Phenotypic and resistome analysis of antibiotic and heavy metal resistance in the Antarctic bacterium Pseudomonas sp. AU10.

Resistance to antibiotics and heavy metals in Antarctic bacteria has been investigated due to anthropogenic impact on the continent. However, there is still much to learn about the genetic determinants of resistance in native bacteria. In this study, we investigated antibiotic, heavy metal, and metalloid resistance in Pseudomonas sp. AU10, isolated from King George Island (Antarctica), and analyzed its genome to look for all the associated genetic determinants (resistome). We found that AU10 displayed resistance to Cr(VI), Cu(II), Mn(II), Fe(II), and As(V), and produced an exopolysaccharide with high Cr(VI)-biosorption capacity. Additionaly, the strain showed resistance to aminopenicillins, cefotaxime, aztreonam, azithromycin, and intermediate resistance to chloramphenicol. Regarding the resistome, we did not find resistance genes in AU10's natural plasmid or in a prophage context. Only a copper resistance cluster indicated possible horizontal acquisition. The mechanisms of resistance found were mostly efflux systems, several sequestering proteins, and a few enzymes, such as an AmpC ß-lactamase or a chromate reductase, which would account for the observed phenotypic profile. In contrast, the presence of a few gene clusters, including the terZABCDE operon for tellurite resistance, did not correlate with the expected phenotype. Despite the observed resistance to multiple antibiotics and heavy metals, the lack of resistance genes within evident mobile genetic elements is suggestive of the preserved nature of AU10's Antarctic habitat. As Pseudomonas species are good bioindicators of human impact in Antarctic environments, we consider that our results could help refine surveillance studies based on monitoring resistances and associated resistomes in these populations.

A general overview of the multifactorial adaptation to cold: biochemical mechanisms and strategies.

Cold environments are more frequent than people think. They include deep oceans, cold lakes, snow, permafrost, sea ice, glaciers, cold soils, cold deserts, caves, areas at elevations greater than 3000 m, and also artificial refrigeration systems. These environments are inhabited by a diversity of eukaryotic and prokaryotic organisms that must adapt to the hard conditions imposed by cold. This adaptation is multifactorial and includes (i) sensing the cold, mainly through the modification of the liquid-crystalline membrane state, leading to the activation of a two-component system that transduce the signal; (ii) adapting the composition of membranes for proper functions mainly due to the production of double bonds in lipids, changes in hopanoid composition, and the inclusion of pigments; (iii) producing cold-adapted proteins, some of which show modifications in the composition of amino acids involved in stabilizing interactions and structural adaptations, e.g., enzymes with high catalytic efficiency; and (iv) producing ice-binding proteins and anti-freeze proteins, extracellular polysaccharides and compatible solutes that protect cells from intracellular and extracellular ice. However, organisms also respond by reprogramming their metabolism and specifically inducing cold-shock and cold-adaptation genes through strategies such as DNA supercoiling, distinctive signatures in promoter regions and/or the action of CSPs on mRNAs, among others. In this review, we describe the main findings about how organisms adapt to cold, with a focus in prokaryotes and linking the information with findings in eukaryotes.

A bacterial cold-active dye-decolorizing peroxidase from an Antarctic Pseudomonas strain.

DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: • An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. • The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. • rDyP-AU10 showed high activity at low temperatures. • rDyP-AU10 is potentially useful for biotechnological applications.

Photo-repair effect of a bacterial Antarctic CPD-photolyase on UVC-induced DNA lesions in human keratinocytes.

Exposure to ultraviolet radiation from sunlight induces oxidative DNA lesions and bipyrimidine photoproducts that can lead to photo-aging and skin carcinogenesis. CPD-photolyases are flavoproteins that repair cyclobutane pyrimidine dimers using blue light as an energy source. In the present work, we evaluated the photo-repair effect of the recombinant CPD-photolyase PhrAHym from the Antarctic bacterium Hymenobacter sp. UV11 on DNA lesions in human keratinocytes induced by UVC light. By performing immunochemistry assays we observed that PhrAHym repairs in a highly efficient way the CPD-photoproducts and reduces the γH2AX formation. Since this enzyme is non-cytotoxic and repairs UVC-induced DNA lesions in human keratinocytes, we propose that PhrAHym could be used as a biotherapeutic agent against UV-induced skin cancer, photoaging, and related diseases.

A (6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9: recombinant production and in silico features.

