Pan-genomic analysis to redefine species and subspecies based on quantum discontinuous variation: the Klebsiella paradigm.

BACKGROUND: Various methods are currently used to define species and are based on the phylogenetic marker 16S ribosomal RNA gene sequence, DNA-DNA hybridization and DNA GC content. However, these are restricted genetic tools and showed significant limitations. RESULTS: In this work, we describe an alternative method to build taxonomy by analyzing the pan-genome composition of different species of the Klebsiella genus. Klebsiella species are Gram-negative bacilli belonging to the large Enterobacteriaceae family. Interestingly, when comparing the core/pan-genome ratio; we found a clear discontinuous variation that can define a new species. CONCLUSIONS: Using this pan-genomic approach, we showed that Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis are species of the Klebsiella genus, rather than subspecies of Klebsiella pneumoniae. This pan-genomic analysis, helped to develop a new tool for defining species introducing a quantic perspective for taxonomy.

Loss of TSS1 in hypervirulent Coxiella burnetii 175, the causative agent of Q fever in French Guiana.

In French Guiana, the unique Coxiella burnetii circulating genotype 17 causes 24% of community-acquired pneumonia, the highest prevalence ever described. To explain this unusual virulence, we performed a comparative genomic analysis of strain Cb175, which was isolated from a patient from French Guiana. Cb175 has a greater number of mutations in genes involved in metabolism compared with the Nine Mile I strain. We found a 6105bp fragment missing in Cb175, which corresponds to the Type 1 secretion systems (T1SS) hlyCABD operon region. This deletion was detected by a specific qPCR in the 8 other strains available from this territory an in none of 298C.burnetii strains from other areas and other genotypes (8/8 vs 0/298, Fisher's exact test, p<0.0000001). Loss of genes implicated in secretion systems has been observed in other epidemic bacterial strains. Thus, the virulence of Cb175 may be linked to this genome reduction.

Postgenomic analysis of bacterial pathogens repertoire reveals genome reduction rather than virulence factors.

In the pregenomic era, the acquisition of pathogenicity islands via horizontal transfer was proposed as a major mechanism in pathogen evolution. Much effort has been expended to look for the contiguous blocks of virulence genes that are present in pathogenic bacteria, but absent in closely related species that are nonpathogenic. However, some of these virulence factors were found in nonpathogenic bacteria. Moreover, and contrary to expectation, pathogenic bacteria were found to lack genes (antivirulence genes) that are characteristic of nonpathogenic bacteria. The availability of complete genome sequences has led to a new era of pathogen research. Comparisons of genomes have shown that the most pathogenic bacteria have reduced genomes, with less ribosomal RNA and unorganized operons; they lack transcriptional regulators but have more genes that encode protein toxins, toxin-antitoxin (TA) modules, and proteins for DNA replication and repair, when compared with less pathogenic close relatives. These findings questioned the paradigm of virulence by gene acquisition and put forward the notion of genomic repertoire of virulence.

How microbiology helps define the rhizome of life.

In contrast to the tree of life (TOF) theory, species are mosaics of gene sequences with different origins. Observations of the extensive lateral sequence transfers in all organisms have demonstrated that the genomes of all life forms are collections of genes with different evolutionary histories that cannot be represented by a single TOF. Moreover, genes themselves commonly have several origins due to recombination. The human genome is not free from recombination events, so it is a mosaic like other organisms' genomes. Recent studies have demonstrated evidence for the integration of parasitic DNA into the human genome. Lateral transfer events have been accepted as major contributors of genome evolution in free-living bacteria. Furthermore, the accumulation of genomic sequence data provides evidence for extended genetic exchanges in intracellular bacteria and suggests that such events constitute an agent that promotes and maintains all bacterial species. Archaea and viruses also form chimeras containing primarily bacterial but also eukaryotic sequences. In addition to lateral transfers, orphan genes are indicative of the fact that gene creation is a permanent and unsettled phenomenon. Currently, a rhizome may more adequately represent the multiplicity and de novo creation of a genome. We wanted to confirm that the term "rhizome" in evolutionary biology applies to the entire cellular life history. This view of evolution should resemble a clump of roots representing the multiple origins of the repertoires of the genes of each species.

Effect of rickettsial toxin VapC on its eukaryotic host.

