- Research article
- Open Access
Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species
© Kumwenda et al.; licensee BioMed Central Ltd. 2014
- Received: 16 October 2013
- Accepted: 17 September 2014
- Published: 25 September 2014
Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms.
Using Meiothermus silvanus DSM 9946 as a reference genome, high level of coordinated rearrangements has been observed in extremely thermophilic Thermus that may imply existence of yet unknown evolutionary forces controlling adaptive re-organization of whole genomes of thermo-extremophiles. However, no remarkable differences were observed across species on distribution of functionally related genes on the chromosome suggesting constraints imposed by metabolic networks. The metabolic network exhibit evolutionary pressures similar to levels of rearrangements as measured by the cross-clustering index. Using stratigraphic analysis of donor-recipient, intensive gene exchanges were observed from Meiothermus species and some unknown sources to Thermus species confirming a well established DNA uptake mechanism as previously proposed.
Global genome rearrangements were found to play an important role in the evolution of Thermus bacteria at both genomic and metabolic network levels. Relatively higher level of rearrangements was observed in extremely thermophilic Thermus strains in comparison to the thermo-tolerant Thermus scotoductus. Rearrangements did not significantly disrupt operons and functionally related genes. Thermus species appeared to have a developed capability for acquiring DNA through horizontal gene transfer as shown by the donor-recipient stratigraphic analysis.
- Metabolic networks
- Genomic island
Bacteria of the genus Thermus inhabit both natural and man-made thermal environments such as hot springs, deep mines, compost manure, sewage sludge and domestic hot water [1, 2]. Thermus bacteria are of major interest because of their industrially important thermostable enzymes; their ability to reduce heavy metals and switch to anaerobic respiration under oxygen deprived conditions. Enzymes from thermophilic organisms have shown higher activity and stability than mesophilic or synthetic enzymes counterparts currently been used in industry for production of food, detergents, drugs and paper . Thermus scotoductus SA-01 in particular, has been found to reduce heavy metals such Fe(III), Cr(VI), Mn(IV), U(VI) and Co(III) [4, 5]. Reduction of Fe(III) and Mn(VI) can be applied in biotechnology for eradicating heavy metal pollution; controlling global warming; removing organic contaminants in ground water; fluxing phosphates and other contaminants from water supplies; and also for clearing clogged wells among many other uses . Fe(III) reduction under anaerobic conditions in swampy areas during flooding diverts electrons away from methane producers thereby reducing global methane fluxes into the atmosphere consequently lowering global warming. Anaerobic respiration is advantageous in bio-fuel production as temperature rises and oxygen depletes due to decomposition of biomass. Cr(IV) is cacogenic; hence its reduction eliminates toxicity in food and air for human health .
Mutations and natural selection have been known to be dominant drivers of microbial evolution until the observation of abrupt changes in traits of an organism such as acquisition of pathogenicity or drug resistance that could not be explained by predominantly known ordinary mechanisms in time. Since then, horizontal gene transfer through which organism acquire foreign DNA to incorporate into their genomes through conjugation, transduction and transformation has been used to explain the ‘quantum leaps’ in traits of organisms that defy neo-Darwinian theories. Genome plasticity due to natural transformations is considered as a major survival technique for Thermus species in extreme temperature environments . It is known that co-expressed and functionally related genes in bacteria are grouped into operons or co-localised on the chromosome creating a network of reusable functional blocks [9, 10]. However, introduction of new genes by horizontal gene transfer and genome rearrangements affect the order of genes and may disrupt operon structure that consequently may lead to metabolic network re-organisation. Genomic recombinations are involved in evolution and speciation of organisms in addition to other mechanisms such as mutations, natural selection and horizontal gene transfer . What triggers rearrangements and determine their locations on the chromosome remains unknown. The extent to which thermal environments affect genome rearrangements on the chromosome or exert evolutionary pressure on the metabolic network is also not clear. Both the retrograde and patchwork theories attempt to explain the evolution of metabolic networks based on gene and operon duplication linking distribution of genes on the chromosome which may be affected by rearrangements and consequently on the structure of the metabolic network . Comparative analysis of genes and genomes in Archea, Bacteria and Eukarya has revealed that different forces and molecular mechanism might have shaped genomes leading to new metabolic capabilities essential for adaptation and survival . Schwarzenlander et al.  and Friedrich et al.  observed high levels of natural transformation and identified a DNA uptake system encoded by 12 competent genes which code for pilin like proteins similar to type IV pilus biogenesis proteins. Eleven of which were identified and implicated in binding naked DNA from the environment, transporting it through the cell wall, outer and inner membranes into the cytoplasm. In T. thermophilus HB27, DNA binding is achieved by pilQ, transported through the outer cell membrane by comEA, pilF and pilA4, through the thick cell wall layers and inner membrane by pilM, pilN, pilO, pilA13 and comEC. Whilst prior work by Gouder et al.  performed a comprehensive analysis of genomic islands possibly acquired through natural transformations, and their functional contribution in Thermus species, this work investigated movement of genomic islands and the ability for Thermus species to acquire external DNA.
