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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.

The novel Gram-negative, aerobic, non-motile, non-spore-forming, short rod bacterium, strain C7T, was isolated from the seawater sample of Menai Straits (Wales, UK) and characterised. Phylogenetic analysis of 16S rRNA gene sequences showed that this strain represented a distinct lineage within the Roseobacter clade of family Rhodobacteracea within Alphaproteobacteria. The members of the genera Pontivivens (P. insulae GYSW-23T), Celeribacter (C. manganoxidans DY2-5T), Donghicola (D. eberneus SW-277T), Roseovarius (R. halotolerans HJ50T and R. pacificus 81-2T), Cribrihabitans (C. marinus CZ-AM5T) and Aestuariihabitans (A. beolgyonensis BB-MW15T) were the closest relatives with 16S rRNA gene sequence identities between 93.4 % and 95.6 %. The strain C7T could utilize a restricted number of complex substrates with a preference for yeast extract and tryptone, consistently with earlier observations that peptides may serve as an important energy and carbon source for bacteria from the Roseobacter clade. Growth occurred in the absence of sodium ions. The isolate C7T is a mesophilic bacterium that optimally grows at 20 ºC. The strain can grow under microaerophilic conditions. The major fatty acid was C18:1 cis d11. The only detected ubiquinone was Q10. The polar lipids of C7T strain were phosphatidylglycerol, two unknown aminolipids and three unknown lipids. The DNA G+C content of the strain was 60.0 mol%. Based on the results of the morphological, physiological and phylogenetic analyses, the new genus, Monaibacterium gen. nov., to include the new species Monaibacterium marinum sp. nov., is proposed. Strain C7T (=DSM 100241T, =LMG 28800T) is the type and only strain of M. marinum.

Crude oil is one of the most important natural assets for humankind, yet it is a major environmental pollutant, notably in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, in which the metabolic potential of indigenous microbial populations towards the large-scale chronic pollution is yet to be defined, particularly in anaerobic and micro-aerophilic sites. Here, we provide an insight into the microbial metabolism in sediments from three chronically polluted marine sites along the coastline of Italy: the Priolo oil terminal/refinery site (near Siracuse, Sicily), harbour of Messina (Sicily) and shipwreck of MT Haven (near Genoa). Using shotgun metaproteomics and community metabolomics approaches, the presence of 651 microbial proteins and 4776 metabolite mass features have been detected in these three environments, revealing a high metabolic heterogeneity between the investigated sites. The proteomes displayed the prevalence of anaerobic metabolisms that were not directly related with petroleum biodegradation, indicating that in the absence of oxygen, biodegradation is significantly suppressed. This suppression was also suggested by examining the metabolome patterns. The proteome analysis further highlighted the metabolic coupling between methylotrophs and sulphate reducers in oxygen-depleted petroleum-polluted sediments.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.