Identification of novel broad host-range promoter sequences functional in diverse Pseudomonadota by a promoter-trap approach.

Finding novel promoter sequences is a cornerstone of synthetic biology. To contribute to the expanding catalog of biological parts, we employed a promoter-trap approach to identify novel sequences within an Antarctic microbial community that act as broad host-range promoters functional in diverse Pseudomonadota. Using Pseudomonas putida KT2440 as host, we generated a library comprising approximately 2,000 clones resulting in the identification of thirteen functional promoter sequences, thereby expanding the genetic toolkit available for this chassis. Some of the discovered promoter sequences prove to be broad host-range as they drove gene expression not only in P. putida KT2440 but also in Escherichia coli DH5α, Cupriavidus taiwanensis R1T, Paraburkholderia phymatum STM 815T, Ensifer meliloti 1021, and an indigenous Antarctic bacterium, Pseudomonas sp. UYIF39. Our findings enrich the existing catalog of biological parts, offering a repertoire of broad host-range promoter sequences that exhibit functionality across diverse members of the phylum Pseudomonadota, proving Antarctic microbial community as a valuable resource for prospecting new biological parts for synthetic biology.

Sphaerotilus uruguayifluvii sp. nov., a novel filamentous bacterium isolated from river water.

Strain C29T, a Gram-staining-negative, straight rod occurring singly, in pairs or short chains, was isolated from floating filamentous biomass of the Uruguay River. The strain was catalase and oxidase positive, chemoorganotrophic, strictly aerobic, non-motile, and grew at pH 6.0-9.0, 15-45 °C, and 0-0.5% (w/v) NaCl. Polyhydroxybutyrate was accumulated in nutrient-limited conditions. Phylogenetic analysis based on the 16S rRNA gene revealed that strain C29T had the highest sequence similarity with Leptothrix discophora SS-1T (97.82%), Ideonella livida TBM-1T (97.82%), Vitreoscilla filiformis L1401-2T (97.52%), Sphaerotilus sulfidivorans D-501T (97.50%) and Sphaerotilus natans DSM 6575T (97.46%). Other type strains with validly published names had similarities below 97.46%. Further phylogenomic analysis showed that strain C29T was affiliated to the family Sphaerotilaceae. Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (dDDH) values with its phylogenetic relatives were lower than 91 and 41%, respectively, revealing that strain C29T represented a new species. The DNA G + C content of strain C29T was 70.9%. The annotation of the genome of the novel strain shows it possessed genes for the degradation of aromatic compounds. It also contained genes that encode sigma factors involved in response regulation of stress resistance, which is an important function for adaptation and survival in natural niches. Based on the results of the phylogenetic and phenotypic analyses, we propose that strain C29T represents a novel species, for which the name Sphaerotilus uruguayifluvii sp. nov. is proposed. The type strain is C29T (= CCM 9043T = DSM 113250T).

Datathons: fostering equitability in data reuse in ecology.

Approaches to rapidly collecting global biodiversity data are increasingly important, but biodiversity blind spots persist. We organized a three-day Datathon event to improve the openness of local biodiversity data and facilitate data reuse by local researchers. The first Datathon, organized among microbial ecologists in Uruguay and Argentina assembled the largest microbiome dataset in the region to date and formed collaborative consortia for microbiome data synthesis.

Pseudomonas violetae sp. nov. and Pseudomonas emilianonis sp. nov., two new species with the ability to degrade TNT isolated from soil samples at Deception Island, maritime Antarctica.

