Acidic Growth Conditions Research Articles

Evolution-aided improvement of the acid tolerance of Levilactobacillus brevis and its application in sourdough fermentation

Levilactobacillus brevis is crucial in food fermentation, particularly in sourdough production. However, the cultivation of L. brevis faces a challenge with accumulation of lactic acid, a major inhibitor. We aimed to increase the acid tolerance of L. brevis, an industrial strain for sourdough fermentation. We used the adaptive laboratory evolution (ALE) to obtain lactic acid tolerant strains. The evolved strain’s fermentation and metabolite profiles, alongside sensory evaluation, were compared with the parental strain by using various analytical techniques. The ALE approach increased lactic acid tolerance in the evolved strain showing an increased growth rate by 1.1 and 1.9 times higher than the parental strain at pH 4.1 and 6.5, respectively. Comprehensive analyses demonstrated its potential application in sourdough fermentation, promising reduced downstream costs. The evolved strain, free from genetically modified organisms concerns, has great potential for industrial use by exhibiting enhanced growth in acidic conditions without affecting consumers’ bread preferences.

Targeting Bacterial Nanocellulose Properties through Tailored Downstream Techniques.

Bacterial nanocellulose (BNC) is a biodegradable polysaccharide with unique properties that make it an attractive material for various industrial applications. This study focuses on the strain Komagataeibacter medellinensis ID13488, a strain with the ability to produce high yields of BNC under acidic growth conditions and a promising candidate to use for industrial production of BNC. We conducted a comprehensive investigation into the effects of downstream treatments on the structural and mechanical characteristics of BNC. When compared to alkaline-treated BNC, autoclave-treated BNC exhibited around 78% superior flexibility in average, while it displayed nearly 40% lower stiffness on average. An SEM analysis revealed distinct surface characteristics, indicating differences in cellulose chain compaction. FTIR spectra demonstrated increased hydrogen bonding with prolonged interaction time with alkaline solutions. A thermal analysis showed enhanced thermal stability in alkaline-treated BNC, withstanding temperatures of nearly 300 °C before commencing degradation, compared to autoclaved BNC which starts degradation around 200 °C. These findings provide valuable insights for tailoring BNC properties for specific applications, particularly in industries requiring high purity and specific mechanical characteristics.

Archaea membranes in response to extreme acidic environments

Bipolar tetraether lipids (BTL), such as glycerol dialkyl calditol tetraether (GDNT) and glycerol dialkyl glycerol tetraether (GDGT), are the dominating lipid species in thermoacidophiles that inhabit at pH ≤ 4 and temperatures ≥65°C. BTL containing archaea membranes respond to environmental pH changes by varying the number of cyclopentane rings in the isoprenoids, the amount of GDNT relative to GDGT, the ratio of tetraethers to diethers, and the level of glycosylation in polar headgroups. These structural and compositional adjustments can alter the hydrogen bond networks in the membrane polar headgroup regions and the packing tightness and rigidity in the membrane hydrophobic core. It is likely that these changes in non-covalent interactions among archaea lipids are made to retain low membrane volume fluctuations and their low sensitivity to temperature, as illustrated in the case of liposomes made of the polar lipid fraction E (PLFE) of Sulfolobus acidocaldarius. As such, a low passive proton permeability and a near neutral intracellular pH can be maintained, and, as a result, optimal activities of soluble and membrane-bound proteins in thermoacidophiles can be retained in acidic growth conditions at elevated growth temperatures.

Acidic growth conditions stabilize the ribosomal RNA gene cluster and extend lifespan through noncoding transcription repression.

Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, jam, and liqueur. Blackcurrant extract is known to relieve cells from DNA damage caused by hydrogen peroxide (H2 O2 ), methyl methane sulfonate (MMS), and ultraviolet (UV) radiation. We found that blackcurrant extract (BCE) stabilizes the ribosomal RNA gene cluster (rDNA), one of the most unstable regions in the genome, through repression of noncoding transcription in the intergenic spacer (IGS) which extended the lifespan in budding yeast. Reduced formation of extrachromosomal circles (ERCs) after exposure to fractionated BCE suggested that acidity of the growth medium impacted rDNA stability. Indeed, alteration of the acidity of the growth medium to pH ~4.5 by adding HCl increased rDNA stability and extended the lifespan. We identified RPD3 as the gene responsible for this change, which was mediated by the RPD3L histone deacetylase complex. In mammals, as inflammation sites in a tissue are acidic, DNA maintenance may be similarly regulated to prevent genome instability from causing cancer.

