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Simonetta Gribaldo - One of the best experts on this subject based on the ideXlab platform.
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one or two membranes diderm Firmicutes challenge the gram positive gram negative divide
Molecular Microbiology, 2020Co-Authors: Daniela Megrian, Christophe Beloin, Najwa Taib, Jerzy Witwinowski, Simonetta GribaldoAbstract:How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.
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outer membrane proteome of veillonella parvula a diderm firmicute of the human microbiome
Frontiers in Microbiology, 2017Co-Authors: Daniel Poppleton, Veronique Hourdel, Jennifer Flechsler, Mariette Matondo, Christophe Beloin, Andreas Klingl, Magalie Duchateau, Simonetta GribaldoAbstract:Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun’s lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of Veillonella parvula. We initially conducted a thorough bioinformatics analysis of all 1844 predicted proteins from V. parvula DSM 2008’s genome using twelve different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE) protein tags and by subcellular fractionation, both of which were analysed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA), the Lpt system component LptD, which is responsible for insertion of LPS into the OM, and several copies of the major OmpM protein. The annotation of V. parvula’s OM proteome markedly extends previous inferences on the nature of the cell envelope of Negativicutes. Finally, many OM hypothetical proteins were identified, which are priority targets for further characterization.
Alberto A Iglesias - One of the best experts on this subject based on the ideXlab platform.
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regulatory properties of the adp glucose pyrophosphorylase from the clostridial Firmicutes member ruminococcus albus
Journal of Bacteriology, 2018Co-Authors: Antonela Estefania Cereijo, Matias Damian Asencion Diez, Miguel A Ballicora, Alberto A IglesiasAbstract:ABSTRACT ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus. Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation. IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus. Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.
Pilar Junier - One of the best experts on this subject based on the ideXlab platform.
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Under-detection of endospore-forming Firmicutes in metagenomic data
Computational and structural biotechnology journal, 2015Co-Authors: Sevasti Filippidou, Tina Wunderlin, Thomas Junier, C.-c. Lo, Po-e Li, Patrick S G Chain, Pilar JunierAbstract:Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methods of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group (spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.
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Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments.
Environmental Microbiology Reports, 2014Co-Authors: Tina Wunderlin, Thomas Junier, Nicole Jeanneret, Ludovic Roussel-delif, Pilar JunierAbstract:: We present a method for the physical isolation of endospores from environmental samples allowing the specific targeting of endospore-forming bacteria for sequencing (endospore-enriched community). The efficiency of the method was tested on lake sediment samples. After 16S rRNA gene amplicon sequencing, the composition in the endospore-enriched community was compared with the community from untreated control samples (whole community). In the whole community, Firmicutes had a relative abundance of 8% and 19% in the two different lake sediments. In contrast, in the endospore-enriched community, Firmicutes abundance increased to 90.6% and 83.9%, respectively, confirming the efficiency of the endospore enrichment. The relative abundance of other microbial groups that form spore-like resisting states (i.e. actinobacteria, cyanobacteria and myxococcales) was below 2% in the endospore-enriched community, indicating that the method is adapted to true endospores. Representatives from two out of the three known classes of Firmicutes (Bacilli and Clostridia) were detected and supposedly asporogenic groups (e.g. Ethanoligenes and Trichococcus) could be detected. The method presented here is a leap forward for ecological studies of endospore-forming Firmicutes. It can be applied to other types of samples in order to reveal the diversity and metabolic potential of this bacterial group in the environment.
Daniela Megrian - One of the best experts on this subject based on the ideXlab platform.
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one or two membranes diderm Firmicutes challenge the gram positive gram negative divide
Molecular Microbiology, 2020Co-Authors: Daniela Megrian, Christophe Beloin, Najwa Taib, Jerzy Witwinowski, Simonetta GribaldoAbstract:How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.
Antonela Estefania Cereijo - One of the best experts on this subject based on the ideXlab platform.
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regulatory properties of the adp glucose pyrophosphorylase from the clostridial Firmicutes member ruminococcus albus
Journal of Bacteriology, 2018Co-Authors: Antonela Estefania Cereijo, Matias Damian Asencion Diez, Miguel A Ballicora, Alberto A IglesiasAbstract:ABSTRACT ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus. Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation. IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus. Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.