Two motile, rod-shaped, Gram-stain-negative bacterial strains, TNT11T and TNT19T, were isolated from soil samples collected at Deception Island, Antarctica. According to the 16S rRNA gene sequence similarity, both strains belong to the genus Pseudomonas. Further genomic analyses based on ANI and dDDH suggested that these strains were new species. Growth of strain TNT11T is observed at 0-30 â„ƒ (optimum, 20 â„ƒ), pH 4.0-9.0 (optimum, pH 6.0) and in the presence of 0-5.0% NaCl (optimum, 1% NaCl), while for TNT19T is observed at 0-30 â„ƒ (optimum between 15 and 20 â„ƒ), pH 5.0-9.0 (optimum, pH 6.0) and in the presence of 0-5.0% NaCl (optimum between 0 and 1% NaCl). The fatty acid profile consists of the major compounds; C16:0 and C16:1 ω6 for TNT11T, and C16:0 and C12:0 for TNT19T. Based on the draft genome sequences, the DNA G + C content for TNT11T is 60.43 mol% and 58.60 mol% for TNT19T. Based on this polyphasic study, TNT11T and TNT19T represent two novel species of the genus Pseudomonas, for which the proposed names are Pseudomonas violetae sp. nov. and Pseudomonas emilianonis sp. nov., respectively. The type strains are Pseudomonas violetae TNT11T (= RGM 3443T = LMG 32959T) and Pseudomonas emilianonis TNT19T (= RGM 3442T = LMG 32960T). Strains TNT11T and TNT19T were deposited to CChRGM and BCCM/LMG with entry numbers RGM 3443/LMG 32959 and RGM 3442/LMG 32960, respectively.

Characterisation of 'Candidatus Methylobacter titanis' sp. nov., a putative novel species of Methylobacter clade 2 and their distribution in sediments of freshwater lakes in maritime Antarctica.

Global warming has a strong impact on the polar regions, in particular, the Antarctic Peninsula and nearby islands. Methane (CH4) is a major factor in climate change and mitigation of CH4 emissions can be accomplished through microbial oxidation by methanotrophic bacteria. Understanding this biological process is crucial given the shortage of research carried out in this geographical area. The aim of this study was to characterise psychrophilic enrichment cultures of aerobic methanotrophs obtained from lake sediments of the Fildes Peninsula (King George Island, South Shetland Islands) and revealing the distribution of the genus Methylobacter in different lake sediments of the peninsula. Four stable methanotrophic enrichment cultures were obtained and analysed by metagenome-assembled genomes (MAGs). The phylogeny of methanotroph MAGs recovered from these enrichment cultures based on the 16S rRNA gene showed that K-2018 MAG008 and D1-2020 MAG004Ts clustered within the Methylobacter clade 2, with high similarity to Methylobacter tundripaludum SV96T (97.88 and 98.56% respectively). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with M. tundripaludum were < 95% (84.8 and 85.0%, respectively) and < 70% (30.2 and 30.3%, respectively), suggesting that they represent a putative novel species for which the name 'Ca. Methylobacter titanis' is proposed. This is the first species of clade 2 of the genus Methylobacter obtained from Antarctica. The bacterial diversity assessed by 16S rRNA gene sequencing of 21 samples of different lakes (water column and sediments) revealed 54 ASVs associated with methanotrophs and the genus Methylobacter as the most abundant. These results suggest that aerobic methanotrophs belonging to the Methylobacter clade 2 would be the main responsible for CH4 oxidation in these sediments.

Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics.

One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and post-translational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for function-driven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

Diversity and Effect of Increasing Temperature on the Activity of Methanotrophs in Sediments of Fildes Peninsula Freshwater Lakes, King George Island, Antarctica.

