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Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG-BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use.

We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C 8 ), decanoic acid (C 10 ) or dodecanoic acid (C 12 ). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C 12 -cTurg-1 and C 8 -cTurg-2 . These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus , Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens . The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys 1 -Cys 6 , Cys 2 -Cys 5 , and Cys 3 -Cys 4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin A Mox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC 50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC 50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.

Arasin 1 from the spider crab Hyas araneus is a proline-rich antimicrobial peptide (PR-AMP), which kills target bacteria by a non-membranolytic mechanism. By using a fluorescent derivative of the peptide, we showed that arasin 1 rapidly penetrates into Escherichia coli cells without membrane damage. To unravel its mode of action, a knockout gene library of E. coli was screened and two types of mutants with a less susceptible phenotype to the arasin 1 fragment (1-23) were found. The first bore the mutation of sbmA , a gene coding for an inner membrane protein involved in the uptake of different antibiotic peptides. The second mutation was located in the ygdD gene, coding for a conserved inner membrane protein of unknown function. Functional studies showed that YgdD is required for the full susceptibility to arasin 1(1-25), possibly by supporting its uptake and/or intracellular action. These results indicated that different bacterial proteins are exploited by arasin 1(1-25) to exert its antibacterial activity and add new insights on the complex mode of action of PR-AMPs.

Antimicrobial peptides (AMPs) are important effector molecules in innate immunity. Here we briefly summarize characteristic traits of AMPs and their mechanisms of antimicrobial activity. Echinoderms live in a microbe-rich marine environment and are known to express a wide range of AMPs. We address two novel AMP families from coelomocytes of sea urchins: cysteine-rich AMPs (strongylocins) and heterodimeric AMPs (centrocins). These peptide families have conserved preprosequences, are present in both adults and pluteus stage larvae, have potent antimicrobial properties, and therefore appear to be important innate immune effectors. Strongylocins have a unique cysteine pattern compared to other cysteine-rich peptides, which suggests a novel AMP folding pattern. Centrocins and SdStrongylocin 2 contain brominated tryptophan residues in their native form. This review also includes AMPs isolated from other echinoderms, such as holothuroidins, fragments of beta-thymosin, and fragments of lectin (CEL-III). Echinoderm AMPs are crucial molecules for the understanding of echinoderm immunity, and their potent antimicrobial activity makes them potential precursors of novel drug leads., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) play a crucial role in innate immunity. We have previously reported the isolation and characterization of the AMPs, strongylocins 1 and 2, and centrocin 1, from coelomocyte extracts of Strongylocentrotus droebachiensis. Here we show that these AMPs were expressed in phagocytes. In addition, transcripts of strongylocin 1 were detected in vibratile cells and/or colorless spherule cells, while transcripts of strongylocin 2 were found in red spherule cells. Results from immunoblotting and immunocytochemistry studies showed that centrocin 1 was produced by phagocytes and stored in granular vesicles. Co-localization of centrocin 1 and phagocytosed bacteria suggests that the granular vesicles containing centrocin 1 may be involved in the formation of phagolysosomes. We also analyzed the temporal and spatial expression of AMPs throughout larval development. Strongylocins were expressed in the early pluteus stage, while centrocin 1 was expressed in the mid pluteus stage. The spatial expression pattern showed that centrocin 1 was mainly located in blastocoelar cells (BCs) around the stomach and the esophagus. In addition, a few patrolling BCs were detected in some larval arms. Together, these results suggest that AMPs are expressed in different types of coelomocytes and that centrocin 1 is involved in response against bacteria. Furthermore, the expression of AMPs in larval pluteus stage, especially in BCs, indicates that AMPs and BCs are engaged in the larval immune system., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K m and k cat of 39 μM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.

Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH(2) terminus of the peptide and the fragment arasin 1(1-23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1-23) were shown to be non-toxic to human red blood cells and arasin 1(1-23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1-23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1-23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1-23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1-23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC.

