Analysis of Co-evolving Gene Families Using Mutually Exclusive Orthologous Modules

Coevolutionary networks can encapsulate information about the dynamics of presence and absence of gene families in organisms. Analysis of such networks should reveal fundamental principles underlying the evolution of cellular systems and the functionality of sets of genes. In this study, we describe a new approach for analyzing coevolutionary networks. Our method detects Mutually Exclusive Orthologous Modules (MEOMs). A MEOM is composed of two sets of gene families, each including gene families that tend to appear in the same organisms, such that the two sets tend to mutually exclude each other (if one set appears in a certain organism the second set does not). Thus, a MEOM reflects the evolutionary replacement of one set of genes by another due to reasons such as lineage/environmental specificity, incompatibility, or functional redundancy. We use our method to analyze a coevolutionary network that is based on 383 microorganisms from the three domains of life. As we demonstrate, our method is useful for detecting meaningful evolutionary clades of organisms as well as sets of proteins that interact with each other. Among our results, we report that: 1) MEOMs tend to include gene families whose cellular functions involve transport, energy production, metabolism, and translation, suggesting that changes in the metabolic environments that require adaptation to new sources of energy are central triggers of complex/pathway replacement in evolution. 2) Many MEOMs are related to outer membrane proteins, such proteins are involved in interaction with the environment and could thus be replaced as a result of adaptation. 3) MEOMs tend to separate organisms with large phylogenetic distance but they also separate organisms that live in different ecological niches. 4) Strikingly, although many MEOMs can be identified, there are much fewer cases where the two cliques in the MEOM completely mutually exclude each other, demonstrating the flexibility of protein evolution. 5) CO dehydrogenase and thymidylate synthase and the glycine cleavage genes mutually exclude each other in archaea; this may represent an alternative route for generation of methyl donors for thymidine synthesis.

Identification and characterization of proteins supporting dehalorespiration in Desulfitobacterium hafniense strain TCE1

