Development of Genetic Tools for the Manipulation of the Planctomycetes

Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.ER-M and DPD are supported by the Spanish Ministry of Economy and Competitivity (Grant BFU2013-40866-P) and the Junta de Andalucía (CEIC Grant C2A program to DPD). IC and ES are supported by the Spanish Ministry of Economy and Competitivity (Grant BIO2014-57545-R).Peer reviewedPeer Reviewe

Transitional forms between the three domains of life and evolutionary implications

The question as to the origin and relationship between the three domains of life is lodged in a phylogenetic impasse. The dominant paradigm is to see the three domains as separated. However, the recently characterized bacterial species have suggested continuity between the three domains. Here, we review the evidence in support of this hypothesis and evaluate the implications for and against the models of the origin of the three domains of life. The existence of intermediate steps between the three domains discards the need for fusion to explain eukaryogenesis and suggests that the last universal common ancestor was complex. We propose a scenario in which the ancestor of the current bacterial Planctomycetes, Verrucomicrobiae and Chlamydiae superphylum was related to the last archaeal and eukaryotic common ancestor, thus providing a way out of the phylogenetic impasse

Efficient and automated large-scale detection of structural relationships in proteins with a flexible aligner

BACKGROUND: The total number of known three-dimensional protein structures is rapidly increasing. Consequently, the need for fast structural search against complete databases without a significant loss of accuracy is increasingly demanding. Recently, TopSearch, an ultra-fast method for finding rigid structural relationships between a query structure and the complete Protein Data Bank (PDB), at the multi-chain level, has been released. However, comparable accurate flexible structural aligners to perform efficient whole database searches of multi-domain proteins are not yet available. The availability of such a tool is critical for a sustainable boosting of biological discovery. RESULTS: Here we report on the development of a new method for the fast and flexible comparison of protein structure chains. The method relies on the calculation of 2D matrices containing a description of the three-dimensional arrangement of secondary structure elements (angles and distances). The comparison involves the matching of an ensemble of substructures through a nested-two-steps dynamic programming algorithm. The unique features of this new approach are the integration and trade-off balancing of the following: 1) speed, 2) accuracy and 3) global and semiglobal flexible structure alignment by integration of local substructure matching. The comparison, and matching with competitive accuracy, of one medium sized (250-aa) query structure against the complete PDB database (216,322 protein chains) takes about 8 min using an average desktop computer. The method is at least 2–3 orders of magnitude faster than other tested tools with similar accuracy. We validate the performance of the method for fold and superfamily assignment in a large benchmark set of protein structures. We finally provide a series of examples to illustrate the usefulness of this method and its application in biological discovery. CONCLUSIONS: The method is able to detect partial structure matching, rigid body shifts, conformational changes and tolerates substantial structural variation arising from insertions, deletions and sequence divergence, as well as structural convergence of unrelated proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0866-8) contains supplementary material, which is available to authorized users

Peroxisome retention involves Inp1-dependent peroxisome-plasma membrane contact sites in yeast

© 2020 Krikken et al.Retention of peroxisomes in yeast mother cells requires Inp1, which is recruited to the organelle by the peroxisomal membrane protein Pex3. Here we show that Hansenula polymorpha Inp1 associates peroxisomes to the plasma membrane. Peroxisome–plasma membrane contact sites disappear upon deletion of INP1 but increase upon INP1 overexpression. Analysis of truncated Inp1 variants showed that the C terminus is important for association to the peroxisome, while a stretch of conserved positive charges and a central pleckstrin homology-like domain are important for plasma membrane binding. In cells of a PEX3 deletion, strain Inp1-GFP localizes to the plasma membrane, concentrated in patches near the bud neck and in the cortex of nascent buds. Upon disruption of the actin cytoskeleton by treatment of the cells with latrunculin A, Inp1-GFP became cytosolic, indicating that Inp1 localization is dependent on the presence of an intact actin cytoskeleton.China Scholarship Council (NO AWARD)

Comparative Genomics of Peroxisome Biogenesis Proteins:Making Sense of the PEX Proteins

