibility of ascribing a function to it. This investigation into ftsE and the 76 minute region has given the results described below. \ud \ud A localised mutagenesis procedure was performed on the 76 minute region and a screen for temperature-sensitive growth yielded 28 temperature- sensitive mutants. There were three classes of mutants based on their microscopic appearance at the restrictive temperature, that is filamentous, rpoH-like (having short filaments with inclusions), and pleomorphic. The mutations carried by these mutants were mapped by complementation with a range of plasmids carrying the known essential genes from the 76 minute region. Of the mutations giving rise to a filamentous phenotype, nine mutations were found to map in ftsE, five in ftsX, two appeared to carry mutations in both ftsE and ftsX, and two appeared to be complemented but were not fully mapped. All nine of the mutations giving rise to an rpoH-like phenotype were mapped to rpoH. The remaining two mutations that gave a pleomorphic phenotype were not complemented by any of the available plasmids. No temperature-sensitive lethal mutations were isolated in ftsY or ORF4, although this does not constitute proof that these genes are non-essential. \ud \ud The ftsE missense mutants were analysed phenotypically and were not easily classified into groups on the basis of temperature-sensitivity, microscopic appearance, salt reversibility of phenotype, or the requirement for de novo protein synthesis for recovery of septation following a period of incubation at the restrictive temperature. The ftsE mutant alleles were cloned and the DNA sequenced. This showed the mutations to be clustered in a region of extensive homology with the ABC superfamily of proteins. Nucleotide binding of maxicell radiolabelled FtsE and FtsY proteins was investigated using dye-ligand chromatography columns. The results were very ambiguous due to the low amounts of the labelled proteins in the maxicell lysates but they are suggestive of a possible interaction between ATP and these proteins. These findings are consistent with the premise that FlsE will, like other ABC proteins, hydrolyse ATP and couple this to an essential biological process. \ud \ud One of the mutations (Ts33) that was not complemented by any of the available constructs carrying DNA from the 76 minute region was mapped by transduction to the 'silent' region clockwise of 76 minutes, and appeared to lie very close to pit. It is therefore thought to define a new essential gene. This confirms the existence of more essential genes near 76 minutes. A cosmid library was constructed and ten cosmids were isolated that contained Ts33 complementary DNA. \ud |