Laure Prat

Respiration is a fundamental catalytic process in the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. The major difference between these organisms is that various organic and inorganic substrates can be used to donate or accept electrons in the bacterial and archaeal kingdoms, in various ranges of redox potentials in order to drive aerobic (with oxygen as electron acceptor) and anaerobic respiratory pathways. During the last few decades, several bacterial strains have been reported to use chlorinated compounds as terminal electron acceptor under anaerobic conditions, in a process called dehalorespiration. Among more than thirty dehalorespiring bacterial strains isolated to date, the Dehalococcoides group and the Gram-positive genus Desulfitobacterium comprise the largest number of reductively dehalogenating pure cultures. While most Dehalococcoides isolates appeared as highly specialized bacteria that depend strictly on dehalorespiration for growth, members of the genus Desulfitobacterium are very versatile microorganisms with regards to the electron donors and acceptors utilized. Globally, this remarkable prokaryotic respiratory flexibility is guaranteed by an elaborate reservoir of complex redox enzymes. Regarding the specific electron transport chain involved in dehalorespiration, rather little knowledge has been obtained so far with the exception of the key enzyme, the reductive dehalogenase, well described in several dehalorespiring bacteria, and of little information on b-type cytochromes and menaquinones. The overall objective of this thesis was to identify new possible components of the electron transport chain by comparative proteomic analysis, to investigate the presence/absence of a regulatory pathway leading to the biosynthesis of the reductive dehalogenase, and finally to characterize newly identified proteins that might support the dehalorespiration process. A comparative 2D proteomic analysis was performed on Desulfitobacterium hafniense strain TCE1, a bacterium capable of using tetrachloroethene (PCE) as electron acceptor. Soluble and membrane-associated proteins were analyzed from cells cultivated on different couples of electron donors and acceptors, lactate – fumarate (LF), lactate – PCE (LP), hydrogen – fumarate (HF), and hydrogen – PCE (HP). Cells growing on LP or HP revealed 72 and 93 protein spots in membrane fraction, and 49 and 12 spots in soluble fraction that correspond to increasingly expressed proteins compared to their fumarate-grown counterparts. More than 80 proteins were identified by mass spectrometry analysis, among which the key enzyme in the dehalorespiration process, the PCE reductive dehalogenase (PceA), as well as interesting candidates which could support dehalorespiration. The possible role of these proteins was analyzed in further details. Involved in energy and carbon metabolisms, electron transfer flavoproteins (ETF) and proteins related to the Wood-Ljungdahl pathway of CO2 fixation were identified in HP growth conditions. Proteins associated to stress response such as the phage shock protein PspA, and to regulatory pathways, the transcriptional repressor CodY, were also observed and discussed. Although only slightly induced by PCE, an additional protein displaying homology to sulfur-transferases was found in a large abundance in the fraction of membrane-associated proteins. This protein was named PhsE, as the corresponding gene belongs to a gene operon with homology to the molybdoenzyme thiosulfate/polysulfide reductase operons (phs/psr) found in Salmonella typhimurium or Wolinella succinogenes. PhsE protein architecture resembles to the class of tandem-domain rhodaneses, with the particularity to contain two active-site cysteines. It is also characterized by the presence of a typical lipoprotein signal peptide (LSP), which anchors PhsE on the outside of the cytoplasmic membrane. In the genome sequence of D. hafniense strain Y51, phsE (DSY3892) is part of an apparent operon encoding one out of about sixty different members of the complex iron-sulfur molybdoenzyme (CISM) family. PhsA, the catalytic molybdoenzyme, displays significant sequence homology with the thiosulfate/polysulfide reductase (PhsA/PsrA) subfamily. So far no other CISM operon has been described with a sulfur-transferase encoding gene. Despite the numerous functions proposed for rhodanese in cyanide detoxification, Fe/S cluster formation, oxidative stress protection, sulfur mobilization and involvement in general sulfur metabolism, the question of the physiological role of PhsE remains open. The expression of the Phs complex appeared to be influenced by the addition of sulfide in the culture medium, generally used as reducing agent in anaerobic cultures. Sulfide is known to affect the intracellular redox status. Therefore, it is hypothesized that PhsE might be involved in the control of the redox balance of the cell, notably by scavenging potentially toxic reduced sulfur species. To support data obtained at the protein level during proteomic analysis, genes and the corresponding messenger RNA from D. hafniense strain TCE1 were studied. While in silico DNA sequence analysis did not reveal any obvious features regulating the transcription of pceA, puzzling results were obtained in proteomic analysis showing an almost complete absence of PceA in cells grown on fumarate. To characterize this phenomenon, starting from a routinely PCE-grown inoculum, strain TCE1 was repeatedly transferred on medium containing fumarate as electron acceptor. The pool of pceA gene in the total culture DNA decreased significantly already after fifteen successive sub-cultures on fumarate, suggesting a shift in strain TCE1 population upon relief of the PCE selection pressure. Consequently the level of pceA mRNA and PceA protein also followed the same trend. Secondly, a PCE pulse experiment was performed in an anaerobic continuous culture of strain TCE1 on fumarate. It revealed that PCE itself has no power of induction on genetic level, except for the positive effect observed on pspA transcripts which encodes a protein involved in cell membrane integrity. Finally the expression study of the phsFABCDE gene cluster revealed that all phs genes are co-transcribed, and most likely build an operon. Thus phsE seemed to be strictly coregulated with the phs operon, and not under the influence of another promoter. In conclusion, proteomic analysis enabled to investigate the general physiological status of Desulfitobacterium hafniense strain TCE1 during dehalorespiration. A tentative model of the dehalorespiration pathway is proposed in summarizing recent data obtained on the topology of the PCE reductive dehalogenase and on sequence similarity with proteins involved in other anaerobic respiratory pathways. Regarding the possible support of PhsE towards the dehalorespiration, the complicated chemistry of sulfur does not allow to clearly attribute a function to the Phs complex, but there are indications that it might build a 'sulfur' oxidoreductase which could catalyze the conversion of sulfide to polysulfide with concomitant electron transfer to the quinone pool. The sulfide oxidation activity might be useful to D. hafniense to scavenge toxic sulfur species, avoiding that they disturb the redox balance of the cell, and redox-sensitive enzymatic complexes.

EPFL2009

Unraveling Patterns of Site-to-Site Synonymous Rates Variation and Associated Gene Properties of Protein Domains and Families

Slavica Dimitrieva Janeva

In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes.