Against this background, we theorized that there may be several general trends in the whole genome adaptation to the high temperature environment in Thermus extreme thermophiles. Therefore we investigated the extent to which Thermus genomes have been shuffled and disintegrated due to rearrangements; how genome rearrangements affected functionally related genes and consequently exerted evolutionary pressure on the metabolic network. To investigate these questions, we performed a comparative genome analysis of Thermus scotoductus SA-01 (GenBank: NC_014974), Thermus thermophilus HB8 (NC_006461) and Thermus thermophilus HB27 (NC_005835). In some cases the comparative analysis was performed against Meiothermus silvanus DSM 9946 (CP002042), Meiothermus ruber (NC_013946), T. aquaticus Y51MC23 (ABVK02000000), Thermus sp. RL (AIJQ00000000), T. igniterrae ATCC 700962 (AQWU01000001), T. oshimai JL-2 (NC_019386-88), Thermus sp. CCB US3 UF1 (NC_017287 and NC_016634); and also against mesophilic Escherichia coli K-12 (NC_000913) and Bacillus subtilis (NC_000964). Furthermore, relative age and the movement of genomic islands among bacteria genomes have been investigated with the aim of analysing competence levels of Thermus species.
Identification of horizontally transferred genomic islands and single genes
Genomic islands in bacterial genomes were predicted by the SeqWord Genome Browser tool  and its semi-automatic realization SeqWord Sniffer , which are available at the SeqWord project website . To identify genes which can be acquired by DNA uptake and homologous replacement, gene trees were designed for all sets of orthologous genes and their topologies were compared against the consensus species tree. This analysis was implemented using an in-house Python script that utilizes PHYLIP package command line programs PROTDIST, NEIGHBOR and TREEDIST .
Identification of orthologous genes
Pairs of genes in two genomes were considered as orthologs if they reciprocally returned the best BLASTp hits in queries of predicted protein sequences of one genome against the whole set of proteins of another genome by using local implementations of BLAST and FORMATDB algorithms from NCBI  and an in-house BioPython based script for pipelining and output analysis. On the next step, MUSCLE alignment  was used to filter out false positive BLASTp predictions when the alignment covered less than 70% of the protein sequences in a pair of predicted orthologs. Resulting alignment files were used in designing gene trees as described above, but prior to phylogenetic analysis every alignment file was edited by the Gblocks program to remove ambiguous blocks .
Co-localization of functionally related genes on the chromosome
Distances between genes on the chromosome were assigned to four distance categories: 0–1,000; 1,001-10,000; 10,001-100,000; 100,001-1,000,000. A biological meaning of these distance categories is that the first category apply to genes belonging to the same operon, the second category was based on the average gene length and the subsequent categories incremented by an order of these magnitudes . To determine expected distribution of genes on the chromosome, average distances were measured between pairs of genes, which were randomly selected. Expected values were predicted based on a hypothesis of random distribution of genes on the chromosome. Observed values were calculated by computing distances between all functionally related genes in a genome in a pair wise manner and then allocating them to their respective distance categories. These were enzymes which acted on the same metabolites in the same metabolic pathways as predicted by the Pathway Tools software . Co-localization of functionally related genes was estimated as a logarithm of the ratio of observed over expected frequencies of gene pairs calculated for each distance category normalised by genome length to eliminate bias.
Genome Rearrangements and Phylogenetic analysis
Genome rearrangement events (relocations) were detected by finding discontinuities in gene syntenies in bacterial chromosomes aligned by Mauve 2.3.1 . Gene orthology was determined as previously discussed. For ortholog sequence alignment and phylogenetic inference, programs Muscle , Gblocks , neighbor.exe , Maximum Likelihood algorithms implemented in PHYLIP  and Mega5  and SplitsTree for phylogenetic network analysis  were used.