Photolyases are proteins that enzymatically repair the UV-induced DNA damage by a protein-DNA electron transfer mechanism. They repair either cyclobutane pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts or just (6-4)-photoproducts. In this work, we report the production and partial characterization of a recombinant (6-4)-photolyase (SphPhrB97) from a bacterial psychrotolerant Antarctic isolate identified as Sphingomonas sp. strain UV9. The spectrum analysis and the in silico study of SphPhrB97 suggest that this enzyme has similar features as compared to the (6-4)-photolyase from Agrobacterium tumefaciens (4DJA; PhrB), including the presence of three cofactors: FAD, DMRL (6,7-dimethyl-8-(1'-D-ribityl) lumazine), and an Fe-S cluster. The homology model of SphPhrB97 predicts that the DNA-binding pocket (area and volume) is larger as compared to (6-4)-photolyases from mesophilic microbes. Based on sequence comparison and on the homology model, we propose an electron transfer pathway towards the FAD cofactor involving the residues Trp342, Trp390, Tyr40, Tyr391, and Tyr399. The phylogenetic tree performed using curated and well-characterized prokaryotic (6-4)-photolyases suggests that SphPhrB97 may have an ancient evolutionary origin. The results suggest that SphPhrB97 is a cold-adapted enzyme, ready to cope with the UV irradiation stress found in a hostile environment, such as Antarctica.

Fighting plant pathogens with cold-active microorganisms: biopesticide development and agriculture intensification in cold climates.

Cold-adapted (CA) microorganisms (= psychrophiles or psychrotolerants) are key players of many ecological interactions in natural ecosystems. Some of them can colonize the rhizosphere of plants and cause damage to their hosts; others, on the contrary, protect plants from their pathogens through direct and indirect mechanisms, thus promoting plant growth and development. These "protective" microbes are known as biocontrol agents (BCA). BCA either limit or inhibit the growth of plant pathogens, owing to the excretion of a panoply of secondary metabolites (including soluble and volatile antibiotics, siderophores, quorum sensing interfering agents). BCA can also control plant pathogens through indirect mechanisms, including competence for nutrients and space, or else by interfering with their chemical communication. That explains why some of these BCA have been included in the formulation of commercial biopesticides, which are environmentally friendly products containing live cells used to control plant diseases and pests. At present, the development of biopesticides from mesophilic microorganisms is an established technology. Unfortunately, these biopesticides are not active at low temperatures. On the other hand, the information concerning the potential use of CA-BCA for the same goal is at its infancy. Here, we review the current knowledge concerning the isolation, identification, and characterization of CA microbes which act as antagonists of plant pathogens, including the mechanisms they deploy to antagonize plant pathogens. We also illustrate their biotechnological potential to develop CA biopesticides and discuss their utility in the context of mountainous agriculture. KEY POINTS: • Many naturally occurring cold-active microbes antagonize plant pathogens. • The mechanisms of biocontrol exerted by these microbes are either direct or indirect. • Cold-active biocontrol agents can be used to develop biopesticides. • Cold-active biopesticides are crucial for sustainably intensifying agriculture in cold climates.

Production and antiproliferative effect of violacein, a purple pigment produced by an Antarctic bacterial isolate.

We studied the production and the potential use of a purple-pigment produced by an Antarctic bacterial isolate. This pigment was identified as violacein, a metabolite produced by many bacterial strains and reported that it has antiproliferative activity in many cell lines. We analyzed the effect of temperature and the composition of the growth medium on pigment production, achieving the highest yield at 20 °C in Tryptic Soy Broth medium supplemented with 3.6 g/L glucose. We doubled the yield of the pigment production when the process was scaled up in a 5 L bioreactor (77 mg/L of crude pigment). The pigment was purified and identified by mass spectrometry (DI-EI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy as violacein. We performed survival assays that showed that the pure pigment has antiproliferative activity and sensitize HeLa cells (cervix cell carcinoma) to cisplatin. Besides, the pigment did not show genotoxic activity in HeLa cells as found performing micronucleus assays. These results suggest that this pigment may be used as anticancer or sensitizer to cisplatin drug in cervix cancer.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Validating biochemical features at the genome level in the Antarctic bacterium Hymenobacter sp. strain UV11.

