Multidrug resistance among uropathogenic clonal group A E. Coli isolates from Pakistani women with uncomplicated urinary tract infections.

OBJECTIVE: Multi-drug resistance (MDR) has notably increased in community acquired uropathogens causing urinary tract infections (UTIs), predominantly Escherichia coli. Uropathogenic E. coli causes 80% of uncomplicated community acquired UTIs, particularly in pre-menopausal women. Considering this high prevalence and the potential to spread antimicrobial resistant genes, the current study was conducted to investigate the presence of clinically important strains of E. coli in Pakistani women having uncomplicated cystitis and pyelonephritis. Women belonging to low-income groups were exclusively included in the study. Seventy-four isolates from urine samples were processed, phylotyped, and screened for the presence of two Single Nucleotide Polymorphisms (SNPs) particularly associated with a clinically important clonal group A of E. coli (CgA) followed by antibiotic susceptibility testing and genome sequence analysis. RESULTS: Phylogroup B2 was most prevalent in patients and 44% of isolates were positive for the presence of CgA specific SNPs in Fumarate hydratase and DNA gyrase subunit B genes. Antibiotic susceptibility testing showed widespread resistance to trimethoprim-sulfamethoxazole and extended-spectrum beta-lactamase production. The infection analysis revealed the phylogroup B2 to be more pathogenic as compared to the other groups. The genome sequence of E. coli strain U17 revealed genes encoding virulence, multidrug resistance, and host colonization mechanisms. CONCLUSIONS: Our research findings not only validate the significant occurrence of multidrug-resistant clonal group A E. coli (CgA) in premenopausal Pakistani women suffering from cystitis and pyelonephritis but also reveal the presence of genes associated withvirulence, and drug efflux pumps. The detection of highly pathogenic, antimicrobial-resistant phylogroup B2 and CgA E. coli strains is likely to help in understanding the epidemiology of the pathogen and may ultimately help to reduce the impact of these strains on human health. Furthermore, the findings of this study will particularly help to reduce the prevalence of uncomplicated UTIs and the cost associated with their treatment in women belonging to low-income groups.

Synthesis of Bioactive Nickel Nanoparticles Using Bacterial Strains from an Antarctic Consortium.

Marine microorganisms have been demonstrated to be an important source for bioactive molecules. In this paper we report the synthesis of Ni nanoparticles (NiSNPs) used as reducing and capping agents for five bacterial strains isolated from an Antarctic marine consortium: Marinomonas sp. ef1, Rhodococcus sp. ef1, Pseudomonas sp. ef1, Brevundimonas sp. ef1, and Bacillus sp. ef1. The NiSNPs were characterized by Ultraviolet-visible (UV-vis) spectroscopy, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) spectroscopic analysis. The maximum absorbances in the UV-Vis spectra were in the range of 374 nm to 422 nm, corresponding to the Surface plasmon resonance (SPR) of Nickel. DLS revealed NiSNPs with sizes between 40 and 45 nm. All NiSNPs were polycrystalline with a face-centered cubic lattice, as revealed by XRD analyses. The NiSNPs zeta potential values were highly negative. TEM analysis showed that the NiSNPs were either spherical or rod shaped, well segregated, and with a size between 20 and 50 nm. The FTIR spectra revealed peaks of amino acid and protein binding to the NiSNPs. Finally, all the NiSNPs possess significant antimicrobial activity, which may play an important role in the management of infectious diseases affecting human health.

Synthesis of Bioactive Silver Nanoparticles Using New Bacterial Strains from an Antarctic Consortium.

In this study, we report on the synthesis of silver nanoparticles (AgNPs) achieved by using three bacterial strains Rhodococcus, Brevundimonas and Bacillus as reducing and capping agents, newly isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii. After incubation of these bacteria with a 1 mM solution of AgNO3 at 22 °C, AgNPs were synthesized within 24 h. Unlike Rhodococcus and Bacillus, the reduction of Ag+ from AgNO3 into Ag0 has never been reported for a Brevundimonas strain. The maximum absorbances of these AgNPs in the UV-Vis spectra were in the range of 404 nm and 406 nm. EDAX spectra showed strong signals from the Ag atom and medium signals from C, N and O due to capping protein emissions. TEM analysis showed that the NPs were spherical and rod-shaped, with sizes in the range of 20 to 50 nm, and they were clustered, even though not in contact with one another. Besides aggregation, all the AgNPs showed significant antimicrobial activity. This biosynthesis may play a dual role: detoxification of AgNO3 and pathogen protection against both the bacterium and ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common nosocomial pathogens.