Marker-assisted selection of one-kbp insertion at the 5’ UTR region of HvAACT1 gene improves plant performance under acidic soil growth conditions in barley (Hordeum vulgare)

Marker-assisted selection of one-kbp insertion at the 5’ UTR region of HvAACT1 gene improves plant performance under acidic soil growth conditions in barley (Hordeum vulgare)

Algae Cultivation as Measure for the Sanitation of Organic Waste—A Case Study Based on the Alga Galdieria sulphuraria Grown on Food Waste Hydrolysate in a Continuous Flow Culture

Due to its growth under harsh acidic conditions, the microalga Galdieria sulphuraria may offer the opportunity to combine sanitation and the utilization of organic waste streams. To further deepen the knowledge of alternative waste treatment strategies that allow for holistic utilization, the control and removal of microbial contaminants via non-sterile heterotrophic G. sulphuraria on food waste hydrolysate were investigated in a continuous flow bioreactor culture. Furthermore, a substrate reservoir and harvested biomass were stored under non-sterile conditions over a period of 12 days. Despite the non-sterile conditions, the microbial load of the biomass could be kept under control. Neither the pathogen Salmonella sp. nor the coliform bacteria Escherichia coli could be found. Only nine counts per g of biomass were found for species belonging to Enterococcus spp., Enterobacteriacae, and moulds. Aerobic spore formers were counted with 2700 counts per g of biomass. Most of the aerobic mesophilic counts were formed by yeasts (1.5 × 106 vs. 1.3 × 106 counts per g biomass). The results revealed that, when using acidic growth conditions, contamination will not take over the culture; thus, the sterilization of waste materials can be skipped. It is assumed that such an approach can result in efficient processes for future waste-based bioeconomy strategies.

Acidic Growth Conditions Promote Epithelial-to-Mesenchymal Transition to Select More Aggressive PDAC Cell Phenotypes In Vitro.

Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an acidic microenvironment, which contributes to therapeutic failure. So far there is a lack of knowledge with respect to the role of the acidic microenvironment in the invasive process. This work aimed to study the phenotypic and genetic response of PDAC cells to acidic stress along the different stages of selection. To this end, we subjected the cells to short- and long-term acidic pressure and recovery to pHe 7.4. This treatment aimed at mimicking PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing. Our results indicate that short acidic treatment limits growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, potentiating their metastatic potential when re-exposed to pHe 7.4. The RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pHe-selected recovered to pHe 7.4 revealed distinct transcriptome rewiring. We describe an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Our work clearly demonstrates that upon acidosis stress, PDAC cells acquire more invasive cell phenotypes by promoting EMT and thus paving the way for more aggressive cell phenotypes.

Titania nanorods array homojunction with sub-stoichiometric TiO2 for enhanced methylene blue photodegradation

