Mario Varcamonti

Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis

BackgroundApplications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods. Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH. Thus the isolation of new bacteriocins active in foods is desirable.ResultsWe isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490. This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp. both in aerobic and in anaerobic conditions. Bactericidal activity was kept during storage at 4°C and was remarkably stable in a wide pH range. The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa). Antimicrobial activity against B. smithii was observed also when this organism was grown in water buffalo milk.ConclusionsBacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature. These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp. in non-acidic foods. The small size suggests its use on solid foods.

Anti-biofilm activity of an exopolysaccharide from a sponge-associated strain of Bacillus licheniformis

BackgroundSecondary metabolites ranging from furanone to exo-polysaccharides have been suggested to have anti-biofilm activity in various recent studies. Among these, Escherichia coli group II capsular polysaccharides were shown to inhibit biofilm formation of a wide range of organisms and more recently marine Vibrio sp. were found to secrete complex exopolysaccharides having the potential for broad-spectrum biofilm inhibition and disruption.ResultsIn this study we report that a newly identified ca. 1800 kDa polysaccharide having simple monomeric units of α-D-galactopyranosyl-(1→2)-glycerol-phosphate exerts an anti-biofilm activity against a number of both pathogenic and non-pathogenic strains without bactericidal effects. This polysaccharide was extracted from a Bacillus licheniformis strain associated with the marine organism Spongia officinalis. The mechanism of action of this compound is most likely independent from quorum sensing, as its structure is unrelated to any of the so far known quorum sensing molecules. In our experiments we also found that treatment of abiotic surfaces with our polysaccharide reduced the initial adhesion and biofilm development of strains such as Escherichia coli PHL628 and Pseudomonas fluorescens.ConclusionThe polysaccharide isolated from sponge-associated B. licheniformis has several features that provide a tool for better exploration of novel anti-biofilm compounds. Inhibiting biofilm formation of a wide range of bacteria without affecting their growth appears to represent a special feature of the polysaccharide described in this report. Further research on such surface-active compounds might help developing new classes of anti-biofilm molecules with broad spectrum activity and more in general will allow exploring of new functions for bacterial polysaccharides in the environment.

The Marine Isolate Novosphingobium sp. PP1Y Shows Specific Adaptation to Use the Aromatic Fraction of Fuels as the Sole Carbon and Energy Source

Novosphingobium sp. PP1Y, isolated from a surface seawater sample collected from a closed bay in the harbour of Pozzuoli (Naples, Italy), uses fuels as its sole carbon and energy source. Like some other Sphingomonads, this strain can grow as either planktonic free cells or sessile-aggregated flocks. In addition, this strain was found to grow as biofilm on several types of solid and liquid hydrophobic surfaces including polystyrene, polypropylene and diesel oil. Strain PP1Y is not able to grow on pure alkanes or alkane mixtures but is able to grow on a surprisingly wide range of aromatic compounds including mono, bi, tri and tetracyclic aromatic hydrocarbons and heterocyclic compounds. During growth on diesel oil, the organic layer is emulsified resulting in the formation of small biofilm-coated drops, whereas during growth on aromatic hydrocarbons dissolved in paraffin the oil layer is emulsified but the drops are coated only if the mixtures contain selected aromatic compounds, like pyrene, propylbenzene, tetrahydronaphthalene and heterocyclic compounds. These peculiar characteristics suggest strain PP1Y has adapted to efficiently grow at the water/fuel interface using the aromatic fraction of fuels as the sole carbon and energy source.

Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon

The importance of fish in vertebrate evolution has been better recognized in recent years after the intense work carried out on fish genomics. The recent discovery that fish genomes comprise homologs of ribonucleases, studied before only in tetrapods, and the isolation of ribonucleases from zebrafish have suggested an experimental model for studying fish and vertebrate evolution. Thus, the cDNAs encoding the RNases from the Atlantic salmon were expressed, and the recombinant RNases (Ss‐RNase‐1 and Ss‐RNase‐2) were isolated and characterized as both proteins and for their biological activities. Salmon RNases are less active than RNase A in degrading RNA, but are both sensitive to the action of the human cytosolic RNase inhibitor. The two enzymes possess both angiogenic and bactericidal activities. However, catalytically inactivated Ss‐RNases do not exert any angiogenic activity, but preserve their full bactericidal activity, which is surprisingly preserved even when the enzyme proteins are fully denatured. Analyses of the conformational stability of the two RNases has revealed that they are as stable as typical RNases of the superfamily, and Ss‐RNase‐2, the most active as an enzyme, is also the most resistant to thermal and chemical denaturation. The implications of these findings in terms of the evolution of early RNases, in particular of the physiological significance of the angiogenic and bactericidal activities of fish RNases, are analyzed and discussed.

Effect of inactivation of stress response regulators on the growth and survival of Streptococcus thermophilus Sfi39.

