Biology subjects

Chabriere, E.

Publications and source records attributed to Chabriere, E..

8 recordsLinked to original sources

bioRxiv2023

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, [~]1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile.

biochemistry↗

bioRxiv2020

Tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: Is it still a virus?

Since the discovery of Acanthamoeba polyphaga Mimivirus, the first giant virus of amoeba, the historical hallmarks defining a virus have been challenged. Giant virion sizes can reach up to 2.3 {micro}m, making them visible by optical microscopy. They have large genomes of up to 2.5 Mb that encode proteins involved in the translation apparatus. Herein, we investigated possible energy production in Pandoravirus massiliensis, the largest of our giant virus collection. MitoTracker and TMRM mitochondrial membrane markers allowed for the detection of a membrane potential in virions that could be abolished by the use of the depolarizing agent CCCP. An attempt to identify enzymes involved in energy metabolism revealed that 8 predicted proteins of P. massiliensis exhibited low sequence identities with defined proteins involved in the universal tricarboxylic acid cycle (acetyl Co-A synthase; citrate synthase; aconitase; isocitrate dehydrogenase; -ketoglutarate decarboxylase; succinate dehydrogenase; fumarase). All 8 viral predicted ORFs were transcribed together during viral replication, mainly at the end of the replication cycle. Two of these proteins were detected in mature viral particles by proteomics. The product of the ORF132, a predicted protein of P. massiliensis, cloned and expressed in Escherichia coli, provided a functional isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle, which converts isocitrate to -ketoglutarate. We observed that membrane potential was enhanced by low concentrations of Acetyl-CoA, a regulator of the tricarboxylic acid cycle. Our findings show for the first time that energy production can occur in viruses, namely, pandoraviruses, and the involved enzymes are related to tricarboxylic acid cycle enzymes. The presence of a proton gradient in P. massiliensis coupled with the observation of genes of the tricarboxylic acid cycle make this virus a form a life for which it is legitimate to question what is a virus?.

microbiology↗

bioRxiv2020

A mysterious 80 nm amoeba virus with a near-complete "ORFan genome" challenges the classification of DNA viruses

Here we report the discovery of Yaravirus, a new lineage of amoebal virus with a puzzling origin and phylogeny. Yaravirus presents 80 nm-sized particles and a 44,924 bp dsDNA genome encoding for 74 predicted proteins. More than 90% (68) of Yaravirus predicted genes have never been described before, representing ORFans. Only six genes had distant homologs in public databases: an exonuclease/recombinase, a packaging-ATPase, a bifunctional DNA primase/polymerase and three hypothetical proteins. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a novel capsid protein with no significant homology with NCLDV major capsid proteins but with a predicted double-jelly roll domain. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists. Significance statementMost of the known viruses of amoeba have been seen to share many features that eventually prompted authors to classify them into common evolutionary groups. Here we describe Yaravirus, an entity that could represent either the first isolated virus of Acanthamoeba spp. out of the group of NCLDVs or, in alternative evolutive scenario, it is a distant and extremely reduced virus of this group. Contrary to what is observed in other isolated viruses of amoeba, Yaravirus is not represented by a large/giant particle and a complex genome, but at the same time carries an important number of previously undescribed genes, including one encoding a novel major capsid protein. Metagenomic approaches also testified for the rarity of Yaravirus in the environment.

microbiology↗

bioRxiv2019

A metallo-beta-lactamase with both beta-lactamase and ribonuclease activity is linked with traduction in giant viruses

Enzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 g/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45{+/-}0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

microbiology↗

bioRxiv2019

Culture of salivary methanogens assisted by chemically produced hydrogen

Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing the pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher when using the biological method than when using the chemical method at six days and nine days postinoculation (Students test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

microbiology↗

bioRxiv2019

Transfer of CRISPR-like activity of MIMIVIRE into Bacteria

MIMIVIRE is a defence system utilized by lineage A Mimiviruses against Zamilon virophages. It is composed of a helicase, a nuclease and a gene of unknown function here named trcg (for Target Repeat-Containing gene), which contains four 15-bp repeats identical to the Zamilon sequence. Their silencing restored susceptibility to Zamilon, and the CRISPR-Cas4-like activity of the nuclease was recently characterised. We expressed these 3 genes after transformation of a modified strain of Escherichia coli made resistant to ampicillin, chloramphenicol and tetracycline. The virophage repeats were replaced with four repeats of 15 nucleotides identical to a sequence in the tetracycline resistance gene. The induction of the MIMIVIRE genes restored E. coli sensitivity to tetracycline; the tetracycline operon and its supporting plasmid harbouring the chloramphenicol resistance gene vanished. We therefore efficiently transferred the defence system MIMIVIRE from giant Mimivirus against virophage to E. coli to clear it from a plasmid.

microbiology↗

bioRxiv2019

Paradoxical β-lactamase activity of archaeal encoding enzymes

{beta}-lactams targeting the bacterial cell wall are not active on archaea. Here, we figure out that annotation of genes as {beta}-lactamase in Archeae on the basis of homologous genes, initially annotated {beta}-lactamases, is a remnant of the identification of the original activities of this group of enzymes, which in fact, have multiple functions including nuclease, ribonuclease, {beta}-lactamase, or glyoxalase; which may specialized over time. We expressed a class B {beta}-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while a weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The {beta}-lactamase activity was inhibited by a {beta}-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to has been transferred to the Flavobacteriaceae group including Elizabethkingia genus in which the expressed gene shows a more specialized activity toward resistance to tienanmicin but no glyoxalase activity. The expressed class C-like {beta}-lactamase gene, also from Methanosarcina sp., shows also hydrolysis activity and was more closely related to DD-peptidase enzymes than known bacterial class C {beta}-lactamases. Our findings highlight the requalification needness of annotated enzymes as {beta}-lactamases and the specification overtime of multipotent enzymes in different ways in Archaea and bacteria.

microbiology↗

bioRxiv2019

Antioxidants maintain Butyrate production by Human Gut Clostridia in the presence of Oxygen

BackgroundOxygen diffused from the human gut mucosa and shape the microbiota with a radial gradient of microbes according to their oxygen tolerance, while microbial and chemical oxygen consumption maintains the lumen in a deeply anaerobic state. Uncontrolled oxidative stress and hyperoxygenation have been reported as a pathogenic mechanism in Salmonella or Citrobacter rodentium infection, in patients with HIV and in severe acute malnutrition. We recently found that antioxidants allow strict anaerobes, including methanogenic archaea, to thrive in an oxidative environment (aerobic). Here, we tested the metabolomics switching of the 3 most odorous anaerobic microbes isolated from human gut when grown in aerobiosis with antioxidants.\n\nMethodsThree human gut Clostridia, Clostridium sporogenes, Clostridium lituseburense and Clostridium subterminale, isolated by culturomics, were grown in anaerobiosis or in aerobiosis with antioxidants. Gaz and liquid chromatography-Mass spectrometry (GC/MS and LC/MS) were used for metabolomics analysis.\n\nResultsAn unexpected global dichotomic metabolomic switching from thiols, alcohols and short-chain fatty acid esters to a specific aerobic metabolic repertoire with the production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed. Analysis of polar metabolites confirmed the discovery of an unexplored aerobic metabolic repertoire, including the production of specific dipeptides and several lysophospholipids, thus unraveling unsuspected human gut microbiome capacities.\n\nConclusionsAntioxidants unraveled an unexplored aerobic metabolic repertoire of human gut Clostridia. The increased production of butyrate suggests that antioxidants contribute to the maintenance and the active resilience of the human gut microbiome against oxidative aggression, as during Salmonella infection.

microbiology↗