Single-particle cryo-EM reveals conformational variability of the oligomeric VCC β-barrel pore in a lipid bilayer

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.     

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions—thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.     

Sequential and non-sequential pattern of monocarpic senescence in two rice cultivars

Two rice ( Oryza sativa L.) cultivars viz. Ratna (dwarf, photoperiod insensitive) and Masuri (tall, photoperiod sensitive) were selected to analyse their mode of senescence. At the vegetative stage, leaf senescence, expressed as the loss of chlorophyll and protein and a decline in the activities of catalase and alkaline pyrophosphatase, was found to be a function of chronological age (sequential) in both cultivars. With advancing reproductive development, cultivar Masuri retained this sequential mode but cultivar Ratna showed a non-sequential mode of senescence where the flag leaf senesced earlier than the older second leaf, unlike that observed at the vegetative stage. Masuri showed a more rapid senescence than Ratna. In both cultivars, excision of any leaf during anthesis initially retarded the senescence of the remaining leaves on the defoliated plants but soon after, at the grain maturation stage, the leaf senescence started at a higher rate compared with that of the intact control plant. In Ratna, when either the second or the third leaf was removed, the flag leaf senesced faster than that of the unexcised control plant. In Masuri, when either the flag or the third leaf was removed, the second leaf senesced earlier than that of the intact control. In both cultivars, excision of the third leaf showed the least detrimental effect on yield. The greatest detrimental effect on grain yield per plant was observed in Ratna when the flag leaf was removed and in Masuri when the second leaf was removed. Mobilization of metabolites from the source leaf to the sink and the consequent depletion in the leaf as the cause of senescence is discussed.     

Immunocytochemical localization of chloramphenicol acetyltransferase as a single-cell marker of transfected and transgenic cells

A technique is described for immunocytochemical localization of the bacterial gene product chloramphenicol acetyltransferase, which is a commonly used reporter gene in transfected and transgenic cells. The described procedure is capable of localizing the enzyme in individual cells, providing a means of determining the cell type(s) expressing a foreign construct in complex cultures or in tissue sections of transgenic mice.     

Oxidant, mitochondria and calcium: an overview

Mitochondria are active in the continuous generation of reactive oxygen species (ROS), (e.g., superoxide), thereby favouring a situation of mitochondrial oxidative stress. Under oxidative stress--for example, ischaemia-reoxygenation injury to cells--mitochondria form superoxide, which in turn is converted to hydrogen peroxide and the potent reactive species, hydroxyl radical. Alternatively, mitochondrial superoxide may react with nitric oxide to form potent oxidant peroxynitrite and as a consequence, mitochondrial function is altered. An increase in the release of calcium from mitochondria by oxidants stimulates calcium-dependent enzymes such as calcium-dependent proteases, nucleases, and phospholipases, which subsequently trigger apoptosis of the cells. In principle, calcium can leave mitochondria by different ways: by non-specific leakage through the inner membrane by "pore formation," by changes in the membrane lipid phase, by reversal of the uniport influx carrier, by the specific calcium/hydrogen (or sodium) antiport system, by channel-mediated release pathways, or by a combination of two or more of these pathways. Additionally, the release of calcium from mitochondria can also occur either by oxidation of internal nicotinamide adenine nucleotides to ADP ribose and nicotinamide or by oxidation of thiols in membrane proteins. Once calcium efflux has been triggered, a series of common pathways of apoptosis are initiated, each of which may be sufficient to destroy the cell. Apoptosis requires the active participation of cellular components, and several genes have been suggested to control apoptosis. The proto-oncogene bcl-2 suppresses apoptosis through mitochondrial effects. Overexpression of bcl-2 in the mitochondrial membrane inhibits calcium efflux, but the underlying mechanisms are not clearly known. Further studies are needed to explore the nature of the apoptosis-inducing pathways, the precise mechanisms of calcium efflux, the molecular partners of bcl-2 oncoproteins at the level of the outer-inner membrane contact sites, the molecular biology of the apoptosis-inducing factor formation and release, and the essential molecular targets of apoptosis-inducing proteases. Clarification of these issues might facilitate the understanding of mitochondrial response on cellular calcium dynamics under oxidant stress.     

Plant Growth-Promoting Rhizobacteria-Mediated Adaptive Responses of Plants Under Salinity Stress

In this recent era, several approaches have been developed to alleviate the adverse effects of salinity stress in different plants. However, some of them are not eco-friendly. In this context, evolving sustainable approaches which enhance the productivity of saline soil without harming the environment are necessary. Many recent studies showed that plant growth-promoting rhizobacteria (PGPR) are known to confer salinity tolerance to plants. Salt-stressed plants inoculated with PGPR enhance the growth and productivity of crops by reducing oxidative damage, maintaining ionic homeostasis, enhancing antioxidant machinery, and regulating gene expressions. The PGPR also regulates the photosynthetic attributes such as net photosynthetic rate, chlorophyll, and carotenoid contents and enhances the salinity tolerance to plants. Moreover, PGPR has a great role in the enhancement of phytohormones and secondary metabolites synthesis in plants under salt stress. This review summarizes the current reports of the application of PGPR in plants under salt stress and discusses the PGPR-mediated mechanisms in plants of salt tolerance. This review also discusses the potential role of PGPR in cross-talk with phytohormones and secondary metabolites to alleviate salt stress and highlights the research gaps where further research is needed.

     

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

In order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL-ethionine after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.