Analysis of metabolic networks and metabolic clustering
The Pathways Tools software  was used to reconstruct metabolic pathways and operons based on genome annotations. The cross-clustering coefficients were calculated based on the method described by Spirin et al. . Two genes encoding enzymes that use the same chemical compound either as a substrate or product were considered as ‘functional neighbors’, or in other words, having a metabolic edge. To simplify the network and avoid creation of unimportant or redundant links, abundant chemicals (such as water, ATP, enzyme co-factors, etc.) with more than 10 links between genes were discarded from consideration. Given that there are metabolic edges from gene i to genes j and k, the cross-clustering coefficient of the node i is the probability of having a genomic edge between its neighbors j and k. Nodes j and k have a genomic edge between them if they are co-localized in the same operon of the chromosomal DNA or the distance between them is not greater than an average length of operons. In this study, the average length of operons was estimated at 10,000 bases. The genome-wide cross-clustering coefficient is calculated as an average for all nodes i for the entire metabolic network. To avoid miss-associations or over-associations the analysis was limited to well annotated genes which participate in 38 common pathways predicted in Thermus scotoductus SA-01, Thermus thermophilus strains HB8 and HB27, E. coli and Bacillus subtilis strain 168.
Bacterial evolution at genomic level involves accumulation of mutations, genome rearrangements and horizontal gene transfer. The contribution of all these different and independent evolutionary events towards speciation and adaptation of thermophilic bacteria of genus Thermus were analysed. Thermus bacteria is of industrial interest due to their ability to withstand extreme abiotic stresses including the high temperature and high-energy irradiation ; and also because of their role in decontamination of the environmental pollutions [32, 33] and ability to synthesize thermostable enzymes for industrial application .
Identification of orthologous genes
Frequencies of the oligomer GCGCGC in coding and non-coding sequences of three Thermus genomes
Thermus/Meiothermus genomes comprise chromosomes, megaplasmids and small plasmids. The number of plasmids per genome differs between strains. For example, T. scotoductus SA-01 comprises one chromosome and one small plasmid TSCp8 (CP001963). Two relative organisms T. thermophilus HB8 and HB27 possess additional large plasmids, but the chromosomes of these organisms are shorter than that in T. scotoductus SA-01. There is an additional small plasmid in T. thermophilus HB8 that resembles TSCp8, but share no homology . For the majority of genes present in the T. scotoductus SA-01 chromosome their orthologous counterparts are found in T. thermophilus chromosomes and plasmids . Functional analysis of genes located on the megaplasmids of T. thermophilus showed that they encoded several metabolic pathways, namely: coenzyme B12 synthesis and metabolism; adenosylcobalamin biosynthesis and adenosylcobalamin salvage pathways; dATP, dGTP and dUTP biosynthetic pathways; neurosporene and siroheme biosynthesis. Other genes encoded different metabolic enzymes: acyl-CoA dehydrogenases, isomerases, oxido-reductase, glucosidases, galactosidases and some others. All these genes are spread on the chromosome of T. scotoductus SA-01 that probably was the case with the common ancestor of Thermus species. Considering evolutionary benefits that lay behind the movement of genes from the chromosome to the plasmid, one obvious benefit may be that two or several smaller replicons are faster replicating and may promote the organism propagation. Another explanation may be that the rate of mutations is higher on plasmids than on the chromosome and the population gets enriched in more variants of genes located on the mega-plasmids than those genes located on the chromosome .
Horizontal gene exchange
The stratigraphic analysis demonstrated that prophages of mycobacteria were the most ancient genomic inserts. Later these genomic islands were acquired by Deinococcus (most likely D. geothermalis lineage) from where they were transmitted to Thermus, Meiothermus and Deinococcus species. But even in Thermus species, these genomic islands were relatively old as to compare to similar inserts in β- and γ-Proteobacteria (Figure 7B, see also Bezuidt et al. ). The majority of genes in genomic islands were annotated as conserved hypotheticals. It impeded inferring of the possible role that these genomic islands might play in the evolution of Thermus. Predicted functional genes in their majority are involved in cell wall polysaccharide biosynthesis that is in consistence with the previously reported observation that this category of enzymes was abundant in mobile genetic elements .
To identify possible horizontal gene transferring events through the sophisticated Thermus DNA uptake system [8, 14], phylogenetic trees were inferred by Neighbour-Joining for all 1,526 recognized groups of orthologous protein shared by 5 sampled Thermus and Meiothermus organisms. In every group, the gene tree was rooted to the sequence from Meiothermus ruber DSM 1279. Topology disagreements between gene trees were inspected by the Treedist program . Mismatches between trees may be explained either by different rates of mutations in distant taxa, or by horizontal gene exchange. It was hypothesized that the differences in the rates of mutations most likely would affect lengths of branches in phylogenetic trees, while the horizontal gene transfer would cause predominantly topological changes. An exception may be if a gene in one of the organisms lost its functionality due to nonsense mutations or gene truncation that also may result in a tree topology alteration. To exclude these situations, only alignments with unambiguously aligned blocks selected by Gblocks comprising 75% or more of the initial alignment were studied.
where S ij is the characteristic parameter calculated for the species j in the tree i; dist i jk is the normalized distance between species j and k in the given tree i; and dist jk cons is the distance between the same species in the consensus tree.
