Low-nutrient Media Research Articles

Selection and application of methanol-utilizing bacteria from tomato leaves for biocontrol of gray mold

Gray mold, caused by Botrytis cinerea, is a significant threat to tomato production. Traditional chemical control methods have become increasingly ineffective because of the development of resistance. This study aimed to isolate methanol-utilizing bacteria from tomato leaves and evaluate their biocontrol potential against gray mold. To obtain bacterial suspensions, tomato leaf samples were collected and washed. We analyzed the microbial communities of these samples using 16S rRNA amplicon sequencing and identified several methylotrophic strains. Among these, 405 isolated strains were cultivated on a solid low-nutrient inorganic salt medium containing methanol, and 7 strains exhibiting considerable antifungal activity against B. cinerea were identified. Greenhouse tests revealed that two strains—SY163 and SY183—significantly reduced the severity of gray mold on tomato leaves. Disease index scores and the area under the disease progress curve values confirmed the efficacy of these strains as biocontrol agents. Statistical analysis indicated the effectiveness of pre- and co-application of these strains with B. cinerea. Phylogenetic analysis identified Serratia rubidaea as the inhibitory strain. The biocontrol activity is likely mediated through the production of antifungal compounds and suppression of B. cinerea sporulation. This study provides the basis for developing a technology of gray mold suppression by controlling the abundance of S. rubidaea in plant microbial communities.

Differential Strategies of Ectomycorrhizal Development between Suillus luteus and Pinus massoniana in Response to Nutrient Changes.

Ectomycorrhizal fungi employ different strategies for mycelial growth and host colonization under varying nutrient conditions. However, key genes associated with mycorrhizal interaction should be influenced solely by the inoculation treatment and not by nutrient variations. To utilize subtle nutrient differences and rapidly screen for key genes related to the interaction between Suillus luteus and Pinus massoniana, we performed an inoculation experiment using culture bottles containing high- and low-nutrient media. Interestingly, S. luteus LS88 promoted the growth of P. massoniana seedlings without mature ectomycorrhiza, and the impact of LS88 inoculation on P. massoniana roots was greater than that of nutrient changes. In this study, the resequenced genome of the LS88 strain was utilized for transcriptome analysis of the strain. The analysis indicated that a unique gene encoding glutathione S-transferase (GST) in LS88 is likely involved in colonizing P. massoniana roots. In this study, the GST gene expression was independent of nutrient levels. It was probably induced by P. massoniana and could be used as a marker for S. luteus colonization degree.

A diffusion-based integrative approach for culturing previously uncultured bacteria from marine sediments

AbstractTraditional cultivation methods with defined growth media can only isolate and cultivate a small number of microbes. However, much higher microbial diversity has been detected by cultivation-independent tools from a range of natural ecosystems. These represent a large unexplored pool of potentially novel taxa. In this study, a diffusion-based integrative cultivation approach (DICA) was developed to efficiently isolate novel taxonomic candidates from marine sediment. DICA combined a newly designed diffusion-based apparatus called a “microbial aquarium” with modified low-nutrient media. To determine the efficiency of DICA, cultivation results were compared with traditional cultivation approach (TCA). Both cultivation approaches resulted in the isolation of numerous representatives from the phyla Pseudomonadota, Actinomycetota, Bacteroidota, and Bacillota. However, the newly developed DICA also led to the successful cultivation of species from rarely cultivated phyla such as Verrucomicrobiota and Balneolota. Based on 16S rRNA analyses, the application of DICA resulted in the successful cultivation of 115 previously uncultured taxa out of a total of 196 isolates. Among these, 39 were identified at the genus level and 4 at the family level, showcasing a novelty ratio of 58%. Conversely, the TCA cultivated 12% (20/165) of novel isolates, with all at species level only. The isolated microbial diversity showed that species recovered by DICA belong to 12 different classes, twice the number produced by TCA. Overall, these results demonstrate that the newly designed DICA produces a high recovery of diverse and previously uncultured bacteria.