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation.

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.

Biogenic Synthesis of Copper Nanoparticles Using Bacterial Strains Isolated from an Antarctic Consortium Associated to a Psychrophilic Marine Ciliate: Characterization and Potential Application as Antimicrobial Agents.

In the last decade, metal nanoparticles (NPs) have gained significant interest in the field of biotechnology due to their unique physiochemical properties and potential uses in a wide range of applications. Metal NP synthesis using microorganisms has emerged as an eco-friendly, clean, and viable strategy alternative to chemical and physical approaches. Herein, an original and efficient route for the microbial synthesis of copper NPs using bacterial strains newly isolated from an Antarctic consortium is described. UV-visible spectra of the NPs showed a maximum absorbance in the range of 380-385 nm. Transmission electron microscopy analysis showed that these NPs are all monodispersed, spherical in nature, and well segregated without any agglomeration and with an average size of 30 nm. X-ray powder diffraction showed a polycrystalline nature and face centered cubic lattice and revealed characteristic diffraction peaks indicating the formation of CuONPs. Fourier-transform infrared spectra confirmed the presence of capping proteins on the NP surface that act as stabilizers. All CuONPs manifested antimicrobial activity against various types of Gram-negative; Gram-positive bacteria; and fungi pathogen microorganisms including Escherichia coli, Staphylococcus aureus, and Candida albicans. The cost-effective and eco-friendly biosynthesis of these CuONPs make them particularly attractive in several application from nanotechnology to biomedical science.

Machine learning models predicting multidrug resistant urinary tract infections using "DsaaS".

BACKGROUND: The scope of this work is to build a Machine Learning model able to predict patients risk to contract a multidrug resistant urinary tract infection (MDR UTI) after hospitalization. To achieve this goal, we used different popular Machine Learning tools. Moreover, we integrated an easy-to-use cloud platform, called DSaaS (Data Science as a Service), well suited for hospital structures, where healthcare operators might not have specific competences in using programming languages but still, they do need to analyze data as a continuous process. Moreover, DSaaS allows the validation of data analysis models based on supervised Machine Learning regression and classification algorithms. RESULTS: We used DSaaS on a real antibiotic stewardship dataset to make predictions about antibiotic resistance in the Clinical Pathology Operative Unit of the Principe di Piemonte Hospital in Senigallia, Marche, Italy. Data related to a total of 1486 hospitalized patients with nosocomial urinary tract infection (UTI). Sex, age, age class, ward and time period, were used to predict the onset of a MDR UTI. Machine Learning methods such as Catboost, Support Vector Machine and Neural Networks were utilized to build predictive models. Among the performance evaluators, already implemented in DSaaS, we used accuracy (ACC), area under receiver operating characteristic curve (AUC-ROC), area under Precision-Recall curve (AUC-PRC), F1 score, sensitivity (SEN), specificity and Matthews correlation coefficient (MCC). Catboost exhibited the best predictive results (MCC 0.909; SEN 0.904; F1 score 0.809; AUC-PRC 0.853, AUC-ROC 0.739; ACC 0.717) with the highest value in every metric. CONCLUSIONS: the predictive model built with DSaaS may serve as a useful support tool for physicians treating hospitalized patients with a high risk to acquire MDR UTIs. We obtained these results using only five easy and fast predictors accessible for each patient hospitalization. In future, DSaaS will be enriched with more features like unsupervised Machine Learning techniques, streaming data analysis, distributed calculation and big data storage and management to allow researchers to perform a complete data analysis pipeline. The DSaaS prototype is available as a demo at the following address: https://dsaas-demo.shinyapps.io/Server/.

Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins' sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25-35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

Synthesis of Bioactive Silver Nanoparticles by a Pseudomonas Strain Associated with the Antarctic Psychrophilic Protozoon Euplotes focardii.

The synthesis of silver nanoparticles (AgNPs) by microorganisms recently gained a greater interest due to its potential to produce them in various sizes and morphologies. In this study, for AgNP biosynthesis, we used a new Pseudomonas strain isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii. After incubation of Pseudomonas cultures with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. Scanning electron (SEM) and transmission electron microscopy (TEM) revealed spherical polydispersed AgNPs in the size range of 20-70 nm. The average size was approximately 50 nm. Energy dispersive X-ray spectroscopy (EDS) showed the presence of a high intensity absorption peak at 3 keV, a distinctive property of nanocrystalline silver products. Fourier transform infrared (FTIR) spectroscopy found the presence of a high amount of AgNP-stabilizing proteins and other secondary metabolites. X-ray diffraction (XRD) revealed a face-centred cubic (fcc) diffraction spectrum with a crystalline nature. A comparative study between the chemically synthesized and Pseudomonas AgNPs revealed a higher antibacterial activity of the latter against common nosocomial pathogen microorganisms, including Escherichia coli, Staphylococcus aureus and Candida albicans. This study reports an efficient, rapid synthesis of stable AgNPs by a new Pseudomonas strain with high antimicrobial activity.

