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 α/ß-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 ß-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. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and 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 than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation.

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.

Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S] cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus.

Cas4 proteins, a core protein family associated with the microbial system of adaptive immunity CRISPR, are predicted to function in the adaptation step of the CRISPR mechanism. Here we show that the Cas4 protein SSO0001 from the archaeon Sulfolobus solfataricus has metal-dependent endonuclease and 5'→3' exonuclease activities against single-stranded DNA, as well as ATP-independent DNA unwinding activity toward double-stranded DNA. The crystal structure of SSO0001 revealed a decameric toroid formed by five dimers with each protomer containing one [4Fe-4S] cluster and one Mn(2+) ion bound in the active site located inside the internal tunnel. The conserved RecB motif and four Cys residues are important for DNA binding and cleavage activities, whereas DNA unwinding depends on several residues located near the [4Fe-4S] cluster. Our results suggest that Cas4 proteins might contribute to the addition of novel CRISPR spacers through the formation of 3'-DNA overhangs and to the degradation of foreign DNA.

Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica.

The uncharacterized α/ß-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.

Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes.

Dehalococcoides mccartyi are obligate organohalide-respiring bacteria that play an important detoxifying role in the environment. They have small genomes (~1.4 Mb) with a core region interrupted by two high plasticity regions (HPRs) containing dozens of genes encoding reductive dehalogenases involved in organohalide respiration. The genomes of eight new strains of D. mccartyi were closed from metagenomic data from a related set of enrichment cultures, bringing the total number of genomes to 24. Two of the newly sequenced strains and three previously sequenced strains contain CRISPR-Cas systems. These D. mccartyi CRISPR-Cas systems were found to primarily target prophages and genomic islands. The genomic islands were identified either as integrated into D. mccartyi genomes or as circular extrachromosomal elements. We observed active circularization of the integrated genomic island containing vcrABC operon encoding the dehalogenase (VcrA) responsible for the transformation of vinyl chloride to non-toxic ethene. We interrogated archived DNA from established enrichment cultures and found that the CRISPR array acquired three new spacers in 11 years. These data provide a glimpse into dynamic processes operating on the genomes distinct to D. mccartyi strains found in enrichment cultures and provide the first insights into possible mechanisms of lateral DNA exchange in D. mccartyi.

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.

Conditional Epistatic Interaction Maps Reveal Global Functional Rewiring of Genome Integrity Pathways in Escherichia coli.

As antibiotic resistance is increasingly becoming a public health concern, an improved understanding of the bacterial DNA damage response (DDR), which is commonly targeted by antibiotics, could be of tremendous therapeutic value. Although the genetic components of the bacterial DDR have been studied extensively in isolation, how the underlying biological pathways interact functionally remains unclear. Here, we address this by performing systematic, unbiased, quantitative synthetic genetic interaction (GI) screens and uncover widespread changes in the GI network of the entire genomic integrity apparatus of Escherichia coli under standard and DNA-damaging growth conditions. The GI patterns of untreated cultures implicated two previously uncharacterized proteins (YhbQ and YqgF) as nucleases, whereas reorganization of the GI network after DNA damage revealed DDR roles for both annotated and uncharacterized genes. Analyses of pan-bacterial conservation patterns suggest that DDR mechanisms and functional relationships are near universal, highlighting a modular and highly adaptive genomic stress response.

Analysis of nuclease activity of Cas1 proteins against complex DNA substrates.

Cas1 genes encode the signature protein of the CRISPR/Cas system, which is present in all CRISPR-containing organisms. Recently, Cas1 proteins (together with Cas2) have been shown to be essential for the formation of new spacers in Escherichia coli, and purified Cas1 proteins from Pseudomonas aeruginosa and E. coli have been shown to possess a metal-dependent endonuclease activity. Here we describe the protocols for the analysis of nuclease activity of purified Cas1 proteins against various DNA substrates including Holliday junctions and other intermediates of DNA recombination and repair.