Circular Bacteriocins: Biosynthesis and Mode of Action

Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.     

Mode of Action of Albocycline, an Inhibitor of Nicotinate Biosynthesis

The antibiotic albocycline blocks the synthesis of nicotinate or nicotinamide in Bacillus subtilis cells. The inhibitory activity of the agent is fully reversed by nicotinic acid, nicotinamide, and, to a moderate extent, also by quinolinate. This suggests that in B. subtilis the antibiotic interferes with a reaction step occurring prior to the formation of quinolinate within the biosynthetic pathway leading to nicotinate.     

Microcin C: Biosynthesis,Mode of Action,and Potential as a Lead in Antibiotics Development

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.     

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

Coelenterate Neuropeptides: Structure, Action and Biosynthesis

Evolutionary "old" nervous systems such as those of coelenteratesare peptidergic: Using various radioimmunoassays we have nowisolated 13 novel neuropeptides from sea anemones and severalothers from hydrozoan polyps and medusae. These peptides areall structurally related and contain the C-terminal sequenceArg-X-NH₂ or Lys-X-NH₂, where X is Ala, Asn, Ile, Phe, Pro orTrp. Three neuropeptides have a novel N-terminal L-3-phenyllactylresidue, which protects against degradation by nonspecific aminopeptidases.The neuropeptides from sea anemones are produced by differentsets of neurones and have excitatory or inhibitory actions onisolated muscle preparations, suggesting that they are neurotransmittersor neuromodulators. We have also cloned the precursor proteinfor the sea-anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH₂).In Calliactis parasitica this precursor harbours 19 copies ofimmature Antho-RFamide (Gln-Gly-Arg-Phe-Gly) together with 7other, putative neuropeptide sequences. The precursor of Anthopleuraelegantissima contains 14 copies of Antho-RFamide and 19 other,putative neuropeptides. This shows that the biosynthetic machineryfor neuropeptides in coelenterates, the lowest animal grouphaving a nervous system, is already very efficient and similarto that of higher invertebrates, such as molluscs and insects,and vertebrates.     

Biosynthesis and action of neurosteroids in the cerebellar Purkinje neuron

The brain is considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Therefore, our studies for this exciting area of brain research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently, genomic and nongenomic actions of neurosteroids have become clear by a series of our studies using an excellent Purkinje cellular model. On the basis of these findings, we summarize the advances made in our understanding of biosynthesis and action of neurosteroids in the cerebellar Purkinje cell.     

Biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell

New findings over the past decade have shown that the brain has the capability of forming steroids de novo from cholesterol, the so-called “neurosteroids”. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we have demonstrated that the Purkinje cell, a cerebellar neuron, is a major site for neurosteroid formation in a variety of vertebrates. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. Since this discovery, organizing actions of neurosteroids are becoming clear by the studies on mammals using the Purkinje cell as an excellent cellular model. In mammals, the Purkinje cell actively synthesizes progesterone de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. The Purkinje cell may also produces estradiol in the neonate. Interestingly, both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such organizing actions may contribute to the formation of cerebellar neuronal circuit during neonatal life. This paper summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of neurosteroids in the developing Purkinje cell.     

Mode of Action of Ponazuril Against Toxoplasma gondii Tachyzoites in Cell Culture

ABSTRACT. Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the inode of action of ponazuril against developing RH strain T. gondii tachyzoites in African green monkey kidney cells. Host cells were infected with  2.0 × 10⁵  tachyzoites and treated with 5 μg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of multinucleate schizonts stages. Up to six tachyzoites were observed partially budded from the surface of these schizonts. Large vacuoles developed in these schizonts and they eventually degenerated.     

Purification,Cloning and Functional Expression of hydroxyphenylpyruvate Reductase Involved in Rosmarinic Acid Biosynthesis in Cell Cultures of Coleus Blumei

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates. The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis. The protein was tryptically digested and the peptides sequenced. Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C. blumei cDNA-library and 5'-RACE-PCR. The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues. The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases. The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate.     

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.     

Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase

Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.     