Differences between Community - and Hospital - acquired urinary tract infections in a tertiary care hospital.

The aim of this retrospective study was to highlight the differences in antibiotic resistance between Hospital-acquired and Community-acquired urinary tract infections (UTIs). Antimicrobial UTIs resistance data were collected from March 2011 to March 2018. Uropathogens were identified from 41,715 patients using routine laboratory methods. Differences in antibiotic resistance between Hospital and Community (non-hospitalized) patients were statistically validated. Odds ratio (OR) and p-values was used to determine whether a particular exposure (hospitalization) was a risk factor for a particular outcome (higher antibiotic resistance). We reported a general increase of unnecessary urine cultures in both community and hospital patients. The most representative microorganism isolated from Community (58.2%) and Hospital (47.6%) was E. coli. UTIs causative bacteria in hospitalized patients was more than twice as resistant to Trimetoprim/sulphamethoxazole (OR 2.26) and Imipenem (OR 2.56), for Gram-positive and Gram-negative, respectively, than in Community patients. Nitrofurantoin was the only agent without differences in resistance rate between community and hospital UTIs. Therefore, physicians could use it as a definitive therapy for uncomplicated cystitis and as a prophylactic agent for recurrent uncomplicated cystitis. With this work we provided a general protocol applicable by physicians to select the most suitable, if necessary, UTIs empiric treatment.

CiliateGEM: an open-project and a tool for predictions of ciliate metabolic variations and experimental condition design.

BACKGROUND: The study of cell metabolism is becoming central in several fields such as biotechnology, evolution/adaptation and human disease investigations. Here we present CiliateGEM, the first metabolic network reconstruction draft of the freshwater ciliate Tetrahymena thermophila. We also provide the tools and resources to simulate different growth conditions and to predict metabolic variations. CiliateGEM can be extended to other ciliates in order to set up a meta-model, i.e. a metabolic network reconstruction valid for all ciliates. Ciliates are complex unicellular eukaryotes of presumably monophyletic origin, with a phylogenetic position that is equal from plants and animals. These cells represent a new concept of unicellular system with a high degree of species, population biodiversity and cell complexity. Ciliates perform in a single cell all the functions of a pluricellular organism, including locomotion, feeding, digestion, and sexual processes. RESULTS: After generating the model, we performed an in-silico simulation with the presence and absence of glucose. The lack of this nutrient caused a 32.1% reduction rate in biomass synthesis. Despite the glucose starvation, the growth did not stop due to the use of alternative carbon sources such as amino acids. CONCLUSIONS: The future models obtained from CiliateGEM may represent a new approach to describe the metabolism of ciliates. This tool will be a useful resource for the ciliate research community in order to extend these species as model organisms in different research fields. An improved understanding of ciliate metabolism could be relevant to elucidate the basis of biological phenomena like genotype-phenotype relationships, population genetics, and cilia-related disease mechanisms.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences.

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI's prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in "transmembrane transporter activity" and "oxidoreductase activity". Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment.

Characterization and comparative analysis of psychrophilic and mesophilic alpha-amylases from Euplotes species: a contribution to the understanding of enzyme thermal adaptation.

The eukaryotic α-amylase isolated from the psychrophilic ciliated protozoon Euplotes focardii (EfAmy) was expressed in Escherichia coli and biochemically characterized. Its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcAmy). The comparison of the amino acid composition and the surface residue composition of the two enzymes indicated a preference for tiny residues and the avoidance of charged, aromatic and hydrophobic residues in EfAmy. Our comparative homology modeling study reveals a lack of surface salt bridges, a decreased number of the surface charged residues, decreased hydrogen bonds and bound ions, and a reduction of aromatic-sulfur interactions, cationic-π interactions and disulfide interactions in EfAmy. In contrast, sequence alignment and homology modeling showed five unconserved prolines located on the surface loops of EcAmy. By analyzing amylolytic activity towards soluble starch as the substrate, we determined the temperature and pH dependence, thermostability and kinetic parameters of these two enzymes. We demonstrated that EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at low temperatures and high thermolability. In contrast, the EcAmy showed mesophilic characteristics with the highest activity at moderate temperatures and a more than 2-fold increased half-life at 50°C compared to EfAmy. The kcat and KM values of EfAmy were higher than those of the mesophilic EcAmy at all tested temperatures. Furthermore, both EfAmy and EcAmy showed maximum activities at pH 9 and maintained high activities in the presence of surfactants. These results suggest the potential applications of EfAmy and EcAmy as ingredients in detergents for industrial applications.