We present experimental data that complement and validate some biochemical features at the genome level in the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11 strain. The genome was sequenced, assembled and annotated. It has 6 096 246 bp, a GC content of 60.6% and 5155 predicted genes. The secretome analysis, by combining in silico predictions with shotgun proteomics data, showed that UV11 strain produces extracellular proteases and carbohydrases with potential biotechnological uses. We observed the formation of outer membrane vesicles, mesosomes and carbon-storage compounds by using transmission electron microscopy. The in silico analysis of the genome revealed the presence of genes involved in the metabolism of glycogen-like molecules and starch. By HPLC-UV-Vis analysis and 1H-NMR spectra, we verified that strain UV11 produces xanthophyll-like carotenoids such as 2'-hydroxyflexixanthin, and the in silico analysis showed that this bacterium has genes involved in the biosynthesis of cathaxanthin, zeaxanthin and astaxanthin. We also found genes involved in the repair of UV-damaged DNA such as a photolyase, the nucleotide excision repair system and the production of ATP-dependent proteases that are important cellular components involved in the endurance to physiological stresses. This information will help us to better understand the ecological role played by Hymenobacter strains in the extreme Antarctic environment.

Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42.

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.

Reference genes for real-time RT-PCR expression studies in an Antarctic Pseudomonas exposed to different temperature conditions.

Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4 °C and 30 °C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species.

Draft Genome Sequence of the UV-Resistant Antarctic Bacterium Sphingomonas sp. Strain UV9.

We report the draft genome sequence of the Antarctic UV-resistant bacterium Sphingomonas sp. strain UV9. The strain has a genome size of 4.25 Mb, a 65.62% GC content, and 3,879 protein-coding sequences. Among others, genes encoding the resolving of the DNA damage produced by the UV irradiation were identified.

A highly efficient and cost-effective recombinant production of a bacterial photolyase from the Antarctic isolate Hymenobacter sp. UV11.

Photolyases are DNA-repairing flavoproteins that are represented in most phylogenetic taxa with the exception of placental mammals. These enzymes reduce the ultraviolet-induced DNA damage; thus, they have features that make them very attractive for dermatological or other medical uses, such as the prevention of human skin cancer and actinic keratosis. In this work, we identified a 50.8 kDa photolyase from the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and its activity was analyzed using different approaches: detection of cyclobutane pyrimidine dimers (CPDs) by immunochemistry, high-performance liquid chromatography and comet assays using Chinese Hamster Ovary (CHO) and immortalized nontumorigenic human epidermal (HaCat) cells. The information supports that the recombinant protein has the ability to repair the formation of CPDs, on both double- and single-stranded DNA. This CPD-photolyase was fully active on CHO and HaCat cell lines, suggesting that this enzyme could be used for medical or cosmetic purposes. Results also suggest that the UV11 CPD-photolyase uses MTHF as chromophore in the antenna domain. The potential use of this recombinant enzyme in the development of new inventions with pharmaceutical and cosmetic applications is discussed during this work.

Identification of Plant Compounds Involved in the Microbe-Plant Communication During the Coinoculation of Soybean with Bradyrhizobium elkanii and Delftia sp. strain JD2.

Delftia sp. strain JD2 is a betaproteobacterium characterized as a plant growth-promoting bacterium with a 'helper' function, enhancing the performance of rhizobial inoculant strains during the coinoculation of alfalfa and clover. In this work we analyzed i) the effect of the coinoculation with Bradyrhizobium elkanii and Delftia sp. strain JD2 strains on the performance of soybean plants and ii) the production of a few secondary plant metabolites that would explain the positive effect of coinoculation on the growth and development of soybean plants. The results showed a beneficial effect of coinoculation on soybean growth, nodulation rate, and pulse yield, with the concomitant benefit for the agricultural economy. In addition, based on a metabolomics approach, we demonstrated that a different pattern of plant metabolites is being produced at different stages of plant growth. The new information suggests that the coinoculation of soybean changes the primary and secondary metabolism of the plant, including changes in the metabolic status of main and secondary nodules within the plant. The relevance of producing a different pattern of photosynthetic and photoprotective pigments, flavonoids, organic acids, and carbohydrates are discussed. Finally, we propose that JD2 could be used together with bradyrhizobia to manipulate the chemical composition of plant tissues, promoting the nutritional benefits and health of soybean.

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m- 2) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.

Trends in amino acid usage across the class Mollicutes.

Many studies have explored the mechanisms involved in relative amino acid usage (RAAU) in a variety of prokaryotes and eukaryotes. A strong bias was observed in highly expressed genes (HEGs) of endosymbiotic bacteria. By means of correspondence analysis, we studied the major trends affecting internal variability of RAAU in Mollicutes. The principal trend is related to the usage of smaller, less aromatic, and GC-rich coded amino acids in HEGs. Given the nature of the genetic code, these properties are linked among them. Expectedly, we found a slow evolutionary rate of HEGs, which is likely driven by purifying selection. On the other hand, the rest of the genes accumulate rapid changes as a result of the extreme mutational bias toward A + T of the genomes and genetic drift, increasing internal variability. Amino acid changes across the phylogeny of the group were traced in order to estimate the mean molecular weight and aromaticity trends in each branch. Finally, we compared amino acid usage bias within and between Mollicutes and the free-living Firmicutes.

