Physiological Characterization Research Articles

Screening for native Trichoderma strains as potential biocontrollers of the olive pathogen Verticillium dahliae

Verticillium dahliae is a soilborne pathogen that causes significant losses in olive crops in northwestern Argentina. Biological control through antagonistic microorganisms such as Trichoderma has great potential in the management of Verticillium wilt of olive. This investigation aims to isolate, identify and characterize native Trichoderma strains for biocontrol of V. dahliae. Thirty-nine Trichoderma strains were isolated from soils of olive orchards and natural areas and they were morphologically and molecularly characterized. Desired attributes for plant growth and bioprotection, such as indole acetic acid (IAA) production, endophytism and antagonistic potential against the pathogen were evaluated. Thirteen Trichoderma species were identified, belonging to the Trichoderma, Longibrachiatum, Virens, and Harzianum clades. Physiological characterization revealed that fourteen strains produced IAA while ten showed endophytic capacity. The antagonistic parameters quantified were very variable: eight strains showed high growth inhibition values (PI > 65%), while six strains reached mycoparasitism (PM) values higher than 90% for both pathogens. Three endophytic strains exhibited IAA production and antagonistic activity against V. dahliae, becoming potential candidates for bioprotection of olive orchards. Olive cultivation in the arid regions of northwestern Argentina is subjected to strong stress conditions mainly due to the type of soils with low organic matter content and water retention. Thus, selected Trichoderma strains with more than one beneficial attribute are a fundamental tool for sustainable olive cultivation, not only as antagonists of emerging pathogens but also as mitigators of abiotic stress conditions that prevail in these environments.

Characterization of bacteriophage BUCT631 lytic for K1 Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Severe infections caused by multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) highlight the need for new therapeutics with activity against this pathogen. Phage therapy is an alternative treatment approach for multidrug-resistant K. pneumoniae infections. Here, we report a novel bacteriophage (phage) BUCT631 that can specifically lyse capsule-type K1 K. pneumoniae. Physiological characterization revealed that phage BUCT631 could rapidly adsorb to the surface of K. pneumoniae and form an obvious halo ring, and it had relatively favorable thermal stability (4–50 ​°C) and pH tolerance (pH ​= ​4–12). In addition, the optimal multiplicity of infection (MOI) of phage BUCT631 was 0.01, and the burst size was approximately 303 ​PFU/cell. Genomic analysis showed that phage BUCT631 has double-stranded DNA (total length of 44,812 bp) with a G ​+ ​C content of 54.1%, and the genome contains 57 open reading frames (ORFs) and no virulence or antibiotic resistance related genes. Based on phylogenetic analysis, phage BUCT631 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae. In addition, phage BUCT631 could quickly inhibit the growth of K. pneumoniae within 2 ​h in vitro and significantly elevated the survival rate of K. pneumoniae infected Galleria mellonella larvae from 10% to 90% in vivo. These studies suggest that phage BUCT631 has promising potential for development as a safe alternative for control and treatment of multidrug-resistant K. pneumoniae infection.

Physiological characterization of Vitis hybrid cv Merlot Khorus under two different water regimes in northeastern Italy

Fungus–resistant varieties are gathering attention all over viticultural areas owing to their contribution to sustainable management. Sustainable viticulture implies lower inputs in terms of pesticides and water. However, investigations on the physiological behavior of fungus–resistant varieties under drought stress have been seldom conducted so far. Under climate change conditions, evaluating precise water use is particularly important to set up proper irrigation. In this study, a two-year experiment was imposed to characterize the physiological behavior of the fungus–resistant variety Merlot Khorus under two contrasting water regimes (full irrigation, WW; water deficit, WD). Berry weight and grape quality in the two treatments was also compared to evaluate the feasibility of adopting deficit irrigation strategies in the region. The study was conducted in the Friuli Venezia Giulia region (Italy). The measured values of stem water potential (Ψstem) did not highlight any severe water stress (> -1.15 MPa) in WD. However, gs and AN decreased to values typical of drought–stressed vines. Concurrently, the experiment assessed that the calculated Kcb of Merlot Khorus in northeast Italy is far different from what has been suggested by standard methodologies and the literature. The grapes under deficit irrigation showed reduced berry weight, while basic maturation parameters and anthocyanins were improved. Overall, the study indicated that Merlot Khorus appears to be a genotype suitable for dry farming or deficit irrigation management in northeastern Italy. Therefore, the results may help winegrowers to improve irrigation strategies.