Rickettsia are intracellular bacteria typically associated with arthropods that can be transmitted to humans by infected vectors. Rickettsia spp. can cause mild to severe human disease with a possible protection effect of corticosteroids when antibiotic treatments are initiated. We identified laterally transferred toxin-antitoxin (TA) genetic elements, including vapB/C, in several Rickettsia genomes and showed that they are functional in bacteria and eukaryotic cells. We also generated a plaque assay to monitor the formation of lytic plaques over time and demonstrated that chloramphenicol accelerates host cell lysis of vapB/C-containing Rickettsia. Whole-genome expression, TUNEL and FISH assays on the infected cells following exposure to the antibiotic revealed early apoptosis of host cells, which was linked to over-transcription of bacterial vapB/C operons and subsequent cytoplasmic VapC toxin release. VapC that is expressed in Escherichia coli and Saccharomyces cerevisiae or microinjected into mammalian cells is toxic through RNase activity and is prevented by dexamethasone. This study provides the first biological evidence that toxin-antitoxin elements act as pathogenic factors in bacterial host cells, confirming comparative genomic evidence of their role in bacterial pathogenicity. Our results suggest that early mortality following antibiotic treatment of some bacterial infections can be prevented by administration of dexamethasone.

The rhizome of Reclinomonas americana, Homo sapiens, Pediculus humanus and Saccharomyces cerevisiae mitochondria.

BACKGROUND: Mitochondria are thought to have evolved from eubacteria-like endosymbionts; however, the origin of the mitochondrion remains a subject of debate. In this study, we investigated the phenomenon of chimerism in mitochondria to shed light on the origin of these organelles by determining which species played a role in their formation. We used the mitochondria of four distinct organisms, Reclinomonas americana, Homo sapiens, Saccharomyces cerevisiae and multichromosome Pediculus humanus, and attempted to identify the origin of each mitochondrial gene. RESULTS: Our results suggest that the origin of mitochondrial genes is not limited to the Rickettsiales and that the creation of these genes did not occur in a single event, but through multiple successive events. Some of these events are very old and were followed by events that are more recent and occurred through the addition of elements originating from current species. The points in time that the elements were added and the parental species of each gene in the mitochondrial genome are different to the individual species. These data constitute strong evidence that mitochondria do not have a single common ancestor but likely have numerous ancestors, including proto-Rickettsiales, proto-Rhizobiales and proto-Alphaproteobacteria, as well as current alphaproteobacterial species. The analysis of the multichromosome P. humanus mitochondrion supports this mechanism. CONCLUSIONS: The most plausible scenario of the origin of the mitochondrion is that ancestors of Rickettsiales and Rhizobiales merged in a proto-eukaryotic cell approximately one billion years ago. The fusion of the Rickettsiales and Rhizobiales cells was followed by gene loss, genomic rearrangements and the addition of alphaproteobacterial elements through ancient and more recent recombination events. Each gene of each of the four studied mitochondria has a different origin, while in some cases, multichromosomes may allow for enhanced gene exchange. Therefore, the tree of life is not sufficient to explain the chimeric structure of current genomes, and the theory of a single common ancestor and a top-down tree does not reflect our current state of knowledge. Mitochondrial evolution constitutes a rhizome, and it should be represented as such.

Phylogenomic analysis of Odyssella thessalonicensis fortifies the common origin of Rickettsiales, Pelagibacter ubique and Reclimonas americana mitochondrion.

BACKGROUND: The evolution of the Alphaproteobacteria and origin of the mitochondria are topics of considerable debate. Most studies have placed the mitochondria ancestor within the Rickettsiales order. Ten years ago, the bacterium Odyssella thessalonicensis was isolated from Acanthamoeba spp., and the 16S rDNA phylogeny placed it within the Rickettsiales. Recently, the whole genome of O. thessalonicensis has been sequenced, and 16S rDNA phylogeny and more robust and accurate phylogenomic analyses have been performed with 65 highly conserved proteins. METHODOLOGY/PRINCIPAL FINDINGS: The results suggested that the O. thessalonicensis emerged between the Rickettsiales and other Alphaproteobacteria. The mitochondrial proteins of the Reclinomonas americana have been used to locate the phylogenetic position of the mitochondrion ancestor within the Alphaproteobacteria tree. Using the K tree score method, nine mitochondrion-encoded proteins, whose phylogenies were congruent with the Alphaproteobacteria phylogenomic tree, have been selected and concatenated for Bayesian and Maximum Likelihood phylogenies. The Reclinomonas americana mitochondrion is a sister taxon to the free-living bacteria Candidatus Pelagibacter ubique, and together, they form a clade that is deeply rooted in the Rickettsiales clade. CONCLUSIONS/SIGNIFICANCE: The Reclinomonas americana mitochondrion phylogenomic study confirmed that mitochondria emerged deeply in the Rickettsiales clade and that they are closely related to Candidatus Pelagibacter ubique.