An untargeted cultivation approach revealed Pseudogemmatithrix spongiicola gen. nov., sp. nov., and sheds light on the gemmatimonadotal mode of cell division: binary fission

Members of the phylum Gemmatimonadota can account for up to 10% of the phylogenetic diversity in bacterial communities. However, a detailed investigation of their cell biology and ecological roles is restricted by currently only six characterized species. By combining low-nutrient media, empirically determined inoculation volumes and long incubation times in a 96-well plate cultivation platform, we isolated two strains from a limnic sponge that belong to this under-studied phylum. The characterization suggests that the two closely related strains constitute a novel species of a novel genus, for which we introduce the name Pseudogemmatithrix spongiicola. The here demonstrated isolation of novel members from an under-studied bacterial phylum substantiates that the cultivation platform can provide access to axenic bacterial cultures from various environmental samples. Similar to previously described members of the phylum, the novel isolates form spherical appendages at the cell poles that were believed to be daughter cells resulting from asymmetric cell division by budding. However, time-lapse microscopy experiments and quantitative image analysis showed that the spherical appendages never grew or divided. Although the role of these spherical cells remains enigmatic, our data suggests that cells of the phylum Gemmatimonadota divide via FtsZ-based binary fission with different division plane localization patterns than in other bacterial phyla.

NaCl-induced modulation of species distribution in a mixed P. aeruginosa / S. aureus /B.cepacia biofilm

Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections.In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations.Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix.Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Homeostasis of cellular amino acids in Acanthamoeba castellanii exposed to different media under amoeba-bacteria coculture conditions

BackgroundAcanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli.MethodA. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii.ResultsUnder short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine.DiscussionUnder short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked.ConclusionWhen A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

First report of Fusarium fujikuroi causes leaf spot in Polygonatum odoratum (Mill.) Druce in China.