Diverse Repertoire and Relationship of Exopolysaccharide Genes in Cold-Adapted Acinetobacter sp. CUI-P1 Revealed by Comparative Genome Analysis.

This study focused on the exploration of microbial communities inhabiting extreme cold environments, such as the Passu and Pisan glaciers of Pakistan, and their potential utilization in industrial applications. Among the 25 initially screened strains, five were found to be suitable candidates for exopolysaccharide (EPS) production, with strain CUI-P1 displaying the highest yield of 7230.5 mg/L compared to the other four strains. The purified EPS from CUI-P1 was tested for its ability to protect probiotic bacteria and E. coli expressing green fluorescence protein (HriGFP) against extreme cold temperatures, and it exhibited excellent cryoprotectant and emulsification activity, highlighting its potential use in the biotechnological industry. Furthermore, the genome of Acinetobacter sp., CUI-P1 comprised 199 contigs, with a genome size of 10,493,143bp and a G + C content of 42%, and showed 98.197% nucleotide identity to the type genome of Acinetobacter baumannii ATCC 17978. These findings offer promising avenues for the application of EPS as a cryoprotectant, an essential tool in modern biotechnology.

Comprehensive insights on environmental adaptation strategies in Antarctic bacteria and biotechnological applications of cold adapted molecules.

Climate change and the induced environmental disturbances is one of the major threats that have a strong impact on bacterial communities in the Antarctic environment. To cope with the persistent extreme environment and inhospitable conditions, psychrophilic bacteria are thriving and displaying striking adaptive characteristics towards severe external factors including freezing temperature, sea ice, high radiation and salinity which indicates their potential in regulating climate change's environmental impacts. The review illustrates the different adaptation strategies of Antarctic microbes to changing climate factors at the structural, physiological and molecular level. Moreover, we discuss the recent developments in "omics" approaches to reveal polar "blackbox" of psychrophiles in order to gain a comprehensive picture of bacterial communities. The psychrophilic bacteria synthesize distinctive cold-adapted enzymes and molecules that have many more industrial applications than mesophilic ones in biotechnological industries. Hence, the review also emphasizes on the biotechnological potential of psychrophilic enzymes in different sectors and suggests the machine learning approach to study cold-adapted bacteria and engineering the industrially important enzymes for sustainable bioeconomy.

Hydrocarbon degradation strategy and pyoverdine production using the salt tolerant Antarctic bacterium Marinomonas sp. ef1.

One of the most concerning environmental problems is represented by petroleum and its derivatives causing contamination of aquatic and underground environments. In this work, the degradation treatment of diesel using Antarctic bacteria is proposed. Marinomonas sp. ef1 is a bacterial strain isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii. Its potential in the degradation of hydrocarbons commonly present in diesel oil were studied. The bacterial growth was evaluated in culturing conditions that resembled the marine environment with 1% (v/v) of either diesel or biodiesel added; in both cases, Marinomonas sp. ef1 was able to grow. The chemical oxygen demand measured after the incubation of bacteria with diesel decreased, demonstrating the ability of bacteria to use diesel hydrocarbons as a carbon source and degrade them. The metabolic potential of Marinomonas to degrade aromatic compounds was supported by the identification in the genome of sequences encoding various enzymes involved in benzene and naphthalene degradation. Moreover, in the presence of biodiesel, a fluorescent yellow pigment was produced; this was isolated, purified and characterized by UV-vis and fluorescence spectroscopy, leading to its identification as a pyoverdine. These results suggest that Marinomonas sp. ef1 can be used in hydrocarbon bioremediation and in the transformation of these pollutants in molecules of interest.

Novel Sources of Biodiversity and Biomolecules from Bacteria Isolated from a High Middle Ages Soil Sample in Palermo (Sicily, Italy).

The urban plan of Palermo (Sicily, Italy) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools. IMPORTANCE One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample (harvested in Palermo, Italy) were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation.

Structural thermal adaptation of ß-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii.

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three ß-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the ß-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii ß-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic ß-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.

Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five ß-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.