Public Library of Science2014

Assembly Mechanisms and Cellular Effects of Bacterial Pore-Forming Toxins

Mirko Bischofberger

The correct assembly of macromolecular protein complexes such as ribosomes or multisubunit membrane channels is essential for their function. Alterations in the process can lead to disease either by loss of function or by acquisition of toxic function. However, the precise mechanisms by which multimeric protein complexes assemble are generally poorly understood owing to the difficulty in reconstituting the process in-vitro. Thus, systems that undergo dynamic assembly and disassembly in solution, such as cytoskeletal proteins, have been particularly attractive. By contrast, the assembly of multi-subunit membrane protein complexes has been notably difficult to study. An interesting, somewhat in between, situation is provided by pore-forming proteins, the best-characterized subclass of which are bacterial pore-forming toxins (PFTs). These proteins can generally be produced recombinantly and in a highly stable soluble monomeric form, but assemble into homo-oligomeric ring-like structures upon addition to cells expressing the appropriate cell surface receptor. These rings concomitantly, or subsequently depending of on the PFT, undergo a conformational change that leads to exposure of hydrophobic surfaces and spontaneous membrane insertion. The built pores then perforate the membrane of the host cell. PFTs do not only occur in bacteria and have a very broad taxonomic distribution, occurring in organisms such as parasites, plants and mammals. However, for pathogenic organisms the PFTs are major virulence factors contributing to the disease caused by the organisms producing them. For this reason bacterial PFTs have been studied extensively during the last century and a lot of insight into their mode of action has been gained. However, despite the increasing knowledge on PFTs, the mechanisms and the kinetics of self-assembly of these complexes remain largely enigmatic. In particular it is unknown whether oligomerization occurs through the sequential addition of monomers or through interaction of multimeric intermediates, whether oligomerization is the rate-limiting step during the pore-formation process and whether the same rules apply to all toxins. This is mostly due to the fact that intermediates have not been visualized by either biochemical or structural methods. Also, with few exceptions, functional assays on the activity of PFTs have always been performed at the level of a population of cells. The aim of this thesis was to measure pore-formation at the single cell level to extract mechanistic information from the analysis of the stochasticity of the pore-formation process. We chose to study three PFTs produced by human pathogens: aerolysin, PFO and Cytolysin A (ClyA). Aerolysin is produced by Aeromonas hydrophila and forms pores of 1-2 nm in diameter, perfringolysin O (PFO) occurs in Clostridium perfringensis and is a cholesterol-dependent cytolysin (CDC) that builds channels of 25-30 nm. Both of these PFTs build structurally similar pores made of transmembrane β-barrels even though the number of strands in the barrel varies by more than 1 order of magnitude. On the contrary, ClyA is a PFT produced by both E. coli and Salmonella enterica strains and forms rings of 12-13 protomers with the membrane spanning domain composed of a-helices. Hence, the pores formed by these three PFTs vary greatly from one another and are representative for the diversity of PFTs. We measured cell permeabilisation at the single cell level using two independent live-cell imaging methods, in erythrocytes and in nucleated cells. Both assays revealed that the assembly mechanism of PFTs is stochastic in its nature and that lowering the initial monomer concentration increases the variability and average time to successfully build first functional pores. The measured stochasticity reflects the chemical kinetics behind the assembly reaction and therefore carries important information about the underlying mechanism. In fact, analysis of the stochasticity of the processes led to the robust determination for each toxin of the minimal number of independent rate-limiting steps required for pore formation. Furthermore, scaling relationships in our data upon changes in initial toxin concentration constrain the possible underlying mechanisms for assembly and reveal that the steps leading to the aerolysin heptamer are composed of at least seven independent reactions whose rates scale with toxin concentration. Taken together, our findings suggest that that what is limiting for aerolysin is the conversion for each monomer to an assembly competent state, while for PFO a single limiting step is observed, likely corresponding to membrane insertion. A second aim of this thesis was to clarify the cellular consequences that occur upon pore formation by different PFTs and to find out how PFT-affected cells sense this event. This was done in collaboration with other members of the lab. Our results suggest that the cells monitor the breaches in the plasma membrane by assessing the correct homeostasis of the potassium ion inside the cell through a yet unknown mechanism. Interestingly, the cells seem to "monitor" the integrity of the membrane through the most upstream effect possible after pore formation, namely the flux of ions. Incidentally, the assays we developed in the first part of this thesis to "monitor" initial pore formation events are based on the same principle, namely the immediate loss of gradients that occur upon pore formation.

EPFL2011