The influence of Spirulina platensis on physiological characterization and mitigation of DNA damage in salt-stressed Phaseolus vulgaris L. plants

Salinity severely reduced crop productivity. In this work, we study the effectiveness of Spirulina platensis (100 mg/l) as a foliar growth stimulator and its ability to reduce the harmful effect of salinity on Phaseolus vulgaris L. plants. The experiment was divided into four groups as follows: G1 (control), G2 (200 mM NaCl), G3 (100 mg/l, Spirulina platensis), and G4 (200 mM NaCl + 100 mg/l, Spirulina platensis). Salinity treatment (G2) showed that the plant height, shoot weight (fresh and dry), and number of leaves/plant decreased significantly during the vegetative growth stage; the number of pods/plant, number of seeds/pod, and pod weight (fresh and dry) at the time of harvest; and the contents of carotenoids, chlorophyll a+b, 100-seed weights, photosynthetic activity (assimilation of 14CO2), and total protein content also decreased significantly. The above parameters were enhanced by G4 treatment when compared to salt-stressed plants. Salinity treatment considerably increased the levels of superoxide dismutase (SOD), peroxidase activity (POD), catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), free proline, and total phenol by 24.3, 32.2, 45, 18.6, 56.5, 38.4, and 13.9% respectively, as compared to the control treatment (G1). However, G4 treatment (200 mM NaCl + S. platensis, 100 mg/l) showed a significant decrease in SOD, POD, CAT, GPX, MDA, free proline, and total phenol by 2.4, 4.5, 4.5, 9.5, 20, 9.4, and 2.7%, respectively, as compared to the control plant (G1). Additionally, salinity dramatically reduced total protein by 36% as compared to the control. The G4 treatment (200 mM NaCl + S. platensis, 100 mg/l), on the other hand, increased total protein content by 31.3% when compared to the salinity treatment (G2). The salinity treatment (G2) decreased significantly the macronutrient content of N, P, and K by 26.1, 34.7, and 43.7%, respectively, and on the contrary, increased significantly the Na+ and Cl− levels by 55.8 and 23.1%, respectively, compared to the control plant (G1). In contrast to salt-stressed plants, G3 (S. platensis, 100 mg/l) and G4 (200 mM NaCl + S. platensis, 100 mg/l) applications considerably enhanced the macronutrient content and reduced the Na+ and Cl− concentrations. Obtained results proved that foliar applications of S. platensis at 100 mg/l have a high potential for improving growth, photosynthetic capacity, yield production, decreasing ROS-induced oxidative damage, and reducing DNA damage in salt-stressed Phaseolus vulgaris.

Physiological, Enological and Agronomic Characterization of Pedro Ximénez Grapevine Cultivar under Organic Farming in a Warm Climate Zone

One of the challenges that European agriculture will have to face will be to adapt conventional agriculture to procedures of the new European agricultural policies. In this way, organic farming will have more importance in the coming years. One of the most important crops worldwide is grapevine. The main objective of this research work focuses on the physiological, agronomic, and enological characterization of Pedro Ximénez with the purpose of knowing the viability of the organic cultivation of this cultivar in a warm climate zone. Two experimental plots were selected with two different types of management, organic and conventional. In both plots, photosynthetic capacity (AN), stomatal conductance (gs), and intrinsic water-use efficiency (WUEi) were measured, and physicochemical composition of grape must was analyzed during ripening. In addition, bunch and pruning weight were measured as agronomic parameters. Physiological results were not significantly different between management at a general level in gs and WUEi, being possible to identify a difference in AN just before the harvest. At the level of fruit ripening, significant differences were found between the two managements. At harvest, grape must had a higher sugar concentration, amine nitrogen (α-NH2), and yeast assimilable nitrogen (YAN) in the organic management than in conventional, where higher values of pH and l-malic acid were observed. Taking into account the preliminary results obtained it could be concluded the viability of the organic management for Pedro Ximénez crop in a warm climate zone.