TiO2 thin films are known to promote photodegradation of dyes and pollutants in water solution via heterogeneous photocatalysis. This ability is guided by the photoexcitation through photons having energies above the band gap. To improve photocatalytic activity, nanostructures with high surface area can be applied, which can ease molecular adsorption/desorption mechanisms, enhance electronic transfer properties and lower excitation energy. For this purpose, square cross-section TiO2 vertically aligned nanorod (TNR) array configuration has been chosen as a semiconductor substrate. On top of it, a thin layer of sub-stoichiometric TiO2−x has been deposited, aiming at inducing a vacancy doped homojunction between two different oxygen rich/deficient TiO2 layers, possibly leading to lower band gap and enhanced photochemical activity. In principle, promotion of electron and holes separation and suppression of charge recombination could occur. Vertically aligned TNRs have been deposited through a hydrothermal growth in acidic conditions on a pre-seeded glass conducting substrate, optimizing the seeding process through spin coating. Sub-stoichiometric TiO2−x layer (50 nm nominal thickness) has been deposited on top of TNRs via radiofrequency magnetron sputtering at three different stoichiometries, tuning the oxygen partial pressure in sputtering argon atmosphere at 10 %, 15 % and 20 %, respectively. Photocatalytic activity has been investigated in the photodegradation of an aqueous solution of methylene blue, both under UV and simulated solar light irradiation at room temperature and atmospheric pressure, resulting in the degradation of methylene blue target molecule up to 99 % under UV and 85 % under simulated solar irradiation after 6 h. These promising achievements unlock new environmental applications for enhanced dye degradation industrial processes.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Lipogenesis mediated by OGR1 regulates metabolic adaptation to acid stress in cancer cells via autophagy.

SUMMARYMalignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.

Acid Tolerant and Acidophilic Microalgae: An Underexplored World of Biotechnological Opportunities.

Despite their large number and diversity, microalgae from only four genera are currently cultivated at large-scale. Three of those share common characteristics: they are cultivated mainly autotrophically and are extremophiles or tolerate “extreme conditions.” Extreme growth conditions aid in preventing contamination and predation of microalgae, therefore facilitating outdoor cultivation. In search for new extremophilic algae suitable for large-scale production, we investigated six microalgal strains able to grow at pH below 3 and belonging to four genera; Stichococcus bacillaris ACUF158, Chlamydomonas acidophila SAG 2045, and Chlamydomonas pitschmannii ACUF238, Viridiella fridericiana ACUF035 and Galdieria sulphuraria ACUF064 and ACUF074. All strains were cultivated autotrophically at light intensity of 100 and 300 μmol m−2 s−1 and pH between 1.9 and 2.9. The autotrophic biomass productivities were compared with one of the most productive microalgae, Chlorella sorokiniana SAG 211-8K, grown at pH 6.8. The acid tolerant strains have their autotrophic biomass productivities reported for the first time. Mixotrophic and heterotrophic properties were investigated when possible. Five of the tested strains displayed autotrophic biomass productivities 10–39% lower than Chlorella sorokiniana but comparable with other commercially relevant neutrophilic microalgae, indicating the potential of these microalgae for autotrophic biomass production under acidic growth conditions. Two acid tolerant species, S. bacillaris and C. acidophila were able to grow mixotrophically with glucose. Chlamydomonas acidophila and the two Galdieria strains were also cultivated heterotrophically with glucose at various temperatures. Chlamydomonas acidophila failed to grow at 37°C, while G. sulphuraria ACUF64 showed a temperature optimum of 37°C and G. sulphuraria ACUF74 of 42°C. For each strain, the biomass yield on glucose decreased when cultivated above their optimal temperature. The possible biotechnological applications of our findings will be addressed.

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment.

Nearly all bacteria are encased in peptidoglycan, an extracytoplasmic matrix of polysaccharide strands crosslinked through short peptide stems. In the Gram-negative model organism Escherichia coli, more than 40 synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to peptidoglycan metabolism remains unclear due to significant apparent redundancy, particularly among the autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases that exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB-and to a lesser extent, AmiC-amidase activity. Combining genetic, biochemical, and computational analyses, we demonstrate that low pH-dependent stimulation of AmiB is mediated through the periplasmic amidase activators NlpD, EnvC, and ActS (formerly known as YgeR). Although NlpD and EnvC promote amidase activity across pH environments, ActS preferentially stimulates AmiB activity in acidic conditions. Altogether, our findings support partially overlapping roles for E. coli amidases and their regulators in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.

