Liquid Phase Electron Microscopy of Bacterial Ultrastructure.

Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometer resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Using graphene encapsulation, scanning transmission electron microscopy (STEM), and energy-dispersive X-ray (EDX) spectroscopy to mitigate these issues provides unprecedented levels of intracellular detail in aqueous specimens. This study demonstrates the potential of LP-STEM to examine and identify internal cellular structures in thick biological samples. Specifically, it highlights the use of LP-STEM to investigate the radiation resistant, gram-positive bacterium, Deinococcus radiodurans using various imaging techniques.

Managing Manganese: The Role of Manganese Homeostasis in Streptococcal Pathogenesis.

Pathogenic streptococci require manganese for survival in the host. In response to invading pathogens, the host recruits nutritional immune effectors at infection sites to withhold manganese from the pathogens and control bacterial growth. The manganese scarcity impairs several streptococcal processes including oxidative stress defenses, de novo DNA synthesis, bacterial survival, and virulence. Emerging evidence suggests that pathogens also encounter manganese toxicity during infection and manganese excess impacts streptococcal virulence by manganese mismetallation of non-cognate molecular targets involved in bacterial antioxidant defenses and cell division. To counter host-imposed manganese stress, the streptococcal species employ a sophisticated sensory system that tightly coordinates manganese stress-specific molecular strategies to negate host induced manganese stress and proliferate in the host. Here we review the molecular details of host-streptococcal interactions in the battle for manganese during infection and the significance of streptococcal effectors involved to bacterial pathophysiology.

Manganese accumulation in probiotic Lactobacillus paracasei ATCC 55544 analyzed by synchrotron X-ray fluorescence microscopy and impact of accumulation on the bacterial viability following encapsulation.

Lactobacillus spp. are known to accumulate large amounts of inorganic manganese, which protects against oxidative damage by scavenging free radicals. The ability of probiotic L. paracasei ATCC 55544 to maintain viability during long-term ambient storage may be enhanced by this microorganism's ability to accumulate manganese, which may act as a free radical scavenger. To investigate this hypothesis, X-ray fluorescence microscopy (XFM) was employed to determine the changes in the elemental composition of L. paracasei during growth in the MRS medium with or without added manganese. Moreover, manganese uptake by cells as a function of physiological growth state, early log vs. stationary phase was evaluated. The semiquantitative X-ray fluorescence microscopy results revealed that lower levels of manganese accumulation occurred during the early log phase of bacterial growth of L. paracasei cells (0.0064 µg/cm2) compared with the stationary phase cells (0.1355 µg/cm2). L. paracasei cells grown in manganese deficient MRS medium resulted in lower manganese uptake by cells (0.0027 µg/cm2). The L. paracasei cells were further embedded in milk powder matrix using a fluidized-bed drying technique and stored at a water activity (aw) of 0.33 at 25 °C for 15 days. The viability counts of L. paracasei cells grown in MRS medium harvested after 18 h growth and embedded in milk powder matrix retained viability of (9.19 ± 0.12 log CFU/g). No viable L. paracasei cells were observed in the case of embedded L. paracasei cells grown in manganese-deficient MRS medium harvested after 18 h growth or in the case of L. paracasei cells harvested after 4 h when grown in MRS medium. The lower level of manganese accumulation was found to be related to the loss of bacterial viability during storage.

Lateral roots but not root hairs contribute to high uptake of manganese and cadmium in rice.

Rice (Oryza sativa L.) can accumulate high manganese (Mn) in the shoots through uptake by the roots, which consist of crown roots, lateral roots and root hairs. We investigated the role of lateral roots and root hairs in Mn and cadmium (Cd) uptake by using two indica rice mutants defective in formation of lateral roots (osiaa11) and root hairs (osrhl1). The uptake of Mn and Cd in osiaa11 was significantly lower than that in wild type 'Kasalath', but there was no difference between wild type and osrhl1. Furthermore, a kinetic study showed that Mn uptake in osiaa11 was much lower than that in wild type and osrhl1 across a wide range of Mn concentrations. The role of lateral roots in Mn and Cd uptake was further confirmed in a japonica rice mutant defective in lateral root formation. We found that expression of Mn transporter gene Natural Resistance-Associated Macrophage Protein 5 (OsNRAMP5), but not of Metal Tolerance Protein 9 (OsMTP9), was lower in osiaa11 than in wild type; however, there were no differences between osrhl1 and the wild type. Immunostaining showed that OsNRAMP5 and OsMTP9 were localized in the exodermis and endodermis of crown roots and lateral roots, but not in the root hairs. Taken together, our results indicate that lateral roots, but not root hairs, play an important role in high Mn and Cd uptake in rice.