Polygonatum odoratum (Mill.) Druce, a member of Liliaceae, is one of the traditional Chinese herbal plants mainly used in Jilin, Hubei, Guangxi, Zhejiang, Liaoning, Hunan and Guangdong provinces. Leaf spot disease of P. odoratum was continuously observed in the Planting Demonstration Garden in Changsha (28 °48 N; 113° 34E), Hunan Province of China, in May 2021 and May 2022. The symptoms initially appeared as tiny reddish-brown spots and continued to expand, resulting in round, oval, or irregular tan lesions with necrotic, film-shaped, or perforated central tissues. Leaf spot disease affects approximately 60-70% of plants. For pathogen isolation, symptomatic leaf samples were collected and disinfected with 70% ethanol for 30 s and 3% sodium hypochlorite for 2 min, followed by rinsing with sterile distilled water. Subsequently, small pieces (3 × 3 mm) of diseased tissues were placed on potato dextrose agar (PDA) and incubated in the dark at 25 °C for 24 h to 36 h. The emerging fungal hyphal tips were transferred to PDA and purified by the single-spore method (Yu, et al., 202). In total, 50 disease spots were isolated, and 10 cultures with the same appearance were obtained. Two strains coded as hnxryzy and hnxryzy01 were randomly selected for identification. After 6 days of culture in PDA, dense pink colonies were observed with a mean radial growth rate of 7.5 mm/day. Strains cultured 6 days on synthetic low nutrient medium, microconidia were oval or ovate (7.5-9.67 µm × 2.49-3.57 µm(n=50)), and macroconidia were sickle-shaped and slightly curved, gradually tapering at both ends, with 2-5 pseudoseptate (10.01-22.14 µm × 2.07-4.22 µm (n=50)). These morphological characteristics were consistent with the description of Fusarium fujikuroi (Fang, et al., 2021). Furthermore, primers ITS1/ITS4, EF728F/EF986R, Bt2a/Bt2b, RPB1-F5/RPB1-R8 and fRPB2-5F2/fRPB2-7cR (Li, et al., 2013, Xie, et al., 2022) were used to amplify the partial region of the internal transcribed spacer (ITS) , the translation elongation factor EF-1α,β-tubulin,polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes from strains hnxryzy and hnxryzy01, respectively. Amplicons were sequenced by Tsingke Biotechnology Co., Ltd. The expected sequences of ITS, EF-1α, β-tubulin, RPB1 and RPB2 of hnxryzy and hnxryzy01 were obtained. The sequence alignment of hnxryzj and hnxryzj01 with the Fusarium ID databased and NCBI shows the following results: The sequences of ITS region, EF-1α, β-tubulin , RPB1 and RPB2 of strain hnxryzy (GenBank accession nos. ON797440, ON820553, ON820554, OP413443, and OP413445, respectively) and strain hnxryzy01 (GenBank accession nos. ON965284, ON968721, ON968722, OP413444, and OP413446, respectively) were 99% to 100% identical to those of F. fujikuroi (GenBank accession numbers CP023090, KC874784, MN490089, MN193916, and MN193888, respectively). Then a phylogenetic tree based on EF-1α, RPB1, and RPB2 sequences was constructed (Torres-Cruz, et al., 2022). The strains hnxryzy and hnxryzy01 were more closely related to F. fujikuroi ( NRRL13566 GenBank accession nos. AF160279, JX171456, and JX171570, respectively), with bootstrap values of 99%. Two sets (5 plants in each set) of potted plants were used in pathogenicity assays. Wounded leaves were sprayed with conidial suspensions (100 µL, 1×107 spores/mL) and sterile water as control. Inoculated plants were covered with plastic bags for 24 h, and maintained at 25 ° C in 12/12 h light/dark conditions in the greenhouse (Yu, et al., 2022). Pathogenicity assays were repeated thrice. Dark brown spots identical to those seen in the field were observed 14 days after inoculation, while the control leaves did not exhibit any symptoms. In this study, the pathogen F. fujikuroi was successfully reisolated from the leaves of inoculated samples showing symptoms, thereby verifying Koch's postulate. To our knowledge, this is the first report of F. fujikuroi inducing leaf spot on P. odoratum in China. Since F. fujikuroi is a common pathogenic fungus that infects different plant species(Qiu, et al., 2020), more attention should be paid to its prevalence in P. odoratum and the potential risk of outbreak in other provinces of China.

Isolation and Characterization of Stress-Tolerant Priestia Species from Cowpea Rhizosphere Under Drought and Nutrient Deficit Conditions

This study aimed to isolate stress-tolerant phytobeneficial bacteria as bio-inoculants for cowpea's sustainable growth under drought and nutrient deficiency conditions. However, the application successful of phytobeneficial bacteria is subject to effective in vitro screening under different physiological conditions. We isolated several Priestia species from cowpea rhizosphere that tolerates polyethylene glycol (PEG6000)-induced drought and nutrient deficiency. Of them, C8 (Priestia filamentosa; basonym: Bacillus filamentosus), followed by C29 (Priestia aryabhattai; basonym: Bacillus aryabhattai), tolerated up to 20% PEG in a low-nutrient medium. In the presence of PEG, Priestia filamentosa and Bacillus aryabhattai exhibited optimal growth in different temperatures and pH but failed to survive at extreme temperatures of 45°C and pH 11. Priestia filamentosa preferred L-proline and L-glutamate, while L-tryptophan and L-tyrosine were the least utilized. Interestingly, Priestia filamentosa and Bacillus aryabhattai used more complex nitrogen sources, peptone, and yeast extract, than inorganic nitrogen for growth. Most importantly, under drought and nutrient deficiency, Priestia filamentosa exhibited multiple plant growth-promoting traits and more amylase and protease production than C29. Our results indicate that Priestia filamentosa is a potential bacterium to enhance the growth of cowpea plants under stressful conditions.

Mycelium-Composite Materials-A Promising Alternative to Plastics?