Bioformulated Hesperidin-Loaded PLGA Nanoparticles Counteract the Mitochondrial-Mediated Intrinsic Apoptotic Pathway in Cancer Cells

In this study, a biological approach has carried out to develop the polymer-based nanoparticles using a bioflavonoid of hesperidin. This formulation has been attempted and established for its biomedical application in cancer treatment. Hesperidin loaded PLGA nanoparticles (Hes-PLGA-NPs) were processed by oil in water single emulsion solvent evaporation technique and the physiochemical traits were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), XRD and NMR analysis. DLS and TEM analysis revealed that the preparation yielded a spherical shaped nanoparticle with an average measure of 38.4 nm. The XRD spectrum showed a crystalline type of nanoparticles. The FTIR and NMR investigation exposed that the PLGA also serves up as capping or switching material for pH-dependent drug release. Furthermore, the cationic natures of Hes-PLGA-NPs easily penetrate cancerous cells and efficiently repressed the growth in acidic conditions. Taking into account, the findings of in vitro studies in HEp-2 cell suggested that bioformulated Hes-PLGA-NPs are more competent than native hesperidin and might be used as a potent anti-cancer candidate. Further studies are warranted to expose the toxicity and molecular level mechanisms involved in the anti-cancer activity of the synthesized Hes-PLGA-NPs as nanomedicine.

Variation of antibiotic resistance in Salmonella Enteritidis, Escherichia coliO157:H7, and Listeria monocytogenes after exposure to acid, salt, and cold stress

AbstractBacteria with antibiotic‐resistant could seriously threaten to human health, increasing the treatment cost for infections and negatively affecting treatment outcomes. Stress adaptation is one possible mechanism for the acquisition or enhancement of antibiotic resistance in bacteria as a result of cross‐protection. In this study, the effects of acid, salt, and cold stress on the antibiotic resistance of Salmonella Enteritidis, Listeria monocytogenes, and Escherichia coli O157:H7 were investigated using the disc diffusion method. For S. Enteritidis, acidic growth conditions increased resistance to ciprofloxacin and erythromycin (p < .05), and addition of 4% NaCl to growth media decreased resistance to chloramphenicol (p < .05). Irrespective of pH and the NaCl concentration of the growth medium, refrigerated E. coli O157:H7 showed increased resistance to amoxycillin, ciprofloxacin, gentamicin, streptomycin, and erythromycin (p < .05). Acid‐adapted L. monocytogenes showed decreased the resistance to amoxycillin, ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, streptomycin, and tetracycline (p < .05). In conclusion, prolonged exposure of foodborne pathogens to acid, salt, and cold stress alters their antibiotic resistance. However, the effect of acid, salt, and cold stress on bacterial antibiotic resistance depend on both the bacterial species and the specific antibiotic. Therefore, multiple factors need to be considered for a foodborne antimicrobial resistant risk assessment.

The Novel Enterococcus Phage vB_EfaS_HEf13 Has Broad Lytic Activity Against Clinical Isolates of Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium frequently found in the gastrointestinal tract, oral cavity, and periodontal tissue. Although it is considered a commensal, it can cause bacteremia, endocarditis, endodontic infections, and urinary tract infections. Because antibiotics are cytotoxic not only to pathogens, but also to health-beneficial commensals, phage therapy has emerged as an alternative strategy to specifically control pathogenic bacteria with minimal damage to the normal flora. In this study, we isolated a novel phage, Enterococcus phage vB_EfaS_HEf13 (phage HEf13), with broad lytic activity against 12 strains of E. faecalis among the three laboratory strains and 14 clinical isolates of E. faecalis evaluated. Transmission electron microscopy showed that phage HEf13 has morphological characteristics of the family Siphoviridae. Phage HEf13 was stable at a wide range of temperature (4–60°C) and showed tolerance to acid or alkaline (pH 3–12) growth conditions. Phage HEf13 had a short latent period (25 min) with a large burst size (approximately 352 virions per infected cell). The lytic activity of phage HEf13 at various multiplicities of infection consistently inhibited the growth of diverse clinical isolates of E. faecalis without any lysogenic process. Moreover, phage HEf13 showed an effective lytic activity against E. faecalis on human dentin ex vivo infection model. Whole genome analysis demonstrated that the phage HEf13 genome contains 57,811 bp of double-stranded DNA with a GC content of 40.1% and 95 predicted open reading frames (ORFs). Annotated functional ORFs were mainly classified into four groups: DNA replication/packaging/regulation, phage structure, host cell lysis, and additional functions such as RNA transcription. Comparative genomic analysis demonstrated that phage HEf13 is a novel phage that belongs to the Sap6virus lineage. Furthermore, the results of multiple sequence alignment showed that polymorphism of phage infection protein of E. faecalis (PIPEF) contributes to determine the host specificity of phage HEf13 against various E. faecalis strains. Collectively, these results suggest that phage HEf13 has characteristics of a lytic phage, and is a potential therapeutic agent for treatment or prevention of E. faecalis-associated infectious diseases.