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Physiological and Histological Characterization of the ESB1 TILLING Mutant of Brassica rapa L.: Potential Use in Biofortification and Phytoremediation Programs

Enhanced suberin1 (ESB1) is a protein whose mutation is correlated with an increase in root suberin and altered nutrient concentrations. Here, we show a physiological and histological characterization of esb1 mutant plants of Brassica rapa L. Therefore, the potential use of this mutant in selenium (Se) biofortification and/or cadmium (Cd) phytoremediation programs was also evaluated by applying 20 μM of Na2SeO4 and 0.49 μM of CdCl2 to a nutrient solution. With respect to wild type (WT) plants, an increase in root suberin was observed in esb1 at the level of the exodermis. This increase in root suberin did not affect photosynthesis performance. However, the esb1 mutant showed an increase in transpiration rate and a decrease in water use efficiency. Additionally, root histological changes affected the transport and concentration of some mineral elements. Thus, our results suggest that esb1 mutants of B. rapa would not be useful for Se biofortification because no significant differences were observed between the two genotypes at the leaf level. Nevertheless, the esb1 mutant reduced Cd translocation to the leaves and increased Fe and Cu uptake, so ESB1 mutation could be useful for Cd phytoremediation and Fe and Cu biofortification, although further research is needed. Therefore, this study provides detailed information on the effect of ESB1 mutation in B. rapa and suggests its potential use in biofortification and phytoremediation programs.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the Plant Defensin 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterisation of i) novel amiRNA lines silenced for the five most similar AtPDF1s, and ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under Zn excess conditions, and with increased plant tolerance to different pathogens - one fungus, one oomycete and one bacterium, while the double mutant behaved similarly to the WT. These unexpected results challenge the current paradigm describing the role of PDFs in the plant response to stresses. Additional roles of plant endogenous defensins are discussed, which open new perspectives for their functions.

Streptomyces hilarionis sp. nov. and Streptomyces hayashii sp. nov., two new strains associated with potato scab in Brazil.

Two new actinobacteria, designated strains IBSBF 2807T and IBSBF 2953T, isolated from scab lesions on potato tubers grown in the southern Brazilian states of Rio Grande do Sul and Santa Catarina, respectively, were characterized and identified through a polyphasic approach. Phylogenetic analyses of 16S rRNA sequences revealed that these two strains belong to the genus Streptomyces. Multilocus sequence analysis using five concatenated genes, atpD, gyrB, recA, rpoB and trpB, allocated strains IBSBF 2807T and IBSBF 2953T in distinct branches of Streptomyces phytopathogenic strains. PCR-RFLP analysis of the atpD gene also confirmed that these strains differ from the type strains of Streptomyces associated with potato scab. The morphological, physiological and biochemical characterization, along with the overall genome-related index properties, indicated that these two strains could be distinguished from their closest phylogenetic relatives and each other. According to the data, IBSBF 2807T and IBSBF 2953T represent two new Streptomyces species related to potato scab. The proposed names for these strains are Streptomyces hilarionis sp. nov. (IBSBF 2807T=CBMAI 2674T=ICMP 24297T=MUM 22.66T) and Streptomyces hayashii sp. nov (IBSBF 2953T=CBMAI 2675T=ICMP 24301T=MUM 22.68T).

Genetic and Physiological Diversity of Marine Actinobacteria from the Okha Coast, Gujarat, India

Saline and alkaline habitats of the Okha coastline, Gujarat, India, were explored for the diversity of actinobacteria. Based on the phenotypic and genotypic heterogeneity, nine haloalkaliphilic actinobacterial strains were characterized and identified. The PCR amplification of 16S rDNA using universal and genus-specific primers corroborated the trends that emerged through cultural and physiological characterization. Streptomyces was the most abundant genus amplified with the Streptomyces-specific primers (StreptB/E, StreptB/F), whereas other actinobacterial strains were amplified by the universal primers U1 and U2. Further molecular diversity was investigated by the gradient PCR-DGGE as a fingerprinting tool that generated group-specific DGGE patterns. Based on the nucleotide homology and phylogenetic analysis, strains OK-1 and OK-2 were identified as Streptomyces somaliensis. At the same time, OK-3 and OK-7 were detected as Streptomyces sp., while OK-5, OK-6, OK-8, OK-9, and OK-10 belonged to Nocardiopsis alba. Further, the cluster analysis using the UPGMA method generated 3 clusters based on biochemical characterization, sugar utilization, and enzyme production. The dendrogram based on the DGGE band pattern created with Jaccard-distance revealed two major clades with 33.33% similarity. Further, the study of alpha diversity calculation using phenotypic characteristics discloses highly diverse sugar utilization abilities. Moreover, a stress value of 0.1236 was obtained based on the NMDS analysis of the plots using Bary-Curties dissimilarity. Overall, the distinct phenotypic, metabolic, and molecular profiling illustrated the diversity among marine actinobacteria.