Neurosteroids: a new function in the brain

"Neurosteroids" accumulate in the central nervous system independently of supply by peripheral endocrine glands. Dehydroepiandrosterone (DHA) and pregnenolone (delta 5P) were first found in the rat brain. Then, a steroid biosynthetic pathway was demonstrated in oligodendrocytes, mostly by enzyme immunocytochemistry and biochemical studies in primary cultures of glial cells, where the formation, from appropriate radioactive precursors, of delta 5P, delta 5-pregn-3 beta, 20 alpha-diol (20 alpha-DH delta 5P), progesterone (P), 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 3 alpha-hydroxy-5 alpha-pregnane-20-one (3 alpha, 5 alpha-THP), as well as estrogen-induced nuclear progesterone receptor (PR) was observed. Several biological effects of neurosteroids have been observed, such as electrical stimulation of neurones, involvement in behaviorial activities, modulation of GABAA-receptor (GABAA-R) function (potentiated by 3 alpha, 5 alpha-THP and its 21-hydroxyderivative, antagonized by delta 5P- and DHA-sulfates) and growth/differentiation of glial cells in vitro. Preliminary findings suggest that the neurosteroid concept applies to all mammalian species, including man. Further investigations should assess the pathophysiological significance of the synthesis of neurosteroids and decipher their mechanisms of action via nuclear and membrane receptors.     

Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by Bacillus sphaericus

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.     

Structural and Functional Properties,Biosynthesis, and Patenting Trends of Bacterial Succinoglycan: A Review

The exopolysaccharide succinoglycan is produced mainly by a large number of soil microbes of Agrobacterium, Rhizobium or Pseudomonas genera etc. Structural properties of succinoglycan are unique in terms of its thermal stability and superior viscosifying property. Unlike the other highly commercialized bacterial exopolysaccharides like dextran or xanthan, mass scale application of succinoglycan has not been that much broadly explored yet. Bacterial succinoglycan is found suitable as a viscosifying and emulsifying agent in food industry, in gravel packing or fluid-loss control agent etc. In this present review, the key aspects of succinoglycan study, in particular, developments in structural characterizations, exo/exs operon system involved in biosynthesis pathway, commercial applications in food and other industries and patenting trends have been discussed.     

细菌素的合成与作用机制

细菌素是由细菌产生的抗菌蛋白,可以杀死与产生菌相近的细菌。很多乳酸菌产生不同多样性的细菌素,虽然这些细菌素都是由发酵或非发酵食品中发现的乳酸菌产生的,但是迄今只有乳酸链球菌素(Nisin)作为食品防腐剂被广泛应用。和抗生素不同的是,细菌素由核糖体合成,需经翻译后修饰活化并且通过特定转运系统输到胞外才能发挥其功能,它一般通过作用于靶细胞膜来抑制靶细胞的生长,同时本身合成细菌素的细胞对其产物具有免疫性。细菌素能安全有效地抑制病原体生长,在食品行业中具有广阔的应用前景。     

Biosynthesis and assay of neurosteroids in rats and mice: Functional correlates

Pregnenolone (PREG), synthesized de novo in rodent brain, is the precursor of PREG sulfate (S) and progesterone (PROG). PROG is further converted to 5-pregnane 3, 20-dione (DH PROG) and to 3-hydroxy-5-pregnan-20-one (TH PROG). PROG, DH PROG and TH PROG have been measured in the brain of male and female rats. Neither PROG nor DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy (ADX) and gonadectomy (CX). Trilostane decreased PROG and increased PREG in the brain of CX + ADX rats and mice, in accordance with a precursor to product relationship. As previously described in CX male mice, the neurosteroid DHEA and its analog 3β-methyl-androst-5-en-17-one (CH₃-DHEA) inhibited the aggressive behavior of female mice towards lactating female intruders. The decrease of biting attacks by DHEA was definitely more prominent in females neonatally imprinted with testosterone. The degree of inhibition of aggressive behavior was related to the decrease of PREG S concentrations in brain. The memory-enhancing effects of DHEA S and PREG S in male mice have been previously documented. Infusion of PREG S (12 fmol) into the nucleus basalis magnocellularis (NBM) of the rat after the acquisition trial enhanced memory performance in a two-trial recognition task (TTRT). Conversely, TH PROG (6 fmol), which potentiates GABAergic neurotransmission, disrupted performance when injected before the acquisition trial. Accordingly, we have found a positive correlation between the performances of 2-year-old rats in the TTRT and the concentrations of PREG S in the hippocampus, namely animals which performed best had the highest steroid levels.     

Cholesterol Biosynthesis and Homeostasis in Regulation of the Cell Cycle

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer’s disease, that are associated with impaired cholesterol biosynthesis and homeostasis.