Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum YT

Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum YT, the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum YT having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by ‘ancient’ CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after ~550 generations, the strain exhibited the mutation rate of ≥1.3 × 10−8 single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G:C to A:T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum YT variants from geographically distant populations

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in 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, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. Copyright © 2023 Distaso et al.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement 101000327. M.F. and F.J.P. also acknowledge grants PID2020-112758RB-I00 (M.F.), PDC2021- 121534-I00 (M.F.), TED2021-130544B-I00 (M.F.), and PID2019-105838RB-C31 (F.J.P.) from MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”). M.A.D., T.N.C., R.B., A.N.K., O.V.G., A.F.Y., and P.N.G. are thankful for support fromthe European Regional Development Fund (ERDF) through the Welsh Government to the Centre for Environmental Biotechnology, project number 81280. P.N.G. and A.F.Y. acknowledge the Natural Environment Research Council UK-funded Plastic Vectors project NE/S004548/1 and the Sêr Cymru program partly funded by the ERDF through the Welsh Government for support of the project BioPOL4Life.We are indebted to Connie Tulloch and GwionWilliams for their technical support.Supporting InformationPeer reviewe

Structural and Biochemical Characterization of CRISPR-associated Cas4 Nucleases from a Prokaryotic Defense System

Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.MAS

Characterization and Reconstruction of the RNA-guided Cascade Complex from Escherichia coli

CRISPR immunity utilizes a wide range of effector complexes in order to target and destroy invading nucleic acids. CRISPR Type I systems employ a multi-subunit protein-RNA complex (termed Cascade), with varying protein composition and activity. They have already been harnessed to regulate cellular metabolism, silence genes, and edit genomes in bacterial and human cells. The E. coli Cascade complex (Type I-E) includes 11-subunits (CasA (Cas8), CasB2 (Cas112), CasC6 (Cas76), CasD (Cas5), and CasE (Cas6)) and 61nt crRNA guide making it a versatile tool for bacteria to safeguard against invading generic elements throughout CRISPR immunity. Cascade shows functional flexibility, with its main function as an effector complex through Protospacer Adjacent Motif (PAM)-specific dsDNA targeting, R-loop formation and Cas3 nuclease recruitment for DNA cleaving. Here I describe the characterization of the minimal E. coli Cascade components required for specific RNA binding, dsDNA targeting, R-loop formation and Cas3 recruitment. I show that all eight nucleotides of the crRNA 5-handle are critical for binding to Cascade and target DNA recognition, whereas most of the 21 nucleotides of the 3-handle (up to 20 nt) were dispensable for Cascade activities. Furthermore, the E. coli Cascade tolerates significant alterations in the crRNA spacer length (32 nt) being able to form functional effector complexes (though with altered stoichiometry) with crRNAs containing either elongated (+ 6 nt) or reduced (-12 nt) spacers. I also show that CasCD (Cas7-5) is the minimal Cascade preparation that can specifically bind Type I-E crRNA at efficiency analogous to full Cascade. Moreover, CasCD (Cas7-5) can be programmed using the crRNA sequence to specifically target dsDNA in a PAM-less manner, with both spacer sequence and repeat handles contributing to DNA targeting specificity, with the former having a greater effect. Cascade reconstitution experiments using purified proteins revealed that both CasA (Cas8) and CasB (Cas11) are needed to achieve the downstream functions of R-loop formation and subsequent Cas3 nuclease recruitment. Together, these results highlight the plasticity of the Cascade-crRNA system and indicate that the E. coli Cascade represents a viable alternative to single-protein effector complexes in developing novel genome editing tools.Ph.D

Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum Y-T

Golyshina OV, Tran H, Reva ON, et al. Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum Y-T. SCIENTIFIC REPORTS. 2017;7(1): 3682.Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum Y-T, the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum Y-T having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by 'ancient' CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after -550 generations, the strain exhibited the mutation rate of >= 1.3 x 10(-8) single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G: C to A: T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum Y-T variants from geographically distant populations