Characterization of lactic acid bacteria isolated from street pickles of Dhaka, Bangladesh

Traditionally fermented pickles are a popular street food in Bangladesh famous for their unique flavors and health benefits. Pickles are often prepared by fermentation using lactic acid bacteria (LAB) that can act as probiotics. The study was aimed to isolate and characterize lactic acid bacteria from pickle samples collected from streets of Dhaka city, as well as assess the microbial quality of pickles for food safety.A total of 30 pickle samples of different kinds were collected from streets of Dhaka city. Isolation and identification were conducted using conventional cultural and biochemical tests, followed by molecular confirmation of identity. Antibiotic susceptibility of isolates was investigated against 7 antibiotics of different groups. Antimicrobial activity of LAB isolates was analyzed by well-diffusion assay and phenotypic enterocin activity assay. Physiological characterizations of LAB were performed to determine their tolerance to temperature, salt, pH, bile, carbohydrate fermentation pattern, proteolytic activity and biofilm formation.Fifty isolates were obtained from pickle samples, of which 18% was identified as LAB, including Enterococcus faecalis (6) and Enterococcus faecium (3). The rest included S. aureus (18), E. coli (11), Klebsiella spp. (5), Salmonella (3), Shigella (3) and Pseudomonas aeruginosa (1). Antibiotic resistance pattern revealed higher occurrence of resistance against azithromycin among the non-LAB isolates, but none of the LAB isolates were found to resist any of the antibiotics used. Antimicrobial activity of LAB isolates was not observed against the foodborne isolates. All LAB isolates fermented a wide range of carbohydrates and showed adequate tolerance to salt, pH, temperature and bile. Out of 9 isolates, 5 displayed proteolytic activity, and 6 were found as strong biofilm producer.These results suggest that although the LAB isolates from street pickles collected from Dhaka does not have antimicrobial activities, they still have potential to be used as probiotics. It also shows high occurrence of antibiotic resistant foodborne pathogens in pickles, indicating that consumption of such street food can be serious health hazard.

Development of a phenotyping protocol for combined drought and salinity stress at seedling stage in rice.

The case of combined drought and salinity stress is increasingly becoming a constraint to rice production, especially in coastal areas and river deltas where low rainfall not only reduces soil moisture levels but also reduces the flow of river water, resulting in intrusion of saline sea-water. A standardized screening method is needed in order to systematically evaluate rice cultivars under combined drought+salinity at the same time because sequential stress of salinity followed by drought or vice-versa is not similar to simultaneous stress effects. Therefore, we aimed to develop a screening protocol for combined drought+salinity stress applied to soil-grown plants at seedling stage. The study system used 30-L soil-filled boxes, which allowed a comparison of plant growth under control conditions, individual drought and salinity stress, as well as combined drought+salinity. A set of salinity tolerant and drought tolerant cultivars were tested, together with several popular but salinity and drought-susceptible varieties that are grown in regions prone to combined drought+salinity. A range of treatments were tested including different timings of the drought and salinity application, and different severities of stress, in order to determine the most effective that resulted in visible distinction among cultivars. The challenges related to determining a protocol with repeatable seedling stage stress treatment effects while achieving a uniform plant stand are described here. The optimized protocol simultaneously applied both stresses by planting into saline soil at 75% of field capacity which was then allowed to undergo progressive drydown. Meanwhile, physiological characterization revealed that chlorophyll fluorescence at seedling stage correlated well with grain yield when drought stress was applied to vegetative stage only. The drought+salinity protocol developed here can be used for screening rice breeding populations as part of a pipeline to develop new rice varieties with improved adaptation to combined stresses.

