Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a major metabolic gene

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.     

Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. Differential localization of the Drosophila and human enzymes

CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12-24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.     

Biosimilarity under stress: A forced degradation study of Remicade® and Remsima™

Remsima? (infliximab) is the first biosimilar monoclonal antibody (mAb) approved by the European Medical Agency and the US Food and Drug Administration. Remsima? is highly similar to its reference product, Remicade®, with identical formulation components. The 2 products, however, are not identical; Remsima? has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans. To understand if these attribute differences could be amplified during forced degradation, solutions and lyophilized powders of the 2 products were subjected to stress at elevated temperature (40–60°C) and humidity (dry-97% relative humidity). Stress-induced aggregation and degradation profiles were similar for the 2 products and resulted in loss of infliximab binding to tumor necrosis factor and FcγRIIIa. Appearances of protein aggregates and hydrolysis products were time- and humidity-dependent, with similar degradation rates observed for the reference and biosimilar products. Protein powder incubations at 40°C/97% relative humidity resulted in partial mAb unfolding and increased asparagine deamidation. Minor differences in heat capacity, fluorescence, levels of subvisible particulates, deamidation and protein fragments were observed in the 2 stressed products, but these differences were not statistically significant. The protein solution instability at 60°C, although quite significant, was also similar for both products. Despite the small initial analytical differences, Remicade® and Remsima? displayed similar degradation mechanisms and kinetics. Thus, our results show that the 2 products are highly similar and infliximab's primary sequence largely defines their protein instabilities compared with the limited influence of small initial purity and glycosylation differences in the 2 products.     

Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.     

Ecological characterisation of <Emphasis Type="Italic">Steinernema siamkayai</Emphasis> (Rhabditida: Steinernematidae), a warm-adapted entomopathogenic nematode isolate from India

Our study describes basic ecological properties of Steinernema siamkayai Tiruchirappalli strain from India. The effect of temperature on nematode infectivity and development, laboratory host range and foraging behaviour were determined. The data showed that S. siamkayai is a warm-adapted nematode species with larval mortality observed between 15°C and 37.5°C and nematode reproduction occurring between 20°C and 35°C. All insect species used in this study were susceptible to S. siamkayai under laboratory conditions. Sixty infective juveniles (IJs) per insect were used and the lepidopterans, Galleria mellonella (100%) and Spodoptera exigua (85%), were the most susceptible species followed by the dipteran, Ceratitis capitata (60%), and lepidopteran, Cydia splendana (55%), and the coleopteran, Tenebrio molitor (45%), whereas the coleopteran, Curculio elephas (25%), was the least susceptible species. S. siamkayai infective juveniles (IJs) stood on their tails and jumped and could also attach to a mobile host at a rate of 27 IJs larvae⁻¹ out of 1000 IJs in 10 min. Larval mortality of G. mellonella by S. siamkayai on different substrates (sand, filter paper, filter paper sprinkled with sand) was 100% on all substrates. Number of IJs out of 100 IJs that penetrated into a G. mellonella host at different soil depths was the highest at the surface (44 IJs larva⁻¹) and the lowest at 5 cm depth (13 IJs larva⁻¹) with no larval mortality observed at 10 cm depth. In addition, the symbiotic bacterium of S. siamkayai was identified as Xenorhabdus stockiae based on genotypic and phenotypic characterisation. Bacterial growth was observed between 15°C and 41°C.     

Correction to: Recruitment of Plasma Membrane GABA-A Receptors by Submembranous Gephyrin/Collybistin Clusters

Cellular and Molecular Neurobiology - The original version of this article unfortunately contained editorial errors in supplementary material.     

Proteome analysis of male accessory gland secretions in Leucinodes orbonalis Guenee (Lepidoptera: Crambidae), a Solanum melongena L. pest

Male accessory gland (MAG) proteins are transferred along with the sperm to females at the time of mating and have diverse effects on female reproductive physiology in a wide range of insects. In this study, we sought to identify the MAG proteins in Leucinodes orbonalis Guenee, a Solanum melongena L. pest, by analyzing the MAG proteins of virgin and mated male moths by nano-LC-ESI-MS/MS techniques. A total of 142 and 131 proteins in virgin and mated males were identified, respectively, among which 17 (12.0%) and 10 (7.6%) proteins were found to show secretory signals in virgin and mated males, respectively. These secretory proteins were shown to be involved in several biological processes in insects, including egg development, sperm-related functions/capacitation, defense, metabolism, and protein chaperoning. To the best of our knowledge, this is the first study to perform a proteome analysis of the MAG proteins of L. orbonalis, and offers an opportunity for further investigation of the functions of these proteins. In insects, certain MAG proteins are known to inhibit mating whereas others accelerate egg-laying. Therefore, the identification of these proteins in L. orbonalis may be useful for pest control.