Intracellular Rickettsiales: Insights into manipulators of eukaryotic cells.

The order Rickettsiales comprises obligate intracellular bacteria that are the ancestors of modern eukaryotes. These bacteria infect various vectors and hosts, with some species being pathogenic to man. Rickettsiales have small, degraded genomes and provide a paradigm for increased pathogenicity despite gene loss; significant levels of genetic exchange occur between bacteria that infect the same host and with the eukaryotic hosts themselves. Crosstalk between host and bacteria appears to be mediated by a Type IV secretion system and proteins containing eukaryotic-like repeat motifs. Rickettsiales also manipulate host reproduction and induce host resistance to viruses. Manipulation of its host by Rickettsiales has long been misunderstood because of technical difficulties, but recent advances in understanding bacterial-eukaryotes interactions have been made and are reviewed here.

Genomes of the most dangerous epidemic bacteria have a virulence repertoire characterized by fewer genes but more toxin-antitoxin modules.

BACKGROUND: We conducted a comparative genomic study based on a neutral approach to identify genome specificities associated with the virulence capacity of pathogenic bacteria. We also determined whether virulence is dictated by rules, or if it is the result of individual evolutionary histories. We systematically compared the genomes of the 12 most dangerous pandemic bacteria for humans ("bad bugs") to their closest non-epidemic related species ("controls"). METHODOLOGY/PRINCIPAL FINDINGS: We found several significantly different features in the "bad bugs", one of which was a smaller genome that likely resulted from a degraded recombination and repair system. The 10 Cluster of Orthologous Group (COG) functional categories revealed a significantly smaller number of genes in the "bad bugs", which lacked mostly transcription, signal transduction mechanisms, cell motility, energy production and conversion, and metabolic and regulatory functions. A few genes were identified as virulence factors, including secretion system proteins. Five "bad bugs" showed a greater number of poly (A) tails compared to the controls, whereas an elevated number of poly (A) tails was found to be strongly correlated to a low GC% content. The "bad bugs" had fewer tandem repeat sequences compared to controls. Moreover, the results obtained from a principal component analysis (PCA) showed that the "bad bugs" had surprisingly more toxin-antitoxin modules than did the controls. CONCLUSIONS/SIGNIFICANCE: We conclude that pathogenic capacity is not the result of "virulence factors" but is the outcome of a virulent gene repertoire resulting from reduced genome repertoires. Toxin-antitoxin systems could participate in the virulence repertoire, but they may have developed independently of selfish evolution.

Gene gain and loss events in Rickettsia and Orientia species.

BACKGROUND: Genome degradation is an ongoing process in all members of the Rickettsiales order, which makes these bacterial species an excellent model for studying reductive evolution through interspecies variation in genome size and gene content. In this study, we evaluated the degree to which gene loss shaped the content of some Rickettsiales genomes. We shed light on the role played by horizontal gene transfers in the genome evolution of Rickettsiales. RESULTS: Our phylogenomic tree, based on whole-genome content, presented a topology distinct from that of the whole core gene concatenated phylogenetic tree, suggesting that the gene repertoires involved have different evolutionary histories. Indeed, we present evidence for 3 possible horizontal gene transfer events from various organisms to Orientia and 6 to Rickettsia spp., while we also identified 3 possible horizontal gene transfer events from Rickettsia and Orientia to other bacteria. We found 17 putative genes in Rickettsia spp. that are probably the result of de novo gene creation; 2 of these genes appear to be functional. On the basis of these results, we were able to reconstruct the gene repertoires of "proto-Rickettsiales" and "proto-Rickettsiaceae", which correspond to the ancestors of Rickettsiales and Rickettsiaceae, respectively. Finally, we found that 2,135 genes were lost during the evolution of the Rickettsiaceae to an intracellular lifestyle. CONCLUSIONS: Our phylogenetic analysis allowed us to track the gene gain and loss events occurring in bacterial genomes during their evolution from a free-living to an intracellular lifestyle. We have shown that the primary mechanism of evolution and specialization in strictly intracellular bacteria is gene loss. Despite the intracellular habitat, we found several horizontal gene transfers between Rickettsiales species and various prokaryotic, viral and eukaryotic species. OPEN PEER REVIEW: Reviewed by Arcady Mushegian, Eugene V. Koonin and Patrick Forterre. For the full reviews please go to the Reviewers' comments section.