Evolution of optimal codon choices in the family Enterobacteriaceae.

The Enterobacteriaceae are a large family of Proteobacteria that include many well-known prokaryotic genera, such as Escherichia, Yersinia and Salmonella. The main ideas of synonymous codon usage (CU) evolution and translational selection have been deeply influenced by studies with these bacterial groups. In this work we report the analysis of the CU pattern of completely sequenced bacterial genomes that belong to the Enterobacteriaceae. The effect of selection in translation acting at the levels of speed and accuracy, and phylogenetic trends within this group are described. Preferred (optimal) codons were identified. The evolutionary dynamics of these codons were studied and following a Bayesian approach these preferences were traced back to the common ancestor of the family. We found that there is some level of variation in selection among the analysed micro-organisms that is probably associated with lineage-specific trends. The codon bias was largely conserved across the evolutionary time of the family in highly expressed genes and protein conserved regions, suggesting a major role of negative selection. In this sense, the results support the idea that the extant CU bias is finely tuned over the ancestral well-conserved pool of tRNAs.

The versatility of Delftia sp. isolates as tools for bioremediation and biofertilization technologies.

Two Pb(II)-resistant bacteria isolated from a soil containing 2,500 mg/kg of Pb were identified by 16S rRNA sequencing analysis as Delftia sp. and designated as 3C and 6C. Both isolates grew at a Pb(II) concentration of 62 mg/L and at the stationary phase showed a Pb(II)-sorption capability of 10 ± 1.5 (3C) and 5 ± 0.8 (6C) mg/g of biomass. Biochemical properties related to heavy metal resistance and plant growth promotion were analyzed and compared with the Cr(VI)-resistant plant growth-promoting Delftia sp. JD2, previously reported by our group. Both isolates and JD2 were resistant to Cr(VI), Pb(II) and many antibiotics, produced siderophores and the phytohormone indole-3-acetic, and showed clover growth-promoting activity in greenhouse conditions. Interestingly, the occurrence of integron class 1 was shown in all isolates. Our results add to previous reports and suggest that bacteria of the genus Delftia could be consider as good candidates for the design of technologies for cleaning up contaminated environments and/or the production of biofertilizers.

Antarctic DNA moving forward: genomic plasticity and biotechnological potential.

Antarctica is the coldest, driest, and windiest continent, where only cold-adapted organisms survive. It has been frequently cited as a pristine place, but it has a highly diverse microbial community that is continually seeded by nonindigenous microorganisms. In addition to the intromission of 'alien' microorganisms, global warming strongly affects microbial Antarctic communities, changing the genes (qualitatively and quantitatively) potentially available for horizontal gene transfer. Several mobile genetic elements have been described in Antarctic bacteria (including plasmids, transposons, integrons, and genomic islands), and the data support that they are actively involved in bacterial evolution in the Antarctic environment. In addition, this environment is a genomic source for the identification of novel molecules, and many investigators have used culture-dependent and culture-independent approaches to identify cold-adapted proteins. Some of them are described in this review. We also describe studies for the design of new recombinant technologies for the production of 'difficult' proteins.

Delftia sp. JD2: a potential Cr(VI)-reducing agent with plant growth-promoting activity.

A chromium (Cr)-resistant bacterium isolated from soil containing 6,000 mg/kg of Cr was identified based on 16S rRNA gene sequence analysis as Delftia, and designated as JD2. Growth of JD2 was accompanied with reduction of Cr(VI) to Cr(III) in liquid medium initially containing 100 mg/L Cr(VI), the maximum concentration allowing growth. JD2 showed NADH/NADPH-dependent reductase activity associated with the soluble fraction of cells. The results suggest that JD2 might be a good candidate for the treatment of highly Cr(VI)-contaminated water and/or industrial effluents. The isolate produced indole-3-acetic acid in the presence and absence of Cr(VI) and showed free-living nitrogen-fixing activity possibly attributable to a V-nitrogenase. JD2 did not counteract the harmful effect of Cr(VI) during leguminous plant growth and nodulation by rhizobial strains but functioned as a "helper" bacterium to enhance the performance of rhizobial inoculant strains during inoculation of alfalfa and clover (used as model plants to study plant growth-promoting activity) in the absence of Cr(VI).