Massive comparative genomic analysis reveals convergent evolution of specialized bacteria

Abstract

Background: Genome size and gene content in bacteria are associated with their lifestyles. Obligate intracellular bacteria (i.e., mutualists and parasites) have small genomes that derived from larger free-living bacterial ancestors; however, the different steps of bacterial specialization from free-living to intracellular lifestyle have not been studied comprehensively. The growing number of available sequenced genomes makes it possible to perform a statistical comparative analysis of 317 genomes from bacteria with different lifestyles.

Results: Compared to free-living bacteria, host-dependent bacteria exhibit fewer rRNA genes, more split rRNA operons and fewer transcriptional regulators, linked to slower growth rates. We found a function-dependent and non-random loss of the same 100 orthologous genes in all obligate intracellular bacteria. Thus, we showed that obligate intracellular bacteria from different phyla are converging according to their lifestyle. Their specialization is an irreversible phenomenon characterized by translation modification and massive gene loss, including the loss of transcriptional regulators. Although both mutualists and parasites converge by genome reduction, these obligate intracellular bacteria have lost distinct sets of genes in the context of their specific host associations: mutualists have significantly more genes that enable nutrient provisioning whereas parasites have genes that encode Types II, IV, and VI secretion pathways.

Conclusion: Our findings suggest that gene loss, rather than acquisition of virulence factors, has been a driving force in the adaptation of parasites to eukaryotic cells. This comparative genomic analysis helps to explore the strategies by which obligate intracellular genomes specialize to particular host-associations and contributes to advance our knowledge about the mechanisms of bacterial evolution.

Figure 1

Phylogenetic relationships and converging evolution. The phylogenetic unrooted tree was constructed on the basis of the 16S rRNA gene sequences from the 317 bacteria using the neighbour-joining method [15] within the Phylip package [14]. The tree was visualized using FigTree software . Organisms are colored according to lifestyle: red for mutualists, purple for parasitic, green for facultative host-dependent, and blue for free-living bacteria. The events of split rRNA operons (yellow triangles) and loss of the 100 genes set (orange circles) are coincident with the location of the host-dependent bacteria in different phyla.

Figure 2

Proposed scenario for genome evolution and specialization. Model of evolution involving 3 steps en route to specialization to an intracellular lifestyle. The different steps correspond to the different levels of dependency to eukaryotic cells, the minimum is for the free-living bacteria and the maximum is for obligate intracellular bacteria. Each Step corresponds to a bacterial community sharing common habitat and relationship with eukaryotic cells. Bacterial specialization to an intracellular lifestyle is characterized by gene loss including transcriptional regulators and rRNA operons. Free-living promiscuous bacteria have large genomes because of a high level of gene importation. They also have a large number of rRNA operons. Obligate intracellular bacteria have reduced genomes with few rRNA operons and transcription regulators. When bacterial lineages make the transition from free-living to permanent associations with hosts, they undergo a major loss of genes. Restriction to an intracellular environment limits the opportunity to acquire foreign genes from other bacteria therefore the balance between acquired and lost genes in specialized bacteria is in favour of genome reduction. Irreversible massive gene decay implies that specialization to an intracellular lifestyle is a one-way road. Differential gene loss is noted in mutualistic and parasitic bacterial groups. Data in circles represent the mean (± s.d.) of genome size in megabases (GS), GC content (GC), rRNA operon (Op), and number of genes assigned to transcriptional regulation (TR) in each bacterial community. Numbers on the arrows represent the average number of lost genes ± the standard error in order to compute confidence intervals for the estimated loss ratio (proportion of genes lost with respect to the whole number of genes present at least in one bacterium).

Figure 3

Plot of the first Principal Coordinate Analysis (PCO) axis of COGs content distances. Multivariate analysis graphics of the COGs content of all studied bacteria using R ade4 package. Each bacterium is represented by a symbol linked by a line to the gravity center of the group it belongs to (M, obligate intracellular mutualists, red triangles; P, obligate intracellular parasites, purple triangles; FHA, facultative host- associated, green asterisks; and FL, free-living, blue squares). An ellipse was also drawn for each class, which size increases with the coordinates' dispersion in the class. It is computed such that it would contain 68% of the individuals in the studied class if the distribution were Gaussian. Otherwise, it is just a way to compare dispersion between classes. 1 represents Treponema pallidum; 2 represents Mycobacterium leprae; 3 represents Candidatus Protochlamydia amoebophila UWE25; 4 represents Coxiella burnetii. These species with larger genome sizes and gene contents than the other obligate intracellular bacteria undergo reductive evolution [28,30]. Some of these bacteria have high number of pseudogenes [27,29,31]. The presence of pseudogenes displays an ongoing process of gene degradation.

Figure 4

Functions lost during specialization. The bars represent the mean number of loci in different functional categories based on functional categorizations established for the clusters of Orthologous Groups (COGs).The proportion of genes lost by obligate intracellular compared to free-living bacteria is indicated next to the bars.

Figure 5

Relationship between growth time, operon number and transcriptional regulators per Mb. Bacteria were classified into 3 categories depending on the experimental growth time: fast growing (24–48 hours), median (3 to 7 days) and slow growing fastidious bacteria (more than 7 days). The 284 genomes for which information about time of growth is available are projected on the first two Principal Component Analysis (PCA) axes, which represent 66.2% and 19.5% of the total inertia. OI, obligate intracellular bacteria, dark red triangles; FHA, facultative host-associated, green asterisks; FL, free-living, blue squares.

Figure 6

Comparison of the genome content from mutualistic and parasitic bacteria. Bars correspond to the mean number of genes in a given COG divided by the total number of genes. The significance of results in the figure is represented by triple asterisks (***) indicating p ≤ 0.001, double asterisks (**) indicating p ≤ 0.01 and a single asterisk (*) indicating p ≤ 0.05 (paired Student's t-test).