Bacteria of the Thermus genus, among which there are many industrially valuable strains, are known by their ability to acquire naked DNA fragments from the environment by using a specialized uptake system [8, 14]. Comparison of recently sequenced genomes showed a huge number of rearrangements of chromosomal loci even between closely related organisms  (Figure 3). Several alternative hypotheses were formulated at the beginning of this work such as that the frequent rearrangement may be caused by acquired pro-phages and other genomic islands, or they are controlled by some yet unknown internal mechanisms. It was unclear whether these rearrangements occur in a random manner, or they are controlled by some positive selection mechanisms, and if so, whether these forces related to the adaptive evolution of these bacteria towards survival in harsher environments?
It was found that the global genome rearrangements appeared to play an important role in this process. Whole operons and metabolic pathways were relocated in T. thermophilus onto the mega-plasmids. Probably, mega-plasmids are the places where the evolutionary processes are speeded up. This observation is in line with the definition of chromids – bacterial megaplasmids distinguishable from both bacterial chromosomes and plasmids . In contrast to plasmids, the chromids carry core metabolic genes but they have plasmid replication system that usually is less reliable than the chromosomal one. According to Harrison et al.  the chromids are particularly rich in genus specific genes and appear at the origin of new genus evolution. This hypothesis is supported by the current observation that the T. thermophilus strains may benefit from the transfer of the evolutionary modifying genes onto the plasmid to achieve a higher level of genetic plasticity.
Even on the chromosomes of different Thermus and Meiothermus organisms their genes were significantly re-shuffled. By confronting evolutionary distances between the strains with the amounts of relocations of genomic fragments it was found that the rate of rearrangements is a bit higher in Thermus extreme thermophiles. The increased rate of genomic rearrangements led to some level of disintegration of functional operons in Thermus/Meiothermus that may be considered either as an effect of persistent environmental temperature stresses or as an adaptation process to fit better to extreme environmental conditions by splitting operons to smaller independent regulons. The observed marginal disintegration of operons may be a price that bacteria paid for the development of new more effective metabolic and regulatory pathways. In spite of a huge number of relocations, the functional disintegration of the metabolic network remained marginal as whole operons were more likely to be relocated than single genes or their parts  either because the latter events would be eliminated from the population by the natural selection, or because of a higher occurrence of rearrangement recognition sites between genes and operons. We observed that the genomic DNA composition might influence the rate of rearrangements. Rearrangement breakpoints were more frequent in GC-rich regions enriched with oligomers of specific types, which were rare in coding sequences. Interestingly, the frequency of these oligomers in non-coding genomic regions of T. thermophilus doubled in comparison to T. scotoductus that may explain the observed increase in rates of rearrangements in these organisms and indirectly it contributes to the hypothesis that the rate of genomic rearrangements is guided by the DNA composition and is an adaptive evolutionary process.
Another important factor of genome evolution is horizontal gene transfer that occurs through three different mechanisms: transformation, conjugation and transduction . Large genomic islands found in Thermus organisms were predominantly old prophages similar to those in Deinococcus genomes. As they comprised mostly hypothetical genes, it was difficult to judge the role they possibly played in Thermus evolution. More intensive gene exchange between these micro-organisms occurred through transformation: a mechanism that is mediated by the uptake of DNA fragments from the environment. In Thermus, the DNA transformation is facilitated by availability of the unique DNA uptake system . Genes, which were likely to be acquired horizontally, have been identified in this study by topological incongruence of gene trees compared to the consensus species tree. It was found that the gene acquisition by transformation is more frequent in Thermus rather than Meiothermus organisms but latter ones frequently are donors of genes for T. scotoductus. T. thermophilus strains also acquired DNA from the environment, but mostly from unknown donor organisms. This difference in horizontal gene acquisition between T. scotoductus and T. thermophilus may reflect either the specificity of their DNA uptake systems, or habitat specificity.
It was found that the extremely frequent genomic rearrangements between chromosomal and plasmid loci in Thermus genomes are moderated by internal mechanisms, which very likely contribute to the adaptive evolution of these bacteria. Whole operons more often are transferred as entities, thus the rearrangements usually do not disrupt syntenies of functionally related genes. We did not find any correlation between the rate of rearrangements and acquisitions of horizontally transferred genomic islands, but an increasing trend was observed in rearrangement frequencies in extreme thermophiles. Gene exchange by transformation were found to occur more frequently between thermophilic T. scotoductus and Meiothermus rather than between the extreme thermophiles. It may be explained either by the sharing of common habitats with moderate thermophiles, or by the fact that naked DNA fragments degrade much faster at extremely high temperature environments.
This work was funded SABINA Fellowship Grant and partly by South African National Research Foundation Grant 71261.
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