Global warming has a strong impact on polar regions. Particularly, the Antarctic Peninsula and nearby islands have experienced a marked warming trend in the past 50 years. Therefore, higher methane (CH4) emissions from this area could be expected in the future. Since mitigation of these emissions can be carried out by microbial oxidation, understanding this biological process is crucial since to our knowledge, no related studies have been performed in this area before. In this work, the aerobic CH4 oxidation potential of five freshwater lake sediments of Fildes Peninsula (King George Island, South Shetland Islands) was determined with values from 0.07 to 10 µmol CH4 gdw-1 day-1 and revealed up to 100-fold increase in temperature gradients (5, 10, 15, and 20°C). The structure and diversity of the bacterial community in the sediments were analyzed by next-generation sequencing (Illumina MiSeq) of 16S rRNA and pmoA genes. A total of 4,836 ASVs were identified being Proteobacteria, Actinobacteriota, Acidobacteriota, and Bacteroidota the most abundant phyla. The analysis of the pmoA gene identified 200 ASVs of methanotrophs, being Methylobacter Clade 2 (Type I, family Methylococcaceae) the main responsible of the aerobic CH4 oxidation. Moreover, both approaches revealed the presence of methanotrophs of the classes Gammaproteobacteria (families Methylococcaceae and Crenotrichaceae), Alphaproteobacteria (family Methylocystaceae), Verrucomicrobia (family Methylacidiphilaceae), and the candidate phylum of anaerobic methanotrophs Methylomirabilota. In addition, bacterial phospholipid fatty acids (PLFA) biomarkers were studied as a proxy for aerobic methane-oxidizing bacteria and confirmed these results. Methanotrophic bacterial diversity was significantly correlated with pH. In conclusion, our findings suggest that aerobic methanotrophs could mitigate in situ CH4 emissions in a future scenario with higher temperatures in this climate-sensitive area. This study provides new insights into the diversity of methanotrophs, as well as the influence of temperature on the CH4 oxidation potential in sediments of freshwater lakes in polar regions of the southern hemisphere.

Paenibacillus farraposensis sp. nov., isolated from a root nodule of Arachis villosa.

Strain UY79T was isolated from a root nodule of Arachis villosa, collected at the Esteros de Farrapos National Park, Río Negro, Uruguay. Cells were non-motile Gram-variable rods with central to subterminal oval to ellipsoidal endospores that swell the sporangia. Growth was observed in the range of 15-42 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0-8.0) and with up to 3 % (w/v) NaCl (optimum, 1-2 %). Strain UY79T was facultative anaerobic, catalase-positive and oxidase-negative. According to the results of 16S rRNA gene sequence analysis, UY79T belongs to the genus Paenibacillus and is closely related to P. ottowii MS2379T, P. peoriae BD-57T, P. polymyxa ATCC 842T and P. brasilensis PB172T, exhibiting 99.4, 99.0, 99.0 and 98.9% sequence identity, respectively. Average nucleotide identity and digital DNA-DNA hybridization values with the most closely related type strains were 74.3-88.6% and 38.2-48.7 %, respectively. Major fatty acids (>10 %) were anteiso-C15:0, iso-C15:0, and C16 : 0. Menaquinones MK-7 and MK-6 were the only isoprenoid quinones detected. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid. Spermidine was the predominant polyamine. The DNA G+C content based on the draft genome sequence was 46.34 mol%. Based on the current polyphasic study, UY79T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus farraposensis sp. nov. is proposed. The type strain is UY79T (=CCM 9147T=CGMCC 1.19038T).

Frigoriflavimonas asaccharolytica gen. nov., sp. nov., a novel psychrophilic esterase and protease producing bacterium isolated from Antarctica.

The rod-shaped and Gram-stain-negative bacterial strain 16FT, isolated from an air sample collected at King George Island, maritime Antarctica, was investigated to determine its taxonomic status. Strain 16FT is strictly aerobic, catalase positive, oxidase positive and non-motile. Strain 16FT hydrolyses casein, lecithin, Tween 20, 60 and 80, but not aesculin, gelatin and starch. Growth of strain 16FT is observed at 0-20 °C (optimum 10 °C), pH 5.0-8.0 (optimum pH 6.0), and in the presence of 0-2.0% NaCl (optimum 0.5%). The predominant menaquinone is MK-6, and the major fatty acids comprise anteiso-C15:0 and iso-C15:0. The major polar lipids are phosphatidylethanolamine, ornithine lipid OL2, unidentified phospholipid PL1 and the unidentified lipids L3 and L6 lacking functional groups. The DNA G + C content based on the draft genome sequence is 32.3 mol%. Sequence analysis of the 16S rRNA gene indicates the highest similarity to Kaistella palustris 3A10T (95.4%), Kaistella chaponensis Sa 1147-06 T (95.2%), Kaistella antarctica AT1013T (95.1%), Kaistella carnis NCTC 13525 T (95.1%) and below 95.0% to other species with validly published names. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences places strain 16FT in a distinct branch, indicating a separate lineage within the family Weeksellaceae. Based on the data from our polyphasic approach, 16FT represents a novel species of a new genus, for which the name Frigoriflavimonas asaccharolytica gen. nov, sp. nov. is proposed. The type strain is 16FT (= CCM 8975 T = CGMCC No.1.16844 T).

Hymenobacter caeli sp. nov., an airborne bacterium isolated from King George Island, Antarctica.

A rod-shaped and Gram-stain-negative bacterial strain 9AT, was isolated from an air sample collected at King George Island, maritime Antarctica. Phylogenetic analysis based on 16S rRNA gene sequence reveals that strain 9AT belongs to the genus Hymenobacter and shows the highest similarity to Hymenobacter coccineus CCM 8649T (96.8 %). The DNA G+C content based on the draft genome sequence is 64.9 mol%. Strain 9AT is strictly aerobic, psychrophilic, catalase-positive, oxidase-positive and non-motile. Growth is observed at 0-20 °C (optimum 10 °C), pH 6.0-8.0 (optimum pH 7.0), and in the absence of NaCl. The predominant menaquinone of strain 9AT is MK-7 and the major fatty acids comprise Summed Feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 25.2 %), iso-C15 : 0 (23.2 %), C16 : 1 ω5c (11.6 %), Summed Feature 4 (anteiso-C17 : 1 B/iso-C17 : 1 I) (9.6 %) and anteiso-C15 : 0 (9.6 %). The polar lipid profile consists of the major lipid phosphatidylethanolamine and moderate to minor amounts of phosphatidylserine, unidentified aminolipids, aminophospholipids, aminophosphoglycolipids, polar lipids lacking a functional group and an unidentified phospholipid and a glycolipid. In the polyamine pattern sym-homospermidine is predominant. On the basis of the results obtained, strain 9AT is proposed as a novel species of the genus Hymenobacter, for which the name Hymenobacter caeli sp. nov. is suggested. The type strain is 9AT (=CCM 8971T=LMG 32109T=DSM 111653T).

Hymenobacter artigasi sp. nov., isolated from air sampling in maritime Antarctica.

A rod-shaped and Gram-stain-negative bacterial strain, 1BT, was isolated from an air sample collected at King George Island, maritime Antarctica. Strain 1BT is strictly aerobic, psychrophilic, catalase-positive, oxidase-positive and non-motile. Growth of strain 1BT is observed at 0-20 °C (optimum, 10 °C), pH 6.0-8.0 (optimum, pH 8.0) and in the presence of 0-1.0% NaCl (optimum, 0.5 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences places strain 1BT within the genus Hymenobacter and shows the highest similarity to Hymenobacter antarcticus VUG-A42aaT (97.5 %). The predominant menaquinone of strain 1BT is MK-7 and the major fatty acids (>10 %) comprise summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 32.5 %), iso-C15 : 0 (17.6 %) and anteiso C15 : 0 (12.3 %). The polar lipid profile consists of the major compounds phosphatidylethanolamine, phosphatidylserine, two unidentified aminolipids and one unidentified phospholipid. The DNA G+C content based on the draft genome sequence is 61.2 mol%. Based on the data from the current polyphasic study, 1BT represents a novel species of the genus Hymenobacter, for which the name Hymenobacter artigasi sp. nov. is suggested. The type strain is 1BT (=CCM 8970T=CGMCC 1.16843T).