Plastic waste inefficiently recycled poses a major environmental concern attracting attention from both civil society and decision makers. Counteracting the phenomenon is an important challenge today. New possibilities are being explored to find alternatives to plastics, and one of them refers to mycelium-composite materials (MCM). Our study aimed at investigating the possibility of using wood and litter inhabiting basidiomycetes, an underexplored group of fungi that grow fast and create strong mycelial mats, to produce biodegradable materials with valuable properties, using cheap by-products as a substrate for growth. Seventy-five strains have been tested for their ability to grow on low-nutrient media and to form compact mycelial mats. Eight strains were selected further for evaluation on several raw substrates for producing in vitro myco-composites. The physico-mechanical properties of these materials, such as firmness, elasticity and impermeability, were analyzed. Abortiporus biennis RECOSOL73 was selected to obtain, at the laboratory scale, a real biodegradable product. Our results suggest that the strain used is a promising candidate with real possibilities for scalability. Finally, corroborating our results with scientific available data, discussions are being made over the feasibility of such technology, cost-effectiveness, scalability, availability of raw materials and, not least, where future studies should be directed to.

Induction of Viable but Non-Culturable State in Clinically Relevant Staphylococci and Their Detection with Bacteriophage K

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

Methylmercury formation in biofilms of Geobacter sulfurreducens.

Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens. Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy. Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10-14 L ∙ cell-1 ∙ h-1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms. These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

A Reproducible and Tunable Synthetic Soil Microbial Community Provides New Insights into Microbial Ecology.

Microbial soil communities form commensal relationships with plants to promote the growth of both parties. The optimization of plant-microbe interactions to advance sustainable agriculture is an important field in agricultural research. However, investigation in this field is hindered by a lack of model microbial community systems and efficient approaches for building these communities. Two key challenges in developing standardized model communities are maintaining community diversity over time and storing/resuscitating these communities after cryopreservation, especially considering the different growth rates of organisms. Here, a model synthetic community (SynCom) of 16 soil microorganisms commonly found in the rhizosphere of diverse plant species, isolated from soil surrounding a single switchgrass plant, has been developed and optimized for in vitro experiments. The model soil community grows reproducibly between replicates and experiments, with a high community α-diversity being achieved through growth in low-nutrient media and through the adjustment of the starting composition ratios for the growth of individual organisms. The community can additionally be cryopreserved with glycerol, allowing for easy replication and dissemination of this in vitro system. Furthermore, the SynCom also grows reproducibly in fabricated ecosystem devices (EcoFABs), demonstrating the application of this community to an existing in vitro plant-microbe system. EcoFABs allow reproducible research in model plant systems, offering the precise control of environmental conditions and the easy measurement of plant microbe metrics. Our results demonstrate the generation of a stable and diverse microbial SynCom for the rhizosphere that can be used with EcoFAB devices and can be shared between research groups for maximum reproducibility. IMPORTANCE Microbes associate with plants in distinct soil communities to the benefit of both the soil microbes and the plants. Interactions between plants and these microbes can improve plant growth and health and are therefore a field of study in sustainable agricultural research. In this study, a model community of 16 soil bacteria has been developed to further the reproducible study of plant-soil microbe interactions. The preservation of the microbial community has been optimized for dissemination to other research settings. Overall, this work will advance soil microbe research through the optimization of a robust, reproducible model community.

Identification, cultivation condition of Parasevetibacter sp C101 and antimicrobial activity of the bacterial culture extract