Comparative genomics evidence that only protein toxins are tagging bad bugs.

The term toxin was introduced by Roux and Yersin and describes macromolecular substances that, when produced during infection or when introduced parenterally or orally, cause an impairment of physiological functions that lead to disease or to the death of the infected organism. Long after the discovery of toxins, early genetic studies on bacterial virulence demonstrated that removing a certain number of genes from pathogenic bacteria decreases their capacity to infect hosts. Each of the removed factors was therefore referred to as a "virulence factor," and it was speculated that non-pathogenic bacteria lack such supplementary factors. However, many recent comparative studies demonstrate that the specialization of bacteria to eukaryotic hosts is associated with massive gene loss. We recently demonstrated that the only features that seem to characterize 12 epidemic bacteria are toxin-antitoxin (TA) modules, which are addiction molecules in host bacteria. In this study, we investigated if protein toxins are indeed the only molecules specific to pathogenic bacteria by comparing 14 epidemic bacterial killers ("bad bugs") with their 14 closest non-epidemic relatives ("controls"). We found protein toxins in significantly more elevated numbers in all of the "bad bugs." For the first time, statistical principal components analysis, including genome size, GC%, TA modules, restriction enzymes, and toxins, revealed that toxins are the only proteins other than TA modules that are correlated with the pathogenic character of bacteria. Moreover, intracellular toxins appear to be more correlated with the pathogenic character of bacteria than secreted toxins. In conclusion, we hypothesize that the only truly identifiable phenomena, witnessing the convergent evolution of the most pathogenic bacteria for humans are the loss of metabolic activities, i.e., the outcome of the loss of regulatory and transcription factors and the presence of protein toxins, alone, or coupled as TA modules.

The influence of rickettsiologists on post-modern microbiology.

Many of the definitions in microbiology are currently false. We have reviewed the great denominations of microbiology and attempted to free microorganisms from the theories of the twentieth century. The presence of compartmentation and a nucleoid in Planctomycetes clearly calls into question the accuracy of the definitions of eukaryotes and prokaryotes. Archaea are viewed as prokaryotes resembling bacteria. However, the name archaea, suggesting an archaic origin of lifestyle, is inconsistent with the lifestyle of this family. Viruses are defined as small, filterable infectious agents, but giant viruses challenge the size criteria used for the definition of a virus. Pathogenicity does not require the acquisition of virulence factors (except for toxins), and in many cases, gene loss is significantly inked to the emergence of virulence. Species classification based on 16S rRNA is useless for taxonomic purposes of human pathogens, as a 2% divergence would classify all Rickettsiae within the same species and would not identify bacteria specialized for mammal infection. The use of metagenomics helps us to understand evolution and physiology by elucidating the structure, function, and interactions of the major microbial communities, but it neglects the minority populations. Finally, Darwin's descent with modification theory, as represented by the tree of life, no longer matches our current genomic knowledge because genomics has revealed the occurrence of de novo-created genes and the mosaic structure of genomes, the Rhizome of life is therefore more appropriate.

Defining pathogenic bacterial species in the genomic era.

Actual definitions of bacterial species are limited due to the current criteria of definition and the use of restrictive genetic tools. The 16S ribosomal RNA sequence, for example, has been widely used as a marker for phylogenetic analyses; however, its use often leads to misleading species definitions. According to the first genetic studies, removing a certain number of genes from pathogenic bacteria removes their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, sympatric free-living bacteria often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, such as pathogenic bacteria, escape these bacterial complexes and colonize a niche, thereby gaining a species name. Their specialization allows them to become allopatric, and their gene losses eventually favor reductive genome evolution. A pathogenic species is characterized by a gene repertoire that is defined not only by genes that are present but also by those that are lacking. It is likely that current bacterial pathogens will disappear soon and be replaced by new ones that will emerge from bacterial complexes that are already in contact with humans.