Bacterial resistance against antibiotic treatment has become a major threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternative agents for treatment of infectious diseases. This study characterizes novel synthetic peptides sequentially derived from the AMP centrocin 1, isolated from the green sea urchin, for their applicability as anti-infective agents.The microbicidal effect of centrocin 1 heavy chain (CEN1 HC-Br), its debrominated analogue (CEN1 HC), the C-terminal truncated variants of both peptides, i.e. CEN1 HC-Br (1-20) and CEN1 HC (1-20), as well as the cysteine to serine substituted equivalent CEN1 HC (Ser) was evaluated using minimal microbicidal concentration assay. The anti-inflammatory properties were assessed by measuring the inhibition of secretion of pro-inflammatory cytokines. All the peptides tested exhibited marked microbicidal and anti-inflammatory properties. No difference in efficacy was seen comparing CEN1 HC-Br and CEN1 HC, while the brominated variant had higher cytotoxicity. C-terminal truncation of both peptides reduced salt-tolerability of the microbicidal effect as well as anti-inflammatory actions. Also, serine substitution of cysteine residue decreased the microbicidal effect. Thus, from the peptide variants tested, CEN1 HC showed the best efficacy and safety profile. Further, CEN1 HC significantly reduced bacterial counts in two different animal models of infected wounds, while Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance against this peptide under continued selection pressure. In summary, CEN1 HC appears a promising new antimicrobial agent, and clinical studies are warranted to evaluate the applicability of this AMP for local treatment of infections in man.

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cysteine-rich strongylocins were the first antimicrobial peptides (AMPs) discovered from the sea urchin species, Strongylocentrotus droebachiensis. Homologous putative proteins (called SpStrongylocin) were found in the sister species, S. purpuratus. To demonstrate that they exhibit the same antibacterial activity as strongylocins, cDNAs encoding the 'mature' peptides (SpStrongylocins 1 and 2) were cloned into a direct expression system fusing a protease cleavage site and two purification tags to the recombinant peptide. Both recombinant fusion peptides were expressed in a soluble form in an Escherichia coli strain tolerant to toxic proteins. Enterokinase was used to remove the fusion tags and purified recombinant SpStrongylocins 1 and 2 showed antimicrobial activity against both Gram-negative and Gram-positive bacteria. The results of membrane integrity assays against cytoplasmic membranes of E. coli suggest that both recombinant SpStrongylocins 1 and 2 conduct their antibacterial activity by intracellular killing mechanisms because no increase in membrane permeability was detected., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase. The expression of the gltAB operon encoding the latter enzyme is impaired in B. subtilis ccpA mutant strains. CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism. However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively. In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied. Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources. In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants. A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium. Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium. It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant. The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media. The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism.

Glycolysis is one of the central routes of carbon catabolism in Bacillus subtilis. Several glycolytic enzymes, including the key enzyme glyceraldehyde-3-phosphate dehydrogenase, are encoded in the hexacistronic gapA operon. Expression of this operon is induced by a variety of sugars and amino acids. Under non-inducing conditions, expression is repressed by the CggR repressor protein, the product of the promoter-proximal gene of the operon. Here, it is shown that the amount of glyceraldehyde-3-phosphate dehydrogenase encoded by the second gene of the operon exceeds that of the CggR repressor by about 100-fold. This differential synthesis was attributed to an mRNA processing event that takes place at the 3' end of the cggR open reading frame and to differential segmental stabilities of the resulting cleavage products. The mRNA specifying the truncated cggR gene is quickly degraded, whereas the downstream processing products encompassing gapA are quite stable. This increased stability is conferred by the presence of a stem-loop structure at the 5' end of the processed mRNAs. Mutations were introduced in the region of the cleavage site. A mutation affecting the stability of the stem-loop structure immediately downstream of the processing site had two effects. First, the steady-state transcript pattern was drastically shifted towards the primary transcripts; second, the stability of the processed mRNA containing the destabilized stem-loop structure was strongly decreased. This results in a reduction of the amount of glyceraldehyde-3-phosphate dehydrogenase in the cell. It is concluded that mRNA processing is involved in differential syntheses of the proteins encoded by the gapA operon.

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.