Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of "cryptic" antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) are essential components of innate immunity. Here we show that antimicrobial potency of CAMPs is linearly correlated to the product CmHnL where C is the net charge of the peptide, H is a measure of its hydrophobicity and L its length. Exponents m and n define the relative contribution of charge and hydrophobicity to the antimicrobial potency. Very interestingly the values of m and n are strain specific. The ratio n/(m+n) can vary between ca. 0.5 and 1, thus indicating that some strains are sensitive to highly charged peptides, whereas others are particularly susceptible to more hydrophobic peptides. The slope of the regression line describing the correlation "antimicrobial potency"/"CmHnL product" changes from strain to strain indicating that some strains acquired a higher resistance to CAMPs than others. Our analysis provides also an effective computational strategy to identify CAMPs included inside the structure of larger proteins or precursors, which can be defined as "cryptic" CAMPs. We demonstrate that it is not only possible to identify and locate with very good precision the position of cryptic peptides, but also to analyze the internal structure of long CAMPs, thus allowing to draw an accurate map of the molecular determinants of their antimicrobial activity. A spreadsheet, provided in the Supplementary material, allows performing the analysis of protein sequences. Our strategy is also well suited to analyze large pools of sequences, thus significantly improving the identification of new CAMPs and the study of innate immunity.

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern.

BACKGROUND: Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome. RESULTS: PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y's phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism. CONCLUSIONS: In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Acclimation to hypoxia in Chlamydomonas reinhardtii: can biophotolysis be the major trigger for long-term H2 production?

In anaerobiosis, the microalga Chlamydomonas reinhardtii is able to produce H2 gas. Electrons mainly derive from mobilization of internal reserves or from water through biophotolysis. However, the exact mechanisms triggering this process are still unclear. Our hypothesis was that, once a proper redox state has been achieved, H2 production is eventually observed. To avoid nutrient depletion, which would result in enhanced fermentative pathways, we aimed to induce long-lasting H2 production solely through a photosynthesis : respiration equilibrium. Thus, growing cells were incubated in Tris Acetate Phosphate (TAP) medium under low light and high chlorophyll content. After a 250-h acclimation phase, a 350-h H2 production phase was observed. The light-to-H2 conversion efficiency was comparable to that given in some reports operating under sulphur starvation. Electron sources were found to be water, through biophotolysis, and proteins, particularly through photofermentation. Nonetheless, a substantial contribution from acetate could not be ruled out. In addition, photosystem II (PSII) inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) showed that it actively contributed to maintaining a redox balance during cell acclimation. In appropriate conditions, PSII may represent the major source of reducing power to feed the H2 evolution process, by inducing and maintaining an ideal excess of reducing power.

Identification and physicochemical characterization of BldR2 from Sulfolobus solfataricