High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments.

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

Microbial associations for bioremediation. What does "microbial consortia" mean?

Microbial associations arise as useful tools in several biotechnological processes. Among them, bioremediation of contaminated environments usually takes advantage of these microbial associations. Despite being frequently used, these associations are indicated using a variety of expressions, showing a lack of consensus by specialists in the field. The main idea of this work is to analyze the variety of microbial associations referred to as "microbial consortia" (MC) in the context of pollutants biodegradation and bioremediation. To do that, we summarize the origin of the term pointing out the features that an MC is expected to meet, according to the opinion of several authors. An analysis of related bibliography was done seeking criteria to rationalize and classify MC in the context of bioremediation. We identify that the microbe's origin and the level of human intervention are usually considered as a category to classify them as natural microbial consortia (NMC), artificial microbial consortia (AMC), and synthetic microbial consortia (SMC). In this sense, NMC are those associations composed by microorganisms obtained from a single source while AMC members come from different sources. SMC are a class of AMC in which microbial composition is defined to accomplish a certain specific task. We propose that the effective or potential existence of the interaction among MC members in the source material should be considered as a category in the classification as well, in combination with the origin of the source and level of intervention. Cross-kingdom MC and new developments were also considered. Finally, the existence of grey zones in the limits between each proposed microbial consortia category is addressed. KEY POINTS: • Microbial consortia for bioremediation can be obtained through different methods. • The use of the term "microbial consortia" is unclear in the specialized literature. • We propose a simplified classification for microbial consortia for bioremediation.

Metagenomic strategies identify diverse integron-integrase and antibiotic resistance genes in the Antarctic environment.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high-throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non-Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside-modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D ß-lactamase (blaOXA-205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.

Characteristics of a Cold-Adapted L-glutaminase with Potential Applications in the Food Industry.

L-glutaminases are enzymes that catalyze the hydrolysis of L-glutamine, producing L-glutamate and ammonium, and they have promising applications in pharmaceutical and food industries. Several investigations have focused on thermo-tolerant L-glutaminases; however, studies on cold-adapted L-glutaminases have not been reported. These enzymes could be useful in the food industry because they display high catalytic activity at low and room temperatures, a valuable feature in processes aimed to save energy. Besides, they can be easily inactivated by warming and are suitable to prevent decomposition of thermo-labile compounds. The objectives of this work were to characterize the L-glutaminase from the Antarctic bacterium Bizionia argentinensis and analyze its capability as flavor enhancer of protein hydrolysates. The enzyme was heterologously expressed and purified from Escherichia coli, obtaining optimum and homogeneous yields. Kinetic parameters Km and Vmax were located at the lower and upper range of values reported for L-glutaminases, suggesting high catalytic efficiency. Optimum temperature was 25 °C, and the enzyme conserved around 90% of maximum activity at 0 °C and in presence of 15% (v/v) ethanol and methanol. In saline conditions, the enzyme conserved around 80% of maximum activity in 3 M NaCl. Analysis of structural model suggested cold-adaptation features such as low Arg/(Arg+Lys) ratio and fewer intramolecular interactions than mesophilic and thermo-tolerant L-glutaminases. This work provides a novel cold-adapted L-glutaminase with promising features in the food industry.

Structure of the putative long tail fiber receptor-binding tip of a novel temperate bacteriophage from the Antarctic bacterium Bizionia argentinensis JUB59.

Tailed bacteriophages are one of the most widespread biological entities on Earth. Their singular structures, such as spikes or fibers are of special interest given their potential use in a wide range of biotechnological applications. In particular, the long fibers present at the termini of the T4 phage tail have been studied in detail and are important for host recognition and adsorption. Although significant progress has been made in elucidating structural mechanisms of model phages, the high-resolution structural description of the vast population of marine phages is still unexplored. In this context, we present here the crystal structure of C24, a putative receptor-binding tip-like protein from Bizionia argentinensis JUB59, a psychrotolerant bacterium isolated from the marine surface waters of Potter Cove, Antarctica. The structure resembles the receptor-binding tip from the bacteriophage T4 long tail fiber yet showing marked differences in its domain organization, size, sequence identity and metal binding nature. We confirmed the viral origin of C24 by induction experiments using mitomycin C. Our results reveal the presence of a novel uncharacterized prophage in the genome of B. argentinensis JUB59, whose morphology is compatible with the order Caudovirales and that carries the nucleotide sequence of C24 in its genome. This work provides valuable information to expand our current knowledge on the viral machinery prevalent in the oceans.

Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts.

The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed.

Metagenomic Analysis of Subtidal Sediments from Polar and Subpolar Coastal Environments Highlights the Relevance of Anaerobic Hydrocarbon Degradation Processes.

In this work, we analyzed the community structure and metabolic potential of sediment microbial communities in high-latitude coastal environments subjected to low to moderate levels of chronic pollution. Subtidal sediments from four low-energy inlets located in polar and subpolar regions from both Hemispheres were analyzed using large-scale 16S rRNA gene and metagenomic sequencing. Communities showed high diversity (Shannon's index 6.8 to 10.2), with distinct phylogenetic structures (<40% shared taxa at the Phylum level among regions) but similar metabolic potential in terms of sequences assigned to KOs. Environmental factors (mainly salinity, temperature, and in less extent organic pollution) were drivers of both phylogenetic and functional traits. Bacterial taxa correlating with hydrocarbon pollution included families of anaerobic or facultative anaerobic lifestyle, such as Desulfuromonadaceae, Geobacteraceae, and Rhodocyclaceae. In accordance, biomarker genes for anaerobic hydrocarbon degradation (bamA, ebdA, bcrA, and bssA) were prevalent, only outnumbered by alkB, and their sequences were taxonomically binned to the same bacterial groups. BssA-assigned metagenomic sequences showed an extremely wide diversity distributed all along the phylogeny known for this gene, including bssA sensu stricto, nmsA, assA, and other clusters from poorly or not yet described variants. This work increases our understanding of microbial community patterns in cold coastal sediments, and highlights the relevance of anaerobic hydrocarbon degradation processes in subtidal environments.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Bioprospection of cold-adapted yeasts with biotechnological potential from Antarctica.

The aim of this study was to investigate the ability to produce extracellular hydrolytic enzymes at low temperature of yeasts isolated from 25 de Mayo island, Antarctica, and to identify those exhibiting one or more of the evaluated enzymatic activities. A total of 105 yeast isolates were obtained from different samples and 66 were identified. They belonged to 12 basidiomycetous and four ascomycetous genera. Most of the isolates were ascribed to the genera Cryptococcus, Mrakia, Cystobasidium, Rhodotorula, Gueomyces, Phenoliferia, Leucosporidium, and Pichia. Results from enzymes production at low temperatures revealed that the Antarctic environment contains metabolically diverse cultivable yeasts, which represent potential tools for biotechnological applications. While most the isolates proved to produce 2-4 of the investigated exoenzymes, two of them evidenced the six evaluated enzymatic activities: Pichia caribbica and Guehomyces pullulans, which were characterized as psycrotolerant and psycrophilic, respectively. In addition, P. caribbica could assimilate several n-alkanes and diesel fuel. The enzyme production profile and hydrocarbons assimilation capacity, combined with its high level of biomass production and the extended exponential growth phase make P. caribbica a promising tool for cold environments biotechnological purposes in the field of cold-enzymes production and oil spills bioremediation as well.

Phenol degradation and heavy metal tolerance of Antarctic yeasts.

In cold environments, biodegradation of organic pollutants and heavy metal bio-conversion requires the activity of cold-adapted or cold-tolerant microorganisms. In this work, the ability to utilize phenol, methanol and n-hexadecane as C source, the tolerance to different heavy metals and growth from 5 to 30 °C were evaluated in cold-adapted yeasts isolated from Antarctica. Fifty-nine percent of the yeasts were classified as psychrotolerant as they could grow in all the range of temperature tested, while the other 41% were classified as psychrophilic as they only grew below 25 °C. In the assimilation tests, 32, 78, and 13% of the yeasts could utilize phenol, n-hexadecane, and methanol as C source, respectively, but only 6% could assimilate the three C sources evaluated. In relation to heavy metals ions, 55, 68, and 80% were tolerant to 1 mM of Cr(VI), Cd(II), and Cu(II), respectively. Approximately a half of the isolates tolerated all of them. Most of the selected yeasts belong to genera previously reported as common for Antarctic soils, but several other genera were also isolated, which contribute to the knowledge of this cold environment mycodiversity. The tolerance to heavy metals of the phenol-degrading cold-adapted yeasts illustrated that the strains could be valuable as inoculant for cold wastewater treatment in extremely cold environments.

Microbial and viral-like rhodopsins present in coastal marine sediments from four polar and subpolar regions.

Rhodopsins are broadly distributed. In this work, we analyzed 23 metagenomes corresponding to marine sediment samples from four regions that share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral rhodopsins, an initial set of 6224 bacterial rhodopsin sequences according to COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment, 123 viral (51) and non-viral (72) sequences (>50 amino acids) were finally included in further analysis. Viral rhodopsin genes were homologs of Phaeocystis globosa virus and Organic lake Phycodnavirus. Non-viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus-Thermus and Cryptophyta and Fungi. A rescreening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 amino acids). Phylogeographic analysis revealed a geographical clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non-viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsin genes related to Actinobacteria and Chloroflexi. This is the first report about the existence of a phylogeographic association of the viral rhodopsin sequences from marine sediments.

Metagenomics unveils the attributes of the alginolytic guilds of sediments from four distant cold coastal environments.

Alginates are abundant polysaccharides in brown algae that constitute an important energy source for marine heterotrophic bacteria. Despite the key role of alginate degradation processes in the marine carbon cycle, little information is available on the bacterial populations involved in these processes. The aim of this work was to gain a better understanding of alginate utilization capabilities in cold coastal environments. Sediment metagenomes from four high-latitude regions of both Hemispheres were interrogated for alginate lyase gene homologue sequences and their genomic context. Sediments contained highly abundant and diverse bacterial assemblages with alginolytic potential, including members of Bacteroidetes and Proteobacteria, as well as several poorly characterized taxa. The microbial communities in Arctic and Antarctic sediments exhibited the most similar alginolytic profiles, whereas brackish sediments showed distinct structures with a higher proportion of novel genes. Examination of the gene neighbourhood of the alginate lyase homologues revealed distinct patterns depending on the potential lineage of the scaffolds, with evidence of evolutionary relationships among alginolytic gene clusters from Bacteroidetes and Proteobacteria. This information is relevant for understanding carbon fluxes in cold coastal environments and provides valuable information for the development of biotechnological applications from brown algae biomass.

Heavy metals in sediments and soft tissues of the Antarctic clam Laternula elliptica: more evidence as a possible biomonitor of coastal marine pollution at high latitudes?

Studies on metal contamination in 25 de Mayo Island, Antarctica, yielded controversial results. In this work, we analyzed Antarctic marine sediments and Antarctic clam (Laternula elliptica) tissues to investigate the possible use of this mollusk as a biomonitor of metals and to identify the sources of metal pollution. Different types of paint from several buildings from Carlini Station were examined to assess their contribution to the local and random metal pollution. Five sediment samples, 105 L. elliptica specimens (40.2-78.0mm length) and four types of paint were analyzed to quantify Cd, Cr, Cu, Fe, Mn, Pb and Zn using inductively coupled plasma-optical emission spectrometry. Metal concentrations in sediments were lower than the global averages of the earth's crust, with the exception of Cd and Cu. These results were related to the contribution of the local fresh-water runoff. The different varieties of paint showed low levels of Cu, Mn, Fe and Zn, whereas a broad range of values were found in the case of Cr and Pb (20-15,100 µg·g(-1) and 153-115,500 µg·g(-1) respectively). The remains of the paint would be responsible for the significant increases in Cr and Pb which are randomly detected by us and by other authors. High levels of Fe and Cd, in comparison to other Antarctic areas, appear to be related to the terrigenous materials transported by the local streams. Accumulation indexes suggested that kidney tissue from L. elliptica could be an adequate material for biomonitoring pollution with Cd, Zn and probably also Pb. In general, relationships between size and metal contents reported by other authors were not verified, suggesting that this issue should be revised.

Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes.

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia-Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.

Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes / Una rica colección de bacterias marinas antárticas (Caleta Potter, islas Shetlands del Sur) revela diversos filotipos endémicos y previamente no descritos

.

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia- Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.

Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes / Una rica colección de bacterias marinas antárticas (Caleta Potter, islas Shetlands del Sur) revela diversos filotipos endémicos y previamente no descritos

.(AU)

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia- Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.(AU)

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ß-strands constituting the central ß-sheet of the αßα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.

Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes. / Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes.

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia-Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.