Genetic characterization and assessment of the biotechnological potential of strains belonging to the genus Arthrospira/Limnospira (Cyanophyceae) deposited in different culture collections

Culture collections of microalgae preserve viable material for conservation, research and biotechnology purposes; however, in the absence of routine authenticity checking, the strains deposited may become inexact references or inadequate starting material for a commercial development. The edible cyanobacteria Arthrospira/Limnospira is recognized as one of the most important industrially cultivated microalgae. Arthrospira/Limnospira strains have been frequently confused and incorrectly denoted as Spirulina, therefore, an accurate taxonomic identification of strains available in culture collections worldwide is imperative. In this study, eleven strains, originally designated as Arthrospira/Limnospira or Spirulina in three microalgae culture collections, were analyzed using their ITS sequence to verify their taxonomic classification. Phylogenetic analysis showed that there were misclassified strains in these collections: seven strains were grouped in one of the two genetic clusters previously described for Arthrospira, while remaining strains were grouped within Spirulina genus, forming two well-supported not previously described genetic clusters. Morphological characterization of Arthrospira strains showed typical characters of the genus; while their physiological and biochemical characterization showed intraspecific variability in growth attributes and pigments, proteins, lipids and fatty acids contents.

Cytobacillus spongiae sp. nov. isolated from sponge Diacarnus spinipoculum.

A novel Gram-stain-positive, aerobic and motile bacterium, designated strain CY-GT, was isolated from a sponge (Diacarnus spinipoculum) collected from the Red Sea. The strain grew at 13-43 °C (optimum 30 °C), pH 5.5-10.0 (optimum pH 9.0) and with 0-8.0 % (w/v) (0-1.37 M) NaCl (optimum 0 %). The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that CY-GT represents a member of the genus Cytobacillus, with the highest sequence identity to Cytobacillus oceanisediminis H2T (97.05 %), followed by Cytobacillus firmus IAM 12464T (96.76 %). The major cellular fatty acids (>5 % of the total) of CY-GT were C15 : 0iso, C16 : 0iso, C16 : 1ω7c alcohol, C16 : 0, C17 : 1iso ω10c and C17 : 0iso. The major polar lipids were glycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major respiratory quinone is menaquinone-7 (MK-7). The cell-wall peptidoglycan contains meso-diaminopimelic acid. The total genome size of CY-GT is 4 789 051 bp. The DNA G+C content is 38.83 mol%. The average nucleotide identity and DNA-DNA hybridization among CY-GT and type strains of other species of the genus Cytobacillus were 76.79-78.97 % and 20.10-24.90 %, respectively. On the basis of the results of phylogenetic analysis, physiological and biochemical characterization, strain CY-GT represents a novel species of the genus Cytobacillus, for which the name Cytobacillus spongiae sp. nov. is proposed. The type strain is CY-GT (=MCCC 1K06383T=KCTC 43348T).

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Survey of the yeast ecology of dehydrated grapes and strain selection for wine fermentation

In this study we investigated the yeast population present on partially dehydrated Nebbiolo grapes destined for ‘Sforzato di Valtellina’, with the aim to select indigenous starters suitable for the production of this wine. Yeasts were enumerated, isolated, and identified by molecular methods (5.8S-ITS-RFLP and D1/D2 domain sequencing). A genetic, physiological (ethanol and sulphur dioxide tolerance, potentially useful enzymatic activities, hydrogen sulphide production, adhesive properties, and killer activity) and oenological (laboratory pure micro-fermentations) characterization was also carried out. Based on relevant physiological features, seven non-Saccharomyces strains were chosen for laboratory-scale fermentations, either in pure or in mixed-culture (simultaneous and sequential inoculum) with a commercial Saccharomyces cerevisiae strain. Finally, the best couples and inoculation strategy were further tested in mixed fermentations in winery. In both laboratory and winery, microbiological and chemical analyses were conducted during fermentation. The most abundant species on grapes were Hanseniaspora uvarum (27.4 % of the isolates), followed by Metschnikowia spp. (21.0 %) and Starmerella bacillaris (12.9 %). Technological characterization highlighted several inter- and intra-species differences. The best oenological aptitude was highlighted for species Starm. bacillaris, Metschnikowia spp., Pichia kluyveri and Zygosaccharomyces bailli. The best fermentation performances in laboratory-scale fermentations were found for Starm. bacillaris and P. kluyveri, due to their ability to reduce ethanol (-0.34 % v/v) and enhance glycerol production (+0.46 g/L). This behavior was further confirmed in winery. Results of this study contribute to the knowledge of yeast communities associated with a specific environment, like those of Valtellina wine region.