Manganese in Plants: From Acquisition to Subcellular Allocation.

Manganese (Mn) is an important micronutrient for plant growth and development and sustains metabolic roles within different plant cell compartments. The metal is an essential cofactor for the oxygen-evolving complex (OEC) of the photosynthetic machinery, catalyzing the water-splitting reaction in photosystem II (PSII). Despite the importance of Mn for photosynthesis and other processes, the physiological relevance of Mn uptake and compartmentation in plants has been underrated. The subcellular Mn homeostasis to maintain compartmented Mn-dependent metabolic processes like glycosylation, ROS scavenging, and photosynthesis is mediated by a multitude of transport proteins from diverse gene families. However, Mn homeostasis may be disturbed under suboptimal or excessive Mn availability. Mn deficiency is a serious, widespread plant nutritional disorder in dry, well-aerated and calcareous soils, as well as in soils containing high amounts of organic matter, where bio-availability of Mn can decrease far below the level that is required for normal plant growth. By contrast, Mn toxicity occurs on poorly drained and acidic soils in which high amounts of Mn are rendered available. Consequently, plants have evolved mechanisms to tightly regulate Mn uptake, trafficking, and storage. This review provides a comprehensive overview, with a focus on recent advances, on the multiple functions of transporters involved in Mn homeostasis, as well as their regulatory mechanisms in the plant's response to different conditions of Mn availability.

SLC39A8 gene encoding a metal ion transporter: discovery and bench to bedside.

SLC39A8 is an evolutionarily highly conserved gene that encodes the ZIP8 metal cation transporter in all vertebrates. SLC39A8 is ubiquitously expressed, including pluripotent embryonic stem cells; SLC39A8 expression occurs in every cell type examined. Uptake of ZIP8-mediated Mn2+, Zn2+, Fe2+, Se4+, and Co2+ represents endogenous functions-moving these cations into the cell. By way of mouse genetic differences, the phenotype of "subcutaneous cadmium-induced testicular necrosis" was assigned to the Cdm locus in the 1970s. This led to identification of the mouse Slc39a8 gene, its most closely related Slc39a14 gene, and creation of Slc39a8-overexpressing, Slc39a8(neo/neo) knockdown, and cell type-specific conditional knockout mouse lines; the Slc39a8(-/-) global knockout mouse is early-embryolethal. Slc39a8(neo/neo) hypomorphs die between gestational day 16.5 and postnatal day 1-exhibiting severe anemia, dysregulated hematopoiesis, hypoplastic spleen, dysorganogenesis, stunted growth, and hypomorphic limbs. Not surprisingly, genome-wide association studies subsequently revealed human SLC39A8-deficiency variants exhibiting striking pleiotropy-defects correlated with clinical disorders in virtually every organ, tissue, and cell-type: numerous developmental and congenital disorders, the immune system, cardiovascular system, kidney, lung, liver, coagulation system, central nervous system, musculoskeletal system, eye, and gastrointestinal tract. Traits with which SLC39A8-deficiency variants are currently associated include Mn2+-deficient hypoglycosylation; numerous birth defects; Leigh syndrome-like mitochondrial redox deficiency; decreased serum high-density lipoprotein-cholesterol levels; increased body mass index; greater risk of coronary artery disease, hypotension, cardiovascular death, allergy, ischemic stroke, schizophrenia, Parkinson disease, inflammatory bowel disease, Crohn disease, myopia, and adolescent idiopathic scoliosis; systemic lupus erythematosus with primary Sjögren syndrome; decreased height; and inadvertent participation in the inflammatory progression of osteoarthritis.

Uptake of Manganese from the Manganese-Lysine Complex in Primary Chicken Intestinal Epithelial Cells.

