Biology subjects

Witwinowski, J.

Publications and source records attributed to Witwinowski, J..

3 recordsLinked to original sources

bioRxiv2023

An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan (PG) for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group lack well-characterised OM attachment systems such as Brauns lipoprotein, Pal and OmpA, but instead have the OmpM protein. OmpM has a periplasmic region containing an S-layer homology domain and a {beta}-barrel region that is embedded in the OM. Here, we have determined the structure of the most abundant OmpM protein from the diderm firmicute Veillonella parvula by single particle cryogenic electron microscopy (cryo-EM). We reveal an OM portion similar to well-characterised trimeric general porins and a mobile periplasmic PG-binding region. Combining the cryo-EM data with crystal structures, structure predictions and molecular dynamics simulations, we show that the periplasmic region of OmpM likely adopts multiple conformations. Single-channel electrophysiology demonstrates ion conductance properties similar to E. coli OmpF. Finally, we demonstrate via functional assays that the four OmpM paralogues are likely to be the only general porins in V. parvula. Together, our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula (and other diderm Firmicutes), suggesting that concurrent loss of two key OM-related functions, tethering and nutrient uptake, promoted the multiple OM loss events that have been inferred in the Terrabacteria.

biochemistry↗

bioRxiv2021

An ancient divide in outer membrane tethering systems in Bacteria

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negatives (diderms) through loss of the outer membrane (OM). However how this happened remains unknown. Considering that tethering of the OM is essential for cell envelope stability in diderm bacteria we hypothesize that its destabilization may have been involved in OM loss. Here, we present an in-depth analysis of the four main OM tethering systems across all Bacteria. We show that their distribution strikingly follows the bacterial phylogeny with a bimodal distribution matching the deepest phylogenetic cleavage between Terrabacteria (a clade encompassing Cyanobacteria, Deinococcus/Thermus, Firmicutes, etc.) and Gracilicutes (a clade encompassing Proteobacteria, Bacteroidetes, Spirochaetes, etc.). Diderm Terrabacteria display as the main system OmpM, a porin that attaches non-covalently to modified peptidoglycan or to secondary cell wall polymers. In contrast, the lipoprotein Pal is restricted to the Gracilicutes along with a more sporadic occurrence of OmpA. While Brauns lipoprotein Lpp is largely considered as the textbook example of OM attachment, it is actually present only in a subclade of Gammaproteobacteria. We propose an evolutionary scenario whereby the last common bacterial ancestor used a system based on OmpM, which was later replaced by one based on the lipoprotein Pal concomitantly to the emergence of the Lol machinery to address lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM tethering system in the Terrabacteria would have allowed the multiple emergences of the monoderm phenotype specifically observed in this clade through OmpM perturbation. We test this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. The resulting mutant is severely affected in growth and displays high sensitivity to OM stress. High resolution imaging and tomogram reconstructions reveal a dramatic - yet non-lethal - phenotype, in which vast portions of the OM detach, producing large vesicles surrounding multiple monoderm-like cells sharing a common periplasm. Complementation by a single OmpM rescues the phenotype to a normal cell envelope. Together, our results highlight an ancient shift in bacterial evolution involving OM tethering systems. They suggest a possible mechanism for OM loss and a high flexibility of the cell envelope in diderm Firmicutes, making them ideal models to further refine our understanding of the mechanisms involved in bacterial OM stability, and opening the way to recapitulate the monoderm/diderm transition in the laboratory.

microbiology↗

bioRxiv2020

Autotransporters drive biofilm formation and auto-aggregation in the diderm Firmicute Veillonella parvula

The Negativicutes are a clade of Firmicutes that have retained the ancestral diderm character and possess an outer membrane. One of the best studied Negativicute, Veillonella parvula, is an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial communities, including the gut and the dental plaque microbiota. Whereas adhesion and biofilm capacity of V. parvula is expected to be crucial for its maintenance and development in these environments, studies of V. parvula adhesion have been hindered by the lack of efficient genetic tools to perform functional analyses in this bacterium. Here, we took advantage of a recently described naturally transformable V. parvula isolate, SKV38, and adapted tools developed for the closely related Clostridia spp. to perform random transposon and targeted mutagenesis to identify V. parvula genes involved in biofilm formation. We show that type V secreted autotransporters -typically found in diderm bacteria-are the main determinants of V. parvula auto-aggregation and biofilm formation, which compete with each other for binding either to cells or to surfaces, with strong consequences on V. parvula biofilm formation capacity. We also show that inactivation of the gene coding for a poorly characterized metal-dependent phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for further molecular characterization of V. parvula interactions with other bacteria and the host within complex microbiota environments.

microbiology↗