During the recent decade, uncultured bacteria have been special interest as potential candidates for discovering novel antibacterial compounds. Two strains C101 and C102 were negative Gram bacteria, only growing on low nutrient media as R2A/3, NB/3, LB/10 and R4/10 compared to the usual. On R2A/3 medium, colonies of the isolates were round, convex, lemon yellow color with the size of 1-1.5 mm after six days of incubation at 28oC. Cells were 0.2-0.3 × 0.8-1.3 µm. The strains C101 and C102 were able to grow at temperature ranging 15-37oC (optimum at 25-28oC), pH 5-8 (optimum in pH 6-7). The sequences of 16S rRNA genes from strain C101 (MT756087) and C102 (MT756088) shared 100% identity. Analysis of full-length 16S rRNA gene sequence of strain C101 via using NCBI Blast, EzTaxon Database revealed the highest similarity of 99.18-100% to uncultured clones, and 97.86% to type species as Parasegetibacter terrae SGM2-10T. Genetic sequence analysis data showed that strain C101 should be considered a novel candidate species of the genus Parasegetibacter. Antibacterial compound was extracted from culture of strain C101 in R4/10 medium for ten days of shaking incubator at 28oC and exhibited susceptible activity to inhibit Bacillus anthracis KEMB 211-146 at a concentration of 2 µg/L and Staphylococcus aureus ATCC 6538 at 4 µg/L; intermediate inhibiting Bacillus subtilis KEMB 51201-001 at 8 µg/L, Staphylococcus epidermidis ATCC 14990 at 8 µg/L, and S. aureus CCARM 3155 at 16 µg/L; inhibition of S. aureus CCARM 3095 at 64 µg/L, S. aureus CCARM 3192 at 32 µg/L, and S. epidermidis CCARM 3710 at 64 µg/L.

Nutrient driven transcriptional changes during phage infection in an aquatic Gammaproteobacterium.

SummaryPhages modulate bacterial metabolism during infection by regulating gene expression, which influences aquatic nutrient cycling. However, the effects of shifting nutrient regimes are less understood. Here, we analyzed transcriptomes of an ecologically relevant Gammaproteobacterium and its lytic phage in high (HNM) and low (LNM) nutrient medium. Despite different infection characteristics, including reduced burst size and longer latent period in LNM, the phage had a fixed expression profile. Bacterial transcription was instead different depending on nutrient regime, with HNM bacteria focusing on growth while LNM bacteria focused on motility and membrane transport. Additionally, phage infection had a larger effect on bacterial gene expression in LNM compared to HNM, e.g. suppressing increased iron uptake and altering expression of phosphorus uptake genes. Overall, phage infection influenced host metabolism more in LNM, which was more similar to natural conditions, emphasizing the importance of considering natural conditions to understand phage and host ecology.

High Frequency Direct Organogenesis, Genetic Homogeneity, Chemical Characterization and Leaf Ultra-Structural Study of Regenerants in Diplocyclos palmatus (L.) C. Jeffrey

Diplocyclos palmatus (L.) C. Jeffrey, commonly referred to as “Shivalingi” or “Lollipop climber” is a valuable medicinal plant with a climbing growth habit used in traditional medicine. It is reputed to have antiarthritic, anti-diabetic properties and to be useful in various skin and reproductive problems. Overexploitation of wild plants and low seed germination have resulted in the decline of the species in the wild. Thus, the present investigation was aimed to establish an effective in vitro propagation procedure for its large-scale production and conservation. Nodal explants, obtained from an established mother plant were grown on MS basal medium augmented with various cytokinins, alone or in combination with auxins, to study the morphogenic response. A maximum of 8.3 shoots/explants with an average shoot length of 7.2 cm were produced after six weeks on MS containing benzylaminopurine 5.0 µM + 1-naphthaleneacetic acid 2.0 µM. After 4 weeks of transfer, microshoots rooted well on a low nutrient medium of ½ MS + 1.0 µM indole-3-butyric acid, with a maximum of 11.0 roots/microshoot and an average root length of 7.4 cm. With an 80% survival rate, the regenerated plantlets were effectively acclimatized to natural conditions. DNA-based molecular markers were used to investigate the genetic uniformity. Scanning Electron Microscopic examination of leaves indicated the adaptation of the plantlets to natural, as evidenced by the formation of normal stomata. Gas chromatography-mass spectrometry analyses of mother and micropropagated plants were performed to identify essential secondary metabolites. The results obtained show that the in vitro propagation system can be adopted for preservation, large-scale production and secondary metabolites’ production in D. palmatus.

