Microbiology 460
Department of MicrobiologySouthern Illinois University at Carbondale
Lecture Topic: Stationary-Phase Adaptive Mutations (SPAM)
Problem is to determine how many mutants arise as a direct and specific response to selection pressure (would not have occurred in its absence) and how many are spontaneous (would have occurred even in the absence of selection).
Replica plating tests cells siblings rather than the cells themselves so it was possible to prepare pure cultures of mutants without ever exposing the cells or their ancestors to any selection pressure. Final proof that at least some forms of bacterial mutation are occurring spontaneously.
But these experiments can't detect the existence of a non-random form of mutation.
Cairns studied mutations where the selection pressure rewards mutants by letting them multiply but also allows all other non-mutant cells to survive so that they can at least have the opportunity to perform directed mutation.
Discovered that during prolonged selection, mutations that were specifically useful to the cell arose continuously in a population of non-dividing cells.
1) Plate Lac- cells on M9 plates without lactose. Overlay with lactose-containing medium. Delayed the overlay by several days and found the same pattern of Lac+ mutagenesis than when overlaid right away. Found accumulation of late Lac+ mutants occurred only in the presence of lactose.
It is now known that this lac mutant is a frameshift mutant and that the Lac operon mutation must be located on an F' plasmid to observe this effect. It appears that the role of the conjugative plasmid is, at least in part, to promote the events that are rare for chromosomal sites - duplication formation and high-level gene amplification. According to the amplification mutagenesis model of adaptive mutation, these events are an essential intermediate in the process of reversion in the Cairns system. Thus, the gene amplification increases the copy number of the mutant Lac gene and therefore increases the frequency of mutation at that site.
2) Used strain in which araC (control element for arabinose operon) is placed upstream of lacZ but separated by short segment of Mu that contains transcription termination signals. If intervening Mu segment is deleted, cells can grow on lactose in the presence of arabinose; called Lac(Ara)+ mutants. In rich media, Lac(Ara)+ cells do not accumulate at a detectable rate. But when selective medium used, cells become Lac(Ara)+ at proportion of 10-8.
3) E. coli has cryptic gene, ebgA, that can hydrolyze lactose if lacZ has been deleted. Activation of ebg requires two mutations, one in the repressor (ebgR) and one in the coding region of ebgA. Each mutation by itself will allow a lacZ mutant to use lactose. Each mutation occurs at frequency of less than 10-8. But yet will see Lac+ colonies of lacZ strain that has been grown to stationary phase on medium containing lactose (these colonies arise thru mutations in both ebgR and ebgA).
Now known that the mutation in ebgR arises first that allows the cells to grow very slowly on lactulose. The second mutation (in the ebgA gene) then arises and allows the double mutants to grow very rapidly. This process of acquired mutations may also occur in tumor cells.
"It is difficult to imagine how bacteria are able to solve complex problems like these - and do so without, at the same time, accumulating a large number of neutral and deleterious mutations - unless they have access to some reversible process of trial and error."
Adaptive mutations appear to be directed in non-growing cells to sites that allow growth.
Now know that SIMS (selection-induced mutations) may be either base substitutions or positive/negative frameshifts in the gene under selection, by excision of mobile genetic elements, or by nonsense suppressor base substitutions in tRNA genes.
Currently known to occur in at least 9 E. coli genes, a Clostridium gene and 3 Saccharomyces genes.
Are SIMS specific to the gene under selection? Yes. The trpB revertants arise only during trp starvation but not during histidine or cysteine starvation.
But if these mutations don't arise during replication, how do SIMS occur?
4 models address this question:
1) Transcription errors that result in functional gene products are captured back into DNA.
2) The transcription event itself is mutagenic such that over- or under-expression of genes increases that apparent mutation rate.
3) DNA mismatch repair is very slow so a useful error can result in a functional gene product which then results in cell division and immortalization of the mismatch.
4) Some of the stationary-phase cells enter a hypermutable state and if a useful mutation arises, replication occurs and the cell can escape from the hypermutable condition.
Known that some SIMS require RecA and RecBCD while other SIMS require UvrABCD. Therefore appears that more than one mechanism may be responsible for SIMS.
Adaptive mutation at some loci (e.g. ebg) requires PhoPQ and is therefore induced. The PhoPQ system behaves as a normal two-component regulatory system. PhoQ is the sensor-kinase member of the pair, while PhoP is the response regulator. PhoQ senses drops in magnesium concentrations outside the cell. When this happens, it autophosphorylates, and then phosphorylates PhoP. PhoP is a classical response regulator, in that it is a positive transcription factor for a host of genes.
This finding is consistent with the view that adaptive mutagenesis is a programmed response to prolonged environmental stress in which a portion of the stressed population transiently enters a hypermutable state as a last desperate effort to find a genetic solution to the current environmental problem.