Stationary Lactococcus cremoris: Energetic State, Protein Synthesis Without Nitrogen and Their Effect on Survival

During storage and ripening of fermented foods, Lactococcus cremoris is predominantly in a non-growing state. L. cremoris can become stationary due to starvation or acidification, and its metabolism in these non-growing states affects the fermented product. Available studies on the response of L. cremoris to acid and starvation stress are based on population level data. We here characterized the energetic state and the protein synthesis capacity of stationary L. cremoris cultures at the single cell level. We show that glucose starved stationary cells are energy-depleted, while acid-induced stationary cells are energized and can maintain a pH gradient over their membrane. In the absence of glucose and arginine, a small pH gradient can still be maintained. Subpopulations of stationary cells can synthesize protein without a nitrogen source, and the subpopulation size decreases with increasing stationary phase length. Protein synthesis capacity during starvation only benefits culturability after 6 days. These results highlight significant differences between glucose starved stationary and acid-induced stationary cells. Furthermore, they show that the physiology of stationary phase L. cremoris cells is multi-facetted and heterogeneous, and the presence of an energy source during stationary phase impacts the cells capacity to adapt to their environment.

Bacteria Respond Stronger Than Fungi Across a Steep Wood Ash-Driven pH Gradient

Soil pH is probably the most important variable explaining bacterial richness and community composition locally as well as globally. In contrast, pH effects on fungi appear to be less pronounced, but also less studied. Here we analyze the community responses of bacteria and fungi in parallel over a local extreme pH gradient ranging from 4 to 8. We established the pH gradient by applying strongly alkaline wood ash in dosages of 0, 3, 9, 15, 30, and 90 t ha–1 to replicated plots in a Picea abies plantation and assessed bacterial and fungal community composition using high throughput amplicon sequencing 1 year after ash application. At the same time, the experiment investigated if returning wood ash to plantation forests pose any immediate threats for the microbial communities. Among the measured environmental parameters, pH was by far the major driver of the microbial communities, however, bacterial and fungal communities responded differently to the pH increment. Whereas both bacterial and fungal communities showed directional changes correlated with the wood ash-induced increase in pH, the bacterial community displayed large changes at wood ash dosages of 9 and 15 t ha–1 while only higher dosages (>30 t ha–1) significantly changed the fungal community. The results confirm that fungi are less sensitive to pH changes than bacteria but also that fertilizing plantation forests with wood ash, viewed through the lens of microbial community changes, is a safe management at standard dosages (typically 3 t ha–1).

Optimization of an ammonia assay based on transmembrane pH-gradient polymersomes

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30–800 μM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

Migration of aerobic bacteria from the duodenum to the pancreas with tumors: a mechanistic understanding

More aerobic bacteria are found in the pancreas with tumors than in the healthy pancreas. We provide a mechanistic understanding of the migration of intestinal bacteria from the duodenum to the pancreas with tumors. Mathematical models of migration of aerobic bacteria from the duodenum to the pancreas with tumors in the hepatopancreatic duct were developed. In addition, the behaviors of GFP E. coli under a pH gradient in a microfluidic device were analyzed. Moreover, upstream migrations of Pseudomonas fluorescens against flow were measured in a polydimethylsiloxane (PDMS) T-shaped cylinder mimicking a pancreatic duct. The simulated bacterial concentration of the pancreas with tumors was higher than that of the healthy pancreas and agreed reasonably well with the literature. Migration of aerobic bacteria in the hepatopancreatic duct is counteracted by bile and pancreatic juice flow but facilitated greatly by bacterial pH taxis from lower pH in duodenum fluid toward slightly alkaline pH in pancreatic juice, favorable for them. Migration of bacteria to the pancreas with tumors is made easier by solid tumors on the pancreatic duct, which compresses the pancreatic duct and thus reduces the fluid flow rate. On the other hand, GFP E. coli migrated under the pH gradient in a microfluidic device from acidic areas toward neutral or slightly alkaline pH, validating pH taxis. Furthermore, Pseudomonas fluorescens migrated upstream from hydrochloride solution but not from bicarbonate solution against bicarbonate flow at >20 µm/s, with an advancing velocity of approximately 60 µm/s, validating the models (244 words).

Instability and Stasis Among the Microbiome of Seagrass Leaves, Roots and Rhizomes, and Nearby Sediments Within a Natural pH Gradient

Seagrass meadows are hotspots of biodiversity with considerable economic and ecological value. The health of seagrass ecosystems is influenced in part by the makeup and stability of their microbiome, but microbiome composition can be sensitive to environmental change such as nutrient availability, elevated temperatures, and reduced pH. The objective of the present study was to characterize the bacterial community of the leaves, bulk samples of roots and rhizomes, and proximal sediment of the seagrass species Cymodocea nodosa along the natural pH gradient of Levante Bay, Vulcano Island, Italy. The bacterial community was determined by characterizing the 16S rRNA amplicon sequencing and analyzing the operational taxonomic unit classification of bacterial DNA within samples. Statistical analyses were used to explore how life-long exposure to different pH/pCO2 conditions may be associated with significant differences in microbial communities, dominant bacterial classes, and microbial diversity within each plant section and sediment. The microbiome of C. nodosa significantly differed among all sample types and site-specific differences were detected within sediment and root/rhizome microbial communities, but not the leaves. These results show that C. nodosa leaves have a consistent microbial community even across a pH range of 8.15 to 6.05. The ability for C. nodosa to regulate and maintain microbial structure may indicate a semblance of resilience within these vital ecosystems under projected changes in environmental conditions such as ocean acidification.

Effect of Exogenous pH on Cell Growth of Breast Cancer Cells

Breast cancer is the most common type of cancer in women and the most life-threatening cancer in females worldwide. One key feature of cancer cells, including breast cancer cells, is a reversed pH gradient which causes the extracellular pH of cancer cells to be more acidic than that of normal cells. Growing literature suggests that alkaline therapy could reverse the pH gradient back to normal and treat the cancer; however, evidence remains inconclusive. In this study, we investigated how different exogenous pH levels affected the growth, survival, intracellular reactive oxygen species (ROS) levels and cell cycle of triple-negative breast cancer cells from MDA-MB-231 cancer cell lines. Our results demonstrated that extreme acidic conditions (pH 6.0) and moderate to extreme basic conditions (pH 8.4 and pH 9.2) retarded cellular growth, induced cell death via necrosis and apoptosis, increased ROS levels, and shifted the cell cycle away from the G0/G1 phase. However, slightly acidic conditions (pH 6.7) increased cellular growth, decreased ROS levels, did not cause significant cell death and shifted the cell cycle from the G0/G1 phase to the G2/M phase, thereby explaining why cancer cells favored acidic conditions over neutral ones. Interestingly, our results also showed that cellular pH history did not significantly affect the subsequent growth of cells when the pH of the medium was changed. Based on these results, we suggest that controlling or maintaining an unfavorable pH (such as a slightly alkaline pH) for cancer cells in vivo could retard the growth of cancer cells or potentially treat the cancer.