Streptococcus thermophilus is an important dairy starter used for the manufacture of fermented milks and cheeses. As for many other lactic acid bacteria, its survival during propagation and preservation of starter cultures and its performance in dairy fermentations are significantly affected by its ability to cope with many environmental stresses. This, in turn, is dependent on the expression of a several genes, which is often controlled by negative response regulators, and has complex relationships with the control of metabolism. In this paper we describe the effect of growth phase, adaptation and inactivation of genes for stress response regulators (hrcA, ctsR and rr01) on the growth, acid production and stress resistance and intracellular protein patterns in the yoghurt strain S. thermophilus Sfi39. Inactivation of stress response regulators significantly affected growth, acid production in milk and stress tolerance. Although mutants showed an increased resistance during the exponential phase compared to the wild type strain, the effect of the mutations was complex and in some cases mutants were unable to mount an acid- or heat-shock response after exposure to mild heat and acid stresses. Significant differences in intracellular protein patterns analyzed by SDS-PAGE and 2-DE/mass spectrometry were found as a function of growth phase, adaptation and mutation, but a detailed transcriptomic and proteomic study would be needed to provide a mechanistic interpretation of the effect of the mutations and to identify a strategy for the construction of food-grade mutants with improved growth and performances.

Expression of the heat shock gene clpL of Streptococcus thermophilus is induced by both heat and cold shock

BackgroundHeat and cold shock response are normally considered as independent phenomena. A small amount of evidence suggests instead that interactions may exist between them in two Lactococcus strains.ResultsWe show the occurrence of molecular relationships between the mechanisms of cold and heat adaptations in Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentation, where it undergoes both types of stress. We observed that cryotolerance is increased when cells are pre-incubated at high temperature. In addition, the production of a protein, identified as ClpL, a member of the heat-shock ATPase family Clp A/B, is induced at both high and low temperature. A knock-out clpL mutant is deficient in both heat and cold tolerance. However lack of production of this protein does not abolish the positive effect of heat pre-treatment towards cryotolerance.ConclusionDual induction of ClpL by cold and heat exposure of cells and reduced tolerance to both temperature shocks in a clpL mutant indicates that the two stress responses are correlated in S. thermophilus. However this protein is not responsible by itself for cryotolerance of cells pre-treated at high temperature, indicating that ClpL is necessary for the two phenomena, but does not account by itself for the relationships between them.

Bioactive Terpenes from Spongia officinalis

The terpene metabolite pattern of Mediterranean Spongia officinalis was chemically investigated. This study resulted in the isolation of a series of sesterterpenes and C21 furanoterpenes, according to the literature data on this sponge. Four new oxidized minor metabolites (compounds 1, 2, 3, and 4) were isolated along with six known compounds of the furospongin series (compounds 5-8, 9, and 10) and three scalarane sesterterpenes (compounds 11-13). Interestingly, tetrahydrofurospongin-2 (6) and dihydrofurospongin-2 (7), which were among the main metabolites, induced biofilm formation by Escherichia coli. All compounds isolated were also assayed for antibacterial and antifungal properties.

A new cryptic cationic antimicrobial peptide from human apolipoprotein E with antibacterial activity and immunomodulatory effects on human cells

Cationic antimicrobial peptides (AMPs) possess fast and broad‐spectrum activity against both Gram‐negative and Gram‐positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein‐derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133–150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad‐spectrum antibacterial activity. As shown for several other AMPs, ApoE (133–150) is structured in the presence of TFE and of membrane‐mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo‐polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133–150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP‐1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti‐inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.

Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.

The bactericidal action on Escherichia coli of ZF‐RNase‐3 is triggered by the suicidal action of the bacterium OmpT protease

ZF‐RNase‐3 is one of the RNases from zebrafish (Danio rerio) with special (i.e. noncatalytic) properties. These include angiogenic and bactericidal activities. Given the interest of fish RNases as host‐defense effectors, we studied the mechanism of the bactericidal action of ZF‐RNase‐3 on Escherichia coli as a model Gram‐negative bacterium. The results obtained indicate that the bactericidal activity of ZF‐RNase‐3 is not lost when its catalytic RNase activity is obliterated. On the other hand, fully denatured ZF‐RNase‐3 conserves its bactericidal activity. When ZF‐RNase‐3 is added to E. coli cultures, it is cleaved at a specific Arg‐Arg peptide bond, thus engendering two peptide fragments. The larger fragment (residues 31–124), produced by proteolysis and reduction of a disulfide, is recognized as the actual bactericidal agent. The protease responsible for the proteolytic attack has been identified with OmpT, an outer membrane E. coli omptin protease. However, the most remarkable result obtained in the present study is the finding that the microbicidal action of ZF‐RNase‐3 can be achieved only with the suicidal cooperation of the bacterium itself.