Development of a Tetracycline-Inducible System for Conditional Gene Expression in Lactococcus lactis and Streptococcus thermophilus.

Inducible gene expression systems are invaluable tools for the functional characterization of genes and in the construction of protein overexpression hosts. Controllable expression is especially important for the study of essential and toxic genes or genes where the level of expression tightly influences their cellular effect. Here, we implemented the well-characterized tetracycline-inducible expression system in two industrially important lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus. Using a fluorescent reporter gene, we show that optimization of the repression level is necessary for efficient induction using anhydrotetracycline in both organisms. Random mutagenesis in the ribosome binding site of the tetracycline repressor TetR in Lactococcus lactis indicated that altering the expression levels of TetR was necessary for efficient inducible expression of the reporter gene. Through this approach, we achieved plasmid-based, inducer-responsive, and tight gene expression in Lactococcus lactis. We then verified the functionality of the optimized inducible expression system in Streptococcus thermophilus following its chromosomal integration using a markerless mutagenesis approach and a novel DNA fragment assembly tool presented herein. This inducible expression system holds several advantages over other described systems in lactic acid bacteria, although more efficient techniques for genetic engineering are still needed to realize these advantages in industrially relevant species, such as S. thermophilus. Our work expands the molecular toolbox of these bacteria, which can accelerate future physiological studies. IMPORTANCE Lactococcus lactis and Streptococcus thermophilus are two industrially important lactic acid bacteria globally used in dairy fermentations and, therefore, are of considerable commercial interest to the food industry. Moreover, due to their general history of safe usage, these microorganisms are increasingly being explored as hosts for the production of heterologous proteins and various chemicals. Development of molecular tools in the form of inducible expression systems and mutagenesis techniques facilitates their in-depth physiological characterization as well as their exploitation in biotechnological applications.

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

Our understanding of in situ microbial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for underrepresented taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of slower-growing Negativicutes and Methanobacteria from stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.

Physiological characterization of instrumental learning in the spinal cord using the Paw Withdrawal Learning paradigm

Following spinal cord injury (SCI), sensory and motor functions are severely disrupted yet the spinal circuitry below the injury site continues to maintain active and functional neuronal properties (Edgerton, 2004). The spinal cord is endogenously capable of several forms of adaptive plasticity, including functional re-training with exercise, instrumental, and Pavlovian learning. The paw withdrawal learning (PaWL) paradigm represents a simple spinal instrumental learning model (Jindrich 2019). Briefly, in mice whose spinal cords are completely transected (ST) at mid-thoracic (T7-T9) level, the tibialis anterior (TA) muscle is given a sub maximal threshold shock to a hind leg when the leg is extended below a 1mm imposed threshold from a baseline starting position. The contingent group mice learn to maintain the shocked leg, in a flexed position while the experimentally coupled noncontingent group of mice do not acquire the flexed position. To demonstrate the acquisition of the flexed paw hold is mediated through muscle-specific proprioceptive inputs and a time dependent modification of the spinal interneuronal network associated with the TA motor pool, we measured the TA muscle activity using acute EMG during PaWL in both Contingent stim and non-contingent stim groups. To demonstrate that TA specific sensory afferents are critical in PaWL, we blocked the proprioceptive muscle and the cutaneous afferents using Lidocaine. During PaWL, the TA muscle showed significant EMG activity when compared to medial gastrocnemius and vastus lateralis muscles. We detected greater TA muscle activity in the Contingent stim compared to the non-contingent groups. The significant increase in the EMG amplitude in the TA muscle and time-frequency and power relationships in the contingent group, but not the non-contingent group, suggests that the successful learning requires a temporal engagement of the spinal sensorimotor network. CSULA Mini-Grant 2020 and Joseph/Gomez-Pinilla R01NS056413 (Diversity Supplement) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.