Biology subjects

Stogios, P. J.

Publications and source records attributed to Stogios, P. J..

7 recordsLinked to original sources

bioRxiv2023

Functional and Structural Characterization of an IclR Family Transcription Factor for the Development of Dicarboxylic Acid Biosensors

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work we characterize the PcaR TF belonging to the IclR family. We present in vitro functional analysis of PcaRs ligand profile and construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this TF. This work provides key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/550818v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@16bcdd2org.highwire.dtl.DTLVardef@95ba9forg.highwire.dtl.DTLVardef@f598borg.highwire.dtl.DTLVardef@1bfe5f2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPcaR is an IclR family transcription regulator responsive to dicarboxylic acids C_LIO_LIPcaR was established as an in vivo biosensor in yeast C_LIO_LICrystal structure of PcaR in the apo form was solved C_LIO_LICrystal structure with PcaR in complex with succinate was solved C_LIO_LISequence alignments unveil ligand-binding positions in the IclR family C_LI

synthetic biology↗

bioRxiv2022

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila

Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLp homologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found in Legionella species. Notably, the toxin HipTLp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipTLp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.

microbiology↗

bioRxiv2022

Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies

Extracytoplasmic solute binding proteins (SBPs) are molecular shuttles involved in the cellular uptake of various small molecules and metal ions including Ni(II). Our previous study with the Ni(II) binding proteins (NiBPs) CjNikZ from Campylobacter jejuni and CcNikZ-II from Clostridium carboxidivorans demonstrated they were able to bind Ni(II) at low micromolar affinity without the need for additional chelators. Here, we determined the crystal structure of apo CcNikZ-II, which revealed a Ni(II) binding site comprised of the highly conserved double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Alanine scanning mutagenesis of the CcNikZ-II Ni(II) binding site identified Glu60 and His511 as essential for high affinity binding to Ni(II). Phylogenetic analysis of >4000 SBP sequences demonstrated the presence of two clusters of proteins containing the HH-prong with CcNikZ-II and CjNikZ. To provide insights into the role of the double His-prong and v-loop sequence in Ni(II) binding of NiBPs, nine purified CcNikZ-II homologues containing the HH-prong and v-loop were screened using an automated screening workflow. Metal binding assays with purified homologous NiBPs revealed high Ni(II) binding affinity without requirement for chelators indicating that the double His prong represents a signature motif for the presence of Ni(II) binding activity in SBPs. The engineered CcNikZ-II variants with the wild type v-loop (TEDKYT) replaced with v-loops from NiBPs with higher affinity showed up to an order of magnitude higher affinity for Ni(II). In addition, the v-loop appears to play a role in metal ion specificity as purified wild type and engineered NiBPs with different v-loop sequences showed distinct metal profiles. This work paves way for metalloprotein engineering of NiBPs towards biocatalytic and metal recovery applications.

biochemistry↗

bioRxiv2022

Thermophilic carboxylesterases from hydrothermal vents of the volcanic island of Ischia active on synthetic and biobased polymers and mycotoxins

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70{o}C of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90{o}C, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80{o}C). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical /{beta} hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 [A] revealed the presence of the N-terminal {beta}-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology. IMPORTANCEHigh-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest "effective volumes" among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 {o}C) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.

microbiology↗

bioRxiv2022

Molecular mechanism of plasmid-borne resistance to sulfonamides

The sulfonamides (sulfas) are the oldest class of synthetic antibacterial that target the essential, conserved dihydropteroate synthase (DHPS) enzyme, encoded by folP, through chemical mimicry of its substrate p-aminobenzoic acid (pABA). Resistance has complicated their clinical utility and is widespread in pathogenic species. Resistance is mediated by acquisition of sul genes on mobile genetic elements, which code for the so-called Sul enzymes that are divergent DHPS enzymes with intrinsic sulfa-insensitivity. Even decades after the discovery of this resistance mechanism, its molecular details have not been understood. In this study, we elucidate the molecular basis for intrinsic resistance of Sul enzymes using x-ray crystallography, enzymology, mutagenesis, intrinsic tryptophan fluorescence, antibiotic susceptibility of a contemporary {Delta}folP strain, and adaptive laboratory evolution of folP. We show that the active sites of Sul enzymes possess a modified pABA-interaction region based on insertion of a Phe-Gly sequence. This insertion is necessary for discrimination between pABA and sulfonamides, more than 1000-fold loss in binding affinity of sulfas to Sul enzymes, and robust pan-sulfonamide resistance. We detect no fitness cost due to this active site modification, as it does not compromise the rate of dihydropteroate biosynthesis and complements the thymidine-auxotrophy of an E. coli folP deletion strain. Lab-evolved sulfa-resistance folP recapitulated this mechanism through the same active site insertion. Finally, we show that this insertion and a nearby loop confer increased active site flexibility of Sul enzymes relative to DHPS. These results provide a molecular foundation for revisiting DHPS-targeted antibacterials to evade resistance.

microbiology↗

bioRxiv2022

Recombinant production of growth factors for application in cell culture

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF-2, IGF-1, PDGF-BB and TGF-{beta}1 in Escherichia coli. Using this expression system, we produced soluble GFs from species including bovine, chicken, and fish. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

cell biology↗

bioRxiv2021

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an AAC(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

microbiology↗