Homology (biology) | EPFL Graph Search

In biology, homology is similarity due to shared ancestry between a pair of structures or genes in different taxa. A common example of homologous structures is the forelimbs of vertebrates, where the wings of bats and birds, the arms of primates, the front flippers of whales and the forelegs of four-legged vertebrates like dogs and crocodiles are all derived from the same ancestral tetrapod structure. Evolutionary biology explains homologous structures adapted to different purposes as the result of descent with modification from a common ancestor. The term was first applied to biology in a non-evolutionary context by the anatomist Richard Owen in 1843. Homology was later explained by Charles Darwin's theory of evolution in 1859, but had been observed before this, from Aristotle onwards, and it was explicitly analysed by Pierre Belon in 1555. In developmental biology, organs that developed in the embryo in the same manner and from similar origins, such as from matching primordia in s

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

Integration of microfluidics and multimodal optics for translational bioanalytics

Daniel Migliozzi

Numerous analytical techniques for life sciences have been at the basis of the improvement of medical diagnostics and treatments. Current challenges in bioanalytics are related to the conception of methods enabling large amount of information to be extracted from biological samples, in order to be translated into concrete solutions for the patient. From this point of view, microfluidics constitutes a key element to automate, accelerate and standardize bioanalytical tests. At the same time, obtaining precise information from the analyte of interest is crucial to specifically address diagnoses and therapies. Since the high flexibility of optical readouts can give access to a multitude of diverse aspects of biological matter in multiplexed fashion, in this thesis we have chosen to integrate microfluidics and multimodal optics to address specific problems in translational bioanalytics: drug screening and tumor diagnostics. In the first part of the work, we approached the problem of drug screening on living organisms, and especially the possibility to provide as much information as possible on potential mechanisms of action and clinical targets. For this, we aimed at developing a method to study the model organism Caenorhabditis elegans, i.e. a small soil worm that is widely used by researchers to study physiopathology at whole-organism scale. In fact, while offering the systemic information that is missing with cellular models, this small organism is much easier to handle and grow in large amounts compared to rodents, for which the costs and the ethical concerns are substantial. Moreover, its large genetic homology with humans supports translational analysis and its genetic manipulation is well mastered to model human diseases. Thus, we developed a microfluidic array to select small separated populations of C. elegans specimens, and combined fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to identify the mode-of-action of an antibiotic. Also, we demonstrated real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic. Furthermore, to enable on-site antibiotic testing, we explored a method to empower low-magnification optical systems with dielectric micro- and macro-spheres for the detection of pathogens in liquid media. Such dielectric objects offer potential solutions to increase the resolution and the sensitivity of imaging systems in a cost-effective fashion. We first characterized the parameters that govern the resolution increase in the case of microsphere-assisted incoherent microscopy and demonstrated it by imaging silicon microstructures and fluorescent bacteria. Then, we developed a method to easily fabricate microfluidic chips that integrate custom designs and precise location of highly-diffractive macro-spheres, and used it for on-chip detection of bacteria in a flow. In the second part of the work, we aimed at developing a technique to assess the status of individual tumor and immune cells in the conventional tissue sections used in the clinics. The first step was to combine fluorescence-based image segmentation with automated microfluidics to quantify, at the single-cell level, the overexpression of two proteins used as biomarkers for breast cancer diagnostics. We first assessed our method on biological controls such as breast cancer cell lines, and then combined it with ...

EPFL2019

TsrE: a sulfur-transferase involved in various anaerobic respiration pathways ?

Christof Holliger, Julien Maillard, Laure Prat

TsrE, a 32kDa sulfur-transferase with two rhodanese domains, was identified as a result of its over-expression during anaerobic tetrachloroethene respiration by the obligate gram-positive anaerobe Desulfitobacterium hafniense. The tsrE gene is part of an operon containing the classical ABCD gene arrangement belonging to the DMSO reductase family, and showing homology with the thiosulfate reductase operon (tsr). Detailed analysis of TsrE sequence features revealed the presence of a N-terminal lipo-box suggesting the insertion of the sulfur-transferase in the cytoplasmic membrane as a lipoprotein. Evidence was obtained for the attachment of TsrE to the membrane where it faces the outside of the cell. Each rhodanese domain of TsrE contains an active-site cysteine with motifs CKSGGR and CAVGAR. Heterologous expression and purification allowed the characterization of TsrE and variants, revealing that despite few differences, both rhodanese motifs are active in sulfur transfer. The influence on TsrE level of various sulfur compounds present in the medium was investigated, indicating a positive effect of thiosulfate, sulfide, sulfite and cysteine, but not sulfate. TsrE was expressed at the highest level, when D. hafniense was growing on thiosulfate as electron acceptor, suggesting that all tsr genes are co-expressed. A speculative function for TsrE is the mobilization of sulfur, although a clear role for TsrE in anaerobic tetrachloroethene respiration remains to be elucidated.

2009