Evidence for adaptation to cave geochemistry

Cave environments have varied geochemistry which may include a variety of metals such a titanium, nickel, iron, magnesium, molybdenum and copper. Uptake of some compounds such as copper will inhibit organism growth while other compounds such as and iron, magnesium are essential to organism biochemistry. We therefore hypothesis that uptake of required metal co-factors whilst excluding harmful componds will affect microbial survival and therefore be under selective pressure and be incorporated into the P. fluorescens strains isolated from caves.

There are three previously sequenced P. fluorescens strains which were isolated from either soil or plant root nodule environments. Comparison of our sequenced genome with these previously sequenced genomes will allow us to determine which genes are be specific to cave environments. This can be done through comparing orthologs between the four strains and determining which are specific to the R124 genome (see literature on [microbial pan-genome][pan-genome]). Furthermore we also identify which of the genes specific to R124 lie in horizontally transfered regions and give further indications of the composition of the environment.

We have the following hypotheses which based on what differences we expect to find in the cave sequenced genomes.

Metal uptake and detoxification

The diversity of metals present in the cave environment will mean that correct selection and uptake of metal cofactors will be under strong selection pressure. Siderophores are iron chelating peptides used by microbes to extract iron from the surrounding environment. A specific class of siderophores used by Pseudomonas species are poverdines.

A comparative genomics analysis by Moon et al. of pyoverdine pathways between Psuedomonas aeruginosa and P. fluorescens SBW25 showed the pyoverdine pathway is conserved between species with many corresponding orthologs identified. Differences were however found in the sequences and organisation of the pyoverdine producing molecules which indicates possible types of pyoverdine production. Furthermore P. aeruoginosa only contained one pyoverdine gene cluster while in P. fluorescens the pyoverdine genes are distributed across 7 loci. In P. fluorescens Pf0-1 and Pf-5 pyoverdine genes were distributed across 3 loci.

The variability in sequence and loci of pyoverdine pathway genes indicates these are candidates for examination to determine the extent of the P. fluorescens adaptation to cave environments in respect to iron uptake as well as other compounds. We therefore predicted that the cave isolated genome will show different large copy number, predicted expression level, or increased sequence similarity rate in siderophore biosynthesis genes compared to other P. fluorescens strains.

Catabolic pathways

The cave environment will likely contain may autotrophs producing biomass from the avaiable energy in the environment. An example of this may be chemolithotrophs which will produce energy from oxidation of environmental compounds. The production of biomass by autotrophs may result in the presence of heterotrophs which will consume the higher energy biomolecules produced by other microbes. We therefore hypothesise that the P. fluorescens R124 genome will contain catabolic pathways capable of metabolising these compounds.

Nitrogen fixation

Nitrogen is an important nutrient required for the production of many biomolecules such as nucleotides and amino acids. There will however be very little nitrogen present in the cave environments except as nitrogen molecules present in the cave air. If the P. fluorescens R124 strain does not catabolise other nitrogen containing biomolecules an alternative they it may fix nitrogen from the air. This will be evident as nitrogen fixing genes.

Anti-microbial compound production

CRISPR regions are thought to related to affecting bacterial defence mechanisms. It may be expected that the plant and cave strains have different numbers of CRISPR regions depending on the numbers of bacterial pathogens in the environment.

Nutrient starvation results in smaller genome size

The more nutrient starved environment will select for smaller genome size in the cave isolated strains. The cave environments may be expected to be relatively nutrient starved with little fluctuation in the environment at the depth samples were isolated. Therefore expected that the genomes of the cave strains will have lost many genes not related to the environment. Furthermore there may be selection pressure to minimise the genome content of the genome to prevent unnecessary transcription/translation which would consume scarce resources.

The nutrient limitation in the environment may be a strong selective pressure in the evolution of cave microbes. These environmental pressures may select for organisms that are oppourtunisitic in taking genetic material from the environment since the additional genetic material could result in a selective fitness advantage. The extra-cellular genetic material incorporated into the genome may be evident from genomic islands and plasmids encoding foreign genes.

The 16S ribosomal DNA of sequenced P. fluorescens stains is approximately 99% similar and is therefore highly conserved. Nevertheless previous analysis of P. fluorescens genomes indicates that there is greater genomic diversity than would be expected given the degree of conservation observed in the rDNA. Phylogenomic analysis of marker genes may reveal a truer representation of the P. fluorescens genetic diversity than is estimated using rDNA alone given the large divergence in the environments from which the strains were isolated. This may indicate greater genomic mutability than is shown by the rDNA alone. Comparison of tree lengths of the two methods of diversity estimation may reveal this.

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