Abstract 1846: Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells

Abstract Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in acidic extracellular microenvironment. Acidity in the microenvironment is a critical stress factor and selection force for the evolution of aggressive tumor types. There are several acid sensing cell surface receptors and ion channels (ASICs) that can sense acidity in the microenvironment; among them proton sensing G protein-coupled receptors (GPCRs) such as OGR1 (GPR68), TDAG8 (GPR65), GPR4 and GPR132 form a major class of acid receptors. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats adiposomes or lipid droplets. These are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Inhibition of fatty acid synthesis was selectively toxic under acidic conditions and attenuated adiposome accumulation. Among the acid sensing receptors, TDAG8 and OGR1 are highly expressed in a panel of breast cancer cell lines compared to non-malignant breast epithelial cells. Highly invasive and acid tolerant MDA-MB-231 cells express significantly higher levels of TDAG8 while MCF7 and T47D cells have high levels of OGR1. We investigated the role of these receptors in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of the major acid sensors in breast cancer cell lines MCF7 and T47D showed that depletion of OGR1, not TDAG8, specifically inhibited acid induced adiposome accumulation. Additionally, inducing the cells with ogerin, an allosteric activator of OGR1 resulted in adiposomogenesis. Further, OGR1 knock out cells showed inhibition in cell growth under acidic growth conditions compared to OGR1 expressing cells. OGR1 knockout cells were defective in acid induced autophagy. OGR1 is coupled with Gq/11 and, upon ligand (H+) binding, triggers activation of phospholipase C stimulating the formation of the second messenger IP3. Adiposome formation was inhibited in presence of PLC inhibitors, Edelfosine or U73122. These results indicate that OGR1 is the major acid sensing GPCR in these cells that mediate adiposomogenesis. Accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities. Citation Format: Smitha Pillai, Michael Langsen, Jonathan Nguyen, Jonathan Wojtkowiak, Marilyn Bui, Robert Gatenby, Robert Gillies. Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1846.

Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488.

Summary Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose‐producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT‐PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT‐PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.

Roseithermus sacchariphilus gen. nov., sp. nov. and proposal of Salisaetaceae fam. nov., representing new family in the order Rhodothermales.

A novel bacterium with cells that were pinkish-cream-coloured, aerobic, rod-shaped, 0.62-1.00 µm wide and 2.3-3.3 µm long, designated as strain MEBiC09517T, was isolated from Buksung-Po, a small port in Incheon, Republic of Korea. Strain MEBiC09517T had low 16S rRNA gene sequence similarity to validly reported strains; among them, Rubrivirgaprofundi SAORIC-476T displayed highest sequence similarity (89.9 %). Nevertheless, the novel strain shared a phylogenetic line with members of the genus Rhodothermus, not the genus Rubrivirga. Optimum growth conditions of strain MEBiC09517T were at 50-55 °C, pH 7 and in 2.0-4.0 % salt concentration. Strain MEBiC09517T was found to be an obligate marine bacterium that requires KCl, MgCl2 and CaCl2 as well as NaCl for growth. A phosphatidylethanolamine, a diphosphatidylglycerol, three glycolipids and four unidentified lipids were the strain's predominant polar lipid components. The fatty acid of the cell wall mainly consisted of carbons with 16 or 18 chain lengths such as C16 : 0, C18 : 0, C18 : 1 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The predominant menaquinone was MK-7. The DNA G+C content is 68.65 mol%. Strain MEBiC09517T differs from genera of the order Rhodothermales in terms of fatty acid composition, growth conditions, and range of carbon source utilization. Based on phylogenetic analysis using the strain's 16S rRNA gene sequence and results of physiological tests, strain MEBiC09517T (KCCM=43267T, JCM=32374T) is proposed as Roseithermus sacchariphilus gen. nov., sp. nov. Additionally, the novel family Salisaetaceae fam. nov. based on phylogenetic analysis and physiological characteristics is suggested.