The influence of the crowding assumptions in biofilm simulations.

Microorganisms are frequently organized into crowded structures that affect the nutrients diffusion. This reduction in metabolite diffusion could modify the microbial dynamics, meaning that computational methods for studying microbial systems need accurate ways to model the crowding conditions. We previously developed a computational framework, termed CROMICS, that incorporates the effect of the (time-dependent) crowding conditions on the spatio-temporal modeling of microbial communities, and we used it to demonstrate the crowding influence on the community dynamics. To further identify scenarios where crowding should be considered in microbial modeling, we herein applied and extended CROMICS to simulate several environmental conditions that could potentially boost or dampen the crowding influence in biofilms. We explore whether the nutrient supply (rich- or low-nutrient media), the cell-packing configuration (square or hexagonal spherical cell arrangement), or the cell growing conditions (planktonic state or biofilm) modify the crowding influence on the growth of Escherichia coli. Our results indicate that the growth rate, the abundance and appearance time of different cell phenotypes as well as the amount of by-products secreted to the medium are sensitive to some extent to the local crowding conditions in all scenarios tested, except in rich-nutrient media. Crowding conditions enhance the formation of nutrient gradient in biofilms, but its effect is only appreciated when cell metabolism is controlled by the nutrient limitation. Thus, as soon as biomass (and/or any other extracellular macromolecule) accumulates in a region, and cells occupy more than 14% of the volume fraction, the crowding effect must not be underestimated, as the microbial dynamics start to deviate from the ideal/expected behaviour that assumes volumeless cells or when a homogeneous (reduced) diffusion is applied in the simulation. The modeling and simulation of the interplay between the species diversity (cell shape and metabolism) and the environmental conditions (nutrient quality, crowding conditions) can help to design effective strategies for the optimization and control of microbial systems.

Use of Yeast Mannoproteins by Oenococcus oeni during Malolactic Fermentation under Different Oenological Conditions.

Oenococcus oeni is the main agent of malolactic fermentation in wine. This fermentation takes place after alcoholic fermentation, in a low nutrient medium where ethanol and other inhibitor compounds are present. In addition, some yeast-derived compounds such as mannoproteins can be stimulatory for O. oeni. The mannoprotein concentration in wine depends on the fermenting yeasts, and non-Saccharomyces in particular can increase it. As a result of the hydrolytic activity of O. oeni, these macromolecules can be degraded, and the released mannose can be taken up and used as an energy source by the bacterium. Here we look at mannoprotein consumption and the expression of four O. oeni genes related to mannose uptake (manA, manB, ptsI, and ptsH) in a wine-like medium supplemented with mannoproteins and in natural wines fermented with different yeasts. We observe a general gene upregulation in response to wine-like conditions and different consumption patterns in the studied media. O. oeni was able to consume mannoproteins in all the wines. This consumption was notably higher in natural wines, especially in T. delbrueckii and S. cerevisiae 3D wines, which presented the highest mannoprotein levels. Regardless of the general upregulation, it seems that mannoprotein degradation is more closely related to the fermenting medium.

Diversity and Distribution of Uncultured and Cultured Gaiellales and Rubrobacterales in South China Sea Sediments.