PEX genes encode proteins involved in peroxisome biogenesis and proliferation. Using a comparative genomics approach, we clarify the evolutionary relationships between the 37 known PEX proteins in a representative set of eukaryotes, including all common model organisms, pathogenic unicellular eukaryotes and human. A large number of previously unknown PEX orthologs were identified. We analyzed all PEX proteins, their conservation and domain architecture and defined the core set of PEX proteins that is required to make a peroxisome. The molecular processes in peroxisome biogenesis in different organisms were put into context, showing that peroxisomes are not static organelles in eukaryotic evolution. Organisms that lack peroxisomes still contain a few PEX proteins, which probably play a role in alternative processes. Finally, the relationships between PEX proteins of two large families, the Pex11 and Pex23 families, were analyzed, thereby contributing to the understanding of their complicated and sometimes incorrect nomenclature. We provide an exhaustive overview of this important eukaryotic organelle

Evolution of the nucleus

Under a Creative Commons license.The nucleus represents a major evolutionary transition. As a consequence of separating translation from transcription many new functions arose, which likely contributed to the remarkable success of eukaryotic cells. Here we will consider what has recently emerged on the evolutionary histories of several key aspects of nuclear biology; the nuclear pore complex, the lamina, centrosomes and evidence for prokaryotic origins of relevant players.Work in our laboratories was supported by the following agencies, and which is gratefully acknowledged; MRC and Wellcome Trust (MR/K008749/1 and 090007/Z/09/Z respectively, to MCF), C2A Junta de Andalucia to DPD and DFG GR1642/4-1 to RG.Open Access funded by Wellcome Trust.Peer Reviewe

Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

© 2020. Published by The Company of Biologists Ltd.The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.This work was supported by a grant from the FP7 People: Marie-Curie Actions Initial Training Networks (ITN) program PerFuMe (Grant Agreement Number 316723) to N.B., D.P.D. and I.J.v.d.K., from the China Scholarship Council (CSC) to F.W., and from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek/Chemical Sciences (NWO/CW) to A.A. (711.012.002)

Hansenula polymorpha Pex37 is a peroxisomal membrane protein required for organelle fission and segregation

Here, we describe a novel peroxin, Pex37, in the yeast Hansenula polymorpha. H. polymorpha Pex37 is a peroxisomal membrane protein, which belongs to a protein family that includes, among others, the Neurospora crassa Woronin body protein Wsc, the human peroxisomal membrane protein PXMP2, the Saccharomyces cerevisiae mitochondrial inner membrane protein Sym1, and its mammalian homologue MPV17. We show that deletion of H. polymorpha PEX37 does not appear to have a significant effect on peroxisome biogenesis or proliferation in cells grown at peroxisome‐inducing growth conditions (methanol). However, the absence of Pex37 results in a reduction in peroxisome numbers and a defect in peroxisome segregation in cells grown at peroxisome‐repressing conditions (glucose). Conversely, overproduction of Pex37 in glucose‐grown cells results in an increase in peroxisome numbers in conjunction with a decrease in their size. The increase in numbers in PEX37‐overexpressing cells depends on the dynamin‐related protein Dnm1. Together our data suggest that Pex37 is involved in peroxisome fission in glucose‐grown cells. Introduction of human PXMP2 in H. polymorpha pex37 cells partially restored the peroxisomal phenotype, indicating that PXMP2 represents a functional homologue of Pex37. H.polymorpha pex37 cells did not show aberrant growth on any of the tested carbon and nitrogen sources that are metabolized by peroxisomal enzymes, suggesting that Pex37 may not fulfill an essential function in transport of these substrates or compounds required for their metabolism across the peroxisomal membrane.This work was supported by a grant from the Marie Curie Initial Training Networks (ITN) program PerFuMe (Grant Agreement Number 316723) to RS, NB, DPD, and IJvdK.Peer reviewe

Genome evolution in morphological adaptation to cave environment in Astyanax mexicanus

Trabajo presentado en el EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021