Organic manganese (Mn) sources can replace inorganic Mn as dietary Mn supplements in poultry. To compare the uptake of Mn from the Mn-lysine complex (MnLys) and MnSO4, we first established the primary chicken intestinal epithelial cells (IECs) model and used it to determine Mn uptake. The MnLys increased the uptake of Mn compared to MnSO4. The uptake of Mn decreased in the IECs with Fe addition in the medium regardless of the Mn sources. The MnLys decreased the Mn2+ efflux transporter ferroportin 1 (FPN1) mRNA level but did not influence the Mn2+ influx transporter divalent metal transporter 1 (DMT1) mRNA expression when compared to MnSO4. The results above indicated that the increase of Mn accumulation for MnLys at least partly was due to the decrease of Mn efflux by reduced FPN1 expression. The addition of N-ethylmaleimide, an L-lysine transport system y+ inhibitor, decreased the uptake of Mn from MnLys but did not affect the uptake of Mn from MnSO4. The cycloheximide, as an L-lysine transport system b0,+ activator, increased the uptake of Mn from MnLys, whereas they did not influence the uptake of Mn from MnSO4. The MnLys increased the system y+ members cationic amino acid transporter (CAT) 1 and CAT2, and system b0,+ components rBAT and b0,+AT mRNA expression when compared to MnSO4. These results suggested that the uptake of MnLys complex might be transported by CAT1/2 and system b0,+, which was different from the ionized Mn2+ uptake pathway. In conclusion, the uptake of Mn from MnLys complex not only might be uptake through the ionized Mn2+ pathway, but also appeared to be transported through the CAT1/2 and system b0,+ in primary chicken IECs.

Phosphate addition diminishes the efficacy of wollastonite in decreasing Cd uptake by rice (Oryza sativa L.) in paddy soil.

Cadmium (Cd) contamination in paddy soils poses food security risks and public health concerns. Exploring effective strategies to reduce rice grain Cd is an urgent need. In this study, field plot experiments were conducted to evaluate the effects of wollastonite application with or without phosphate (P) addition on Cd accumulation in rice (Oryza sativa L.). Co-application of P and wollastonite showed greater efficiency than wollastonite amendments alone in raising soil pH and CEC and decreasing soil Cd availability. Cd concentration in brown rice was decreased by 71% under the wollastonite treatment alone, but was decreased by only 29-39% when wollastonite was coupled with different P amendments. This seeming contradiction could be ascribed to the dramatic decline in the phytoavailability of manganese (Mn) and the increase in molar ratio of iron (Fe) to Mn (Fe/Mn) in Fe plaques on root surfaces in the presence of P additions. Significant negative correlations between Mn and Cd in rice plants and positive correlations between Fe/Mn in Fe plaque and Cd in rice plants indicated that P-induced soil Mn deficiency and reduced Mn in Fe plaque impeded the alleviation of Cd accumulation in rice. Application of wollastonite in Si-deficient paddy soils was effective in reducing rice Cd accumulation while boosting rice yield, but co-application of P and wollastonite was counterproductive and should be avoided. This work emphasized that a better understanding of the relationships between Cd and related mineral nutrient uptake would be helpful in developing more efficient measures to reduce rice grain Cd.

Overexpression of a High-Affinity Nitrate Transporter OsNRT2.1 Increases Yield and Manganese Accumulation in Rice Under Alternating Wet and Dry Condition.

Nitrate and manganese (Mn) are necessary elements for the growth and development of rice in paddy soil. Under physiological conditions, we previously reported that the uptake of Mn in roots can be improved by the addition of external nitrate but not ammonium. To investigate the mechanism(s) of this phenotype, we produced plant lines overexpressing OsNRT2.1 and assessed Mn uptake under alternating wet and dry (AWD) and waterlogged (WL) conditions. Under AWD condition, we observed a 31% reduction in grain yields of wild type (WT) plants compared to WL condition. Interestingly, the overexpression of OsNRT2.1 could recover this loss, as OsNRT2.1 transgenic lines displayed higher grain yields than WT plants. We also observed 60% higher grain Mn in the transgenic lines in AWD condition and approximately 30% higher Mn in the grain of transgenic lines in WL condition. We further found that the overexpression of OsNRT2.1 did not alter Mg and Fe in the seeds in either growth condition. The reasons for the increased Mn content in OsNRT2.1 transgenic seeds in AWD condition could be explained by the elevated expression of OsNRAMP family genes including OsNRAMP3, OsNRAMP5, and OsNRAMP6 in node I, the panicle-neck, and the flag leaves. The mechanism(s) underpinning the upregulation of these genes requires further investigation. Taken together, our results provide a new function of OsNRT2.1 in improving rice yields and grain Mn accumulation during water-saving cultivation patterns. This represents a new strategy for maintaining yield and improving food quality in a sustainable agricultural system.