U075378.pdf (58.65 MB)
The isolation and characterisation of an Escherichia coli mutant resistant to the voltage operated calcium channel inhibitor, verapamil.
thesisposted on 2015-11-19, 08:53 authored by Martin David. Goldberg
In eukaryotes, sophisticated mechanisms have been evolved to ensure that key events in the cell cycle take place at the correct time. The triggering of these events is largely controlled by transient increases in the concentration of intracellular free calcium ions [Ca2+ ]i. If these transient increases m [Ca2+ ]i are prevented by the use of drugs that either block the voltage operated calcium channels (VOCCs), inhibit the release of Ca2+ from the cells' internal reserves, or block the receptor proteins that bind intracellular Ca2+ ions, the cell cycle will stop at specific points. The cell cycle will only recommence following removal of the Ca2+ blockade. In bacteria, many genes have now been identified that are essential for specific cell cycle events. However, no regulator has been identified that actually controls the timing of these events. Many theoretical models have been developed to explain bacterial cell cycle control, but there is little solid evidence to support them. A hypothesis has been developed that states that Ca2+ ions regulate the bacterial cell cycle in a manner analogous to eukaryotes. To test this hypothesis, temperature sensitive mutants of the E. coli strain, N43, resistant to the eukaryotic VOCC inhibitors verapamil and diltiazem were isolated, although detailed characterisation was confined to a verapamil resistant mutant, N43verA1. All the mutants are affected at a specific locus on the E. coli chromosome consisting either of deletions or major genetic rearrangements of three genes, hns, galU and hnrG (renamed rrx in this work). These genes do not constitute the expected receptor, but nevertheless, detailed examination of N43verA1 demonstrated that it appears to be unable to regulate its [Ca2+]I, since it is hypersensitive both to the concentration of Ca2+ in the medium and the Ca2+-chelator, EGTA. Moreover, when N43verA1 was transferred from the permissive to non-permissive temperature, it formed filaments, minicells and "chains of sausages", demonstrating some defect in cell division. An examination of the wild-type and mutant's responses to treatment with verapamil, EGTA or Ca2+ by labelling cell proteins with [35S-] methionine, indicated an extremely complex response. N43verA1 constitutively expresses a number of heat-stable proteins, which are further induced following treatment with verapamil or EGTA. Preliminary data suggests that some of the EGTA inducible proteins possess calmodulin-like properties. The three genes, hns, galU and rrx appear to interact, either at the genetic or protein level. Measurements of hns expression indicated that it is induced by verapamil, but not by EGTA. Overexpression of galU results in hyper-resistance to verapamil and Ca2+, which is modulated by hns and rrx, respectively. It is proposed that these genes in some way regulate the [Ca2+]i, in addition to, or in relation to their role in nucleoid organisation and control of the stationary phase sigma factor, RpoS.
Date of award1995-01-01
Awarding institutionUniversity of Leicester