Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

Update on Dihydropteroate Synthase (DHPS) Mutations in Pneumocystis jirovecii

A Pneumocystis jirovecii is one of the most important microorganisms that cause pneumonia in immunosupressed individuals. The guideline for treatment and prophylaxis of Pneumocystis pneumonia (PcP) is the use of a combination of sulfa drug-containing trimethroprim and sulfamethoxazole. In the absence of a reliable method to culture Pneumocystis, molecular techniques have been developed to detect mutations in the dihydropteroate synthase gene, the target of sulfa drugs, where mutations are related to sulfa resistance in other microorganisms. The presence of dihydropteroate synthase (DHPS) mutations has been described at codon 55 and 57 and found almost around the world. In the current work, we analyzed the most common methods to identify these mutations, their geographical distribution around the world, and their clinical implications. In addition, we describe new emerging DHPS mutations. Other aspects, such as the possibility of transmitting Pneumocystis mutated organisms between susceptible patients is also described, as well as a brief summary of approaches to study these mutations in a heterologous expression system.

Heterologous expression of the human ZIP4 zinc transporter in Saccharomyces cerevisiae

The human (h) transporter, hZIP4 is the primary zinc importer in the intestine and is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the zinc deficiency disease acrodermatitis enteropathica (AE), which disrupts digestive and immune system homeostasis. Structure-function studies of hZIP4 have been greatly hindered by the absence of a robust heterologous expression system. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. Both a wild type and a mutant S. cerevisiae strain, in which the endogenous zinc transporters are deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane domain only (mhZIP4-GFP) protein were successfully produced and targeted to the plasma membrane in yeast.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity

Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

Light-induction of endocannabinoids and activation of Drosophila TRPC channels

Drosophila phototransduction represents a classical model for signaling cascades that culminate with activation of TRP channels. TRP and TRPL are the canonical TRP (TRPC) channels, which are gated by light stimulation of rhodopsin and engagement of Gq and phospholipase Cβ (PLC). Despite decades of investigation, the mechanism of TRP activation in photoreceptor cells is unresolved. Here, using a combination of genetics, lipidomics and Ca2+ imaging, we found that light increased the levels of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG) in vivo. The elevation in 2-LG strictly depended on the PLC encoded by norpA. Moreover, this endocannabinoid upregulated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

Reconstitution of Polyketide-Derived Meroterpenoid Biosynthetic Pathway in Aspergillus oryzae

The heterologous gene expression system with Aspergillus oryzae as the host is an effective method to investigate fungal secondary metabolite biosynthetic pathways for reconstruction to produce un-natural molecules due to its high productivity and genetic tractability. In this review, we focus on biosynthetic studies of fungal polyketide-derived meroterpenoids, a group of bioactive natural products, by means of the A. oryzae heterologous expression system. The heterologous expression methods and the biosynthetic reactions are described in detail for future prospects to create un-natural molecules via biosynthetic re-design.

The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

A rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining cell shape. In contrast with most bacteria, the Mollicutes family is devoid of cell wall. As a consequence, many Mollicutes have undefined morphologies. Spiroplasma species are an exception as they robustly grow with a characteristic helical shape, but how they maintain their morphology remains unclear. Paradoxal to their lack of cell wall, the genome of Spiroplasma contains five homologues of MreB (SpMreBs). Since MreB is a homolog of actin and that short MreB filaments participate in its function, we hypothesize that SpMreBs form a polymeric cytoskeleton. Here, we investigate the function of SpMreB in forming a polymeric cytoskeleton by focusing on the Drosophila endosymbiont Spiroplasma poulsonii. We found that in vivo, Spiroplasma maintain a high concentration of all five MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that strong intracellular levels of SpMreb systematically produced polymeric filaments of various morphologies. Using co-immunoprecipitation and co-expression of fluorescent fusions, we characterized an interaction network between isoforms that regulate the filaments formation. Our results point to a sub-functionalization of each isoform which, when all combined in vivo, form a complex inner polymeric network that shapes the cell in a wall-independent manner. Our work therefore supports the hypothesis where MreB mechanically supports the cell membrane, thus forming a cytoskeleton.

Pheromone Receptors Help Cnaphalocrocis medinalis Avoid Competition in Rice Fields

Cnaphalocrocis medinalis (Guene'e) (Lepidoptera: Pyralidae) is one of the most important insect pests that attack the rice crop, Oryza sativa L., in China, feeding on rice leaves. Chilo suppressalis and Sesamia inferens are two common insects living within the same ecological system that feed on rice stalks. Their behavior could affect C. medinalis's choice of oviposition place, so we tested the electroantennogram (EAG) response of C. medinalis to a conspecific sex pheromone (Z11-18:OH; Z11-18:Ald; Z13-18:OH; Z13-18:Ald) and two other insects' pheromone compounds (Z9-16:Ald; Z11-16Ald; Z11-16:OH;Z11-16:Ac and 16:Ald). The results indicate C. medinalis can detect those pheromones and is sensitive to Z11-16:Ald and Z9-16:Ald. In the heterologous expression system of Xenopus oocytes, we cloned three pheromone receptor genes, CmedPR1, CmedPR2, and CmedPR3. These had the same electroantennogram response, in addition to the response to the conspecific pheromone. CmedPR2 and CmedPR3 displayed strong sensitivity to Z11-16Ald and Z9-16:Ald. These results may contribute to clarifying how C. medinalis recognizes pheromones and interspecies communication.

Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

IntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete β-AR CaV1.2 modulation cascade in a heterologous expression system.ObjectiveOur primary goal was to heterologously reconstitute the complete β-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2.Methods and ResultsWe utilized the Xenopus oocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and β1-AR or β2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate β-AR. Whole-cell Ba2+ currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of α1C and for the CaVβ subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either β1-AR or β2-AR. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR.ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of CaV1.2 activity, crucially depends on CaVβ subunit, and is differently regulated by β1-AR and β2-AR. The reconstitution of the full β-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.