Actinobacteria are ubiquitous in marine ecosystems, and they are regarded as an important, underexplored, potential pharmaceutical resource. The orders Gaiellales and Rubrobacterales are deep taxonomic lineages of the phylum Actinobacteria, both are represented by a single genus and contain only a few species. Although they have been detected frequently by high-throughput sequencing, their functions and characteristics in marine habitats remain unknown due to the lack of indigenous phenotypes. Here, we investigated the status of the orders in South China Sea (SCS) sediments using culture-independent and culture-dependent methods. Gaiellales is the second-most abundant order of Actinobacteria and was widely distributed in SCS sediments at water depths of 42–4,280 m, and four novel marine representatives in this group were successfully cultured. Rubrobacterales was present at low abundance in energy-limited marine habitats. An isolation strategy for Rubrobacterales from marine samples was proposed, and a total of 138 mesophilic Rubrobacterales strains were isolated under conditions of light and culture time combined with high-salinity or low-nutrient media. Marine representatives recovered in this study formed branches with a complex evolutionary history in the phylogenetic tree. Overall, the data indicate that both Gaiellales and Rubrobacterales can adapt to and survive in extreme deep-sea environments. This study lays the groundwork for further analysis of the distribution and diversity of the orders Gaiellales and Rubrobacterales in the ocean and provides a specific culture strategy for each group. The results open a window for further research on the ecological roles of the two orders in marine ecosystems.

MIR210HG regulates glycolysis, cell proliferation, and metastasis of pancreatic cancer cells through miR-125b-5p/HK2/PKM2 axis

ABSTRACT Pancreatic cancer has the worst prognosis of all common cancers. Pancreatic cancer cells have a metabolic advantage due to their swiftly adaptive responses to hypoxic and low-nutrient medium. This advantage contributes to the aggressivity of pancreatic cancer. In this study, lncRNA MIR210HG was abnormally upregulated within pancreatic cancer. It acted as a key oncogenic regulator of pancreatic cancer aggressiveness and glycolysis. Knockdown of MIR210HG significantly inhibited the aggressive phenotype of pancreatic cancer cells and inhibited the growth of xenograft tumours. More importantly, MIR210HG knockdown inhibited pancreatic cancer cell glycolysis via regulating the glycolysis-related hexokinase 2 (HK2) and Pyruvate kinase muscle isozyme M2 (PKM2) expression. Compared with the MIR210HG knockdown group, miR-125b-5p inhibition promoted the aggressive phenotypes and glycolysis of pancreatic cancer cells. Furthermore, the effects of MIR210HG knockdown on HK2 and PKM2 expression, pancreatic cancer cell aggressive phenotypes, and glycolysis were significantly reversed by miR-125b-5p inhibition. In tissue samples, MIR210HG expression was negatively correlated with miR-125b-5p levels and positively correlated with HK2 and PKM2 expression. miR-125b-5p expression was negatively correlated with HK2 and PKM2 expression. In conclusion, MIR210HG affected the phenotypes of pancreatic cancer cells, including proliferation, invasion, migration, and glycolysis, via modulating the miR-125b-5p/HK2/PKM2 axis.

Growth of Flavobacterium columnare Genomovars in the Presence or Lack of Supplemental Cations.

Flavobacterium columnare is a problematic pathogen for the aquaculture industry where isolates are classified by genomovars. Suspended growth in a low nutrient media, like tryptone yeast extract salts, is a common method used for laboratory study. The presence of calcium and magnesium is the factor contributing to growth, virulence, and biofilm formation for F. columnare. Exponential growth occurs within 24h for F. columnare when grown in complete tryptone yeast extract salts medium at 30ºC. Withholding CaCl2 and MgSO4 components from a complete TYES formulation reduced or completely inhibited growth of genomovar I isolates but not the growth of genomovar II, IIB, or III isolates. Only 3 of 20 genomovar I isolates, MS-FC-4, FC-CSF-53, and 023-08-3, could achieve O.D. 540 readings ≥ 0.3 but only after 48-h incubation in cation-restricted TYES. Independently adding CaCl2 or MgSO4 to tryptone and yeast extracts did not result in a genomovar-specific growth phenotype, but generally demonstrated increased clumping with individual isolates presenting abnormal growth. Clumping formed filamentous strings that migrated to the top of the culture tube when isolates were grown in TYE+CaCl2. Several of the F. columnare isolates from all the genomovars exhibited delayed growth when a single cation source was provided. This study demonstrates phenotypic differences between and within genomovars of a single bacterial species when grown under different TYES media conditions.