Abstract 171: Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering

Abstract Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells in tumors adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to a disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, niche construction and remodeling promoted by adaptation to acidosis, which itself is a metabolic adaptation to the early harsh environment in intraductal neoplasms. Early carcinogenesis is an avascular and hypoxic disease leading to extracellular acidosis. Hence, we investigated how acid-adapted tumor cells engineer their niche to support their growth, proliferation, and invasion. We show that relatively rare collagens are produced by acid-adapted cancer cells to create an environment that contributes to their survival. We found collagen and other fibrillar structure in the periluminal region of DCIS lesions using Second Harmonic Generation Microscopy (SHG). We compared the microarray of DCIS samples to normal tissue and observed that Col10a1 and Col11a1 were significantly increased. We have shown previously that DCIS lesions are acidic. To test if these were related, we adapted low grade MCF7 and DCIS-10AT breast cancer cells to growth in acidic conditions and probed them for collagen production using qPCR, western blotting and Immunofluorescence. We found acid adaptation increases number of collagens including Col10a1, Col11a1, and different subtypes of Col4. Further, we developed a window chamber model to study the collagen remodeling real time through intra-vital microscopy. SNARF and collagen imaging simultaneously showed the collagen structural modifications in acidic regions. Further proteomic and secretome analysis of acid adapted cells versus non-adapted cells identified additional enzymes involved in collagen remodeling such as TGM2 and LOXL2 that have been shown to play major role in matrix remodeling. These findings were also validated by western blot and ICC. To test the role of collagens in viability of cancer cells we grew acid adapted and non-adapted MCF7 cells as 3D spheroids and treat them with FAK inhibitors. FAK inhibitors dramatically affected the acid adapted spheroids. The specific role of each of collagen genes in survival and invasion is still under study. We discuss that adaptation to acidosis induces cancer cells to adopt strategies that assist them to be more independent, particularly in harsh conditions such periluminal areas of DCIS that they don't have access to stroma cells; they produce the collagen they need and will remodel it by the enzymes produced by themselves to be able to survive, grow, and invade. Finally these adaptation mechanisms may present a vulnerability to design new therapeutics against the emerging aggressive phenotypes during cancer cell evolution. Citation Format: Ava Niazi, Johnson J. Joseph, Robert J. Gillies, Mehdi Damaghi. Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 171.

Role of Partially Saturated Canthaxanthin and Ergosterol in the Survival of Aspergillus carbonarius Mutant at Extreme Acidic Condition

Unlike the wild type, the mutant Aspergillus carbonarius synthesized a yellow pigment, partially saturated canthaxanthin (PSC) when the growth medium acidified to low pH. Since the pigment found pharmaceutical applications, the possible mechanism involved in its ability to grow at extreme acidic conditions is described. To understand the mutation in the pathway, specific inhibitors affecting carotenoid biosynthesis were used in the medium and PSC synthesis and cell integrity were studied. Results suggested that the possible occurrence of mutation in the isoprenoid pathway for higher production of carotenoid as well as ergosterol caused the mutant to grow in extremely acidic conditions. The results also suggested that the flow of carbon for sterol biosynthesis and that of carotenoids are dependent. The deposition of carotenoids and ergosterol in the cell membrane causing the cells to maintain pH homeostasis under the acidic growth conditions is of significant importance. In A. carbonarius, understanding the cause of stress induced PSC accumulation is essential for efficient expression and production of the pharmaceutically significant carotenoid and this will further facilitate research into the role of carotenoids in stress tolerance of other filamentous fungi.