Compensating for alien genes…

[This post was restored from a WayBackWhen archive of an older incarnation of mentalindigestions.net]

“FROM the perspective of a bacterium, higher eukaryotes are oversexed, unadventurous and reproduce in an inconvenient way.” So says Pål Johnsen and Bruce Levin in their commentary of today’s article for discussion, and nary a truer word said. Of course, one may state that inconvenient as reproduction may be, bacteria clearly have no sense of fun.

There was once an idea that we could address the problem of antibiotic resistant bacterial strains by removing the ailing antibiotic from clinical use. In the absence of selective pressure it was thought that the evolutionary traits that enable the strain to resist the antibiotic would actually put the strain at a competitive disadvantage compared with a strain that doesn’t have such antibiotic resistance. The proposed cause of this? Fitness costs – these are imposed by a resource-expensive set of mutations, or carriage of alien DNA, that make the resistant strain compete less well once its non-resistant brethren are no longer being killed off by the antibiotic.

However, some years ago now experimental evidence suggested that this is not always the case; it may in fact be often not the case. It is worth mentioning at this point that it has been shown that in some circumstances (alt) the number of infections caused by a particular antibiotic-resistant pathogenic bacterium have become fewer on reduction (or removal) of the antibiotic to which that strain is resistant, but to assume this would be the case with all strains/antibiotics is naïve.

It is true, with few exceptions, that initially both plasmid and chromosomally encoded resistances result in fitness losses. However, when resistance has a cost it is possible for compensatory mutations in a cell to ameliorate these costs, usually without the loss of resistance. The type of compensatory mutations that mitigate the fitness cost of acquiring antibiotic resistance, or any other incoming DNA that encodes potentially useful genes, will depend very much upon the environment in which the bacteria finds itself. These include the availability of resources, i.e. the growth environment of the bacteria, the environment of the genes (mobile or chromosomal), or whether the genes are being selected for by an external factor, such as the presence of antibiotics in the case of resistance genes.

So what sort of ‘nips and tucks’ might a bacterial population undergo in order to maintain a battery of costly genes, but that may provide an ongoing advantage? Well, this is the subject of much ongoing research; one example indicated that, in the absence of selective pressure, costly genes are simply silenced – a molecular mechanism often found in higher organisms that prevents a gene from being ‘switched on’. Thus a reservoir of drug resistance determinants may remain in populations that have compensated for their presence, remaining ‘inactive’ until a selective pressure removes the silencing.

A recent study by Peter Lind (et al.), a grad student working in the lab of Dan Andersson at Uppsala, Sweden, addresses a particularly pertinent question of compensatory mutations: those associated with genes acquired by horizontal gene transfer (HGT). HGT bypasses the slow and haphazard process of evolution (via random mutation, selection and recombination) by offering an opportunity for bacteria to receive fully fledged genes encoding pathogenicity factors (genes that make bacteria better at causing disease) as well as genes that encode resistances to disinfectants and/or antibiotics, amongst others. There is no doubt that such incoming DNA may pose significant fitness costs, so Lind et. al. set out to quantify the nature of compensatory mutations on such incoming DNA.

To do this they replaced three core genes encoding ribosomal proteins (key components of the protein-making factories of the cell) in Salmonella typhimurium with orthologues (genes performing a similar function) from various other microbes; some closely-related, and some very much not. The aim was to replicate receipt of DNA by HGT processes that can occur naturally in the environment, but here they used genes that form conserved parts of multi-protein complexes that perform a central role in the cells. These genes actually represent rare targets for HGT, but in these experiments the aim was to force the cells to make compensatory mutations to stabilize them (even if they’re poor substitutes) just to survive. Also, by studying proteins that are implicitly involved in the growth of the cells, they are able to link the stabilisation of these genes to the bacterial growth rate, the latter being a relatively easy property to measure experimentally.

So what of compensatory mutations? Lind points out, ‘as with any mutation, most transferred genes are likely to be neutral or deleterious in an alien cell, and over time become mutationally inactivated or lost.’ Alternatively, even if performing weakly, the gene may persist and its function can be fine-tuned by compensatory mutations to reduce fitness costs and improve gene function. This fine-tuning may take a number of forms: it may be achieved by multiple DNA basepair mutations, each resulting in slight improvement; or alternatively a phenomenon called ‘gene amplification’, which is relatively common, may result in a large number of extra copies of the gene.

This would have two outcomes:

a) It would compensate for the protein not doing its job very well, bymaking more of it; and,

b) provide a large number of extra copies of the DNA, each of which will be subject to mutation, so increasing the rate at which the gene can be ultimately improved.

In Lind’s (et al.) experiments, the Salmonella populations bearing costly genes were subjected to cycles of serial passage, where a small culture of bacteria is grown for 24 h, then diluted 1000-fold into fresh growth medium. Each strain producing the alien ribosomal proteins grew significantly more slowly than the aboriginal population, with the most distantly related proteins exacting the greatest fitness cost, as one might expect. At points between 40 – 250 generations (a bacterial generation being the time taken to double the population), the initial cost of carrying the foreign genes was mitigated. Populations of these improved growers were subjected to DNA sequencing to identify changes to the DNA sequence, as well as to techniques that test the relative abundance of a particular gene and corresponding protein.

What the researchers found is that compensation for fitness costs was achieved by increasing the amount of ribosomal protein several-fold, which was achieved by the cells duplicating the region of DNA encoding the alien genes by 100-fold. Thus Lind et al. show that by increasing the dosage of suboptimal genes, and thus proteins, gene amplifications can compensate for the fitness cost of carrying alien genes; and as mentioned earlier, may be subject to increased mutational frequency that can further refine their activity.

In this study the researcher focussed on how compensatory mutations affected a specific set of alien ribosomal genes. However, there are additional secondary genetic ‘nips and tucks’ that can happen elsewhere on the chromosome, within other genes or regulatory processes, that can compensate for fitness costs imposed by newly introduced genes. For the above set of experiments such mutations were controlled by pre-adapting theSalmonella strains in the experimental (growth) environment, prior to introduction of the alien genes. Echoing Pål Johnsen and Bruce Levin in their commentary, I think it will be particularly interesting to follow up this work with studies on how bacterial populations confront the costs associated with truly novel genes that have no corresponding genes in the host, e.g. those encoding antibiotic resistance.

____________________

THIS is the first of three posts about bacterial antibiotic resistance, given that I will hopefully be returning to research in this area. In the above post I essentially pointed out that bacteria can have their cake, and eat it too. In my next post I will talk about an alternative strategy that could be taken to prevent the spread of antibiotic resistance, and in a final post I will talk about how bacteria can (and do) avoid being killed by antibioticswithout encoding a defined resistance mechanism in the conventional sense.

Lind, P., Tobin, C., Berg, O., Kurland, C., & Andersson, D. (2010). Compensatory gene amplification restores fitness after inter-species gene replacements Molecular Microbiology, 75 (5), 1078-1089 DOI: 10.1111/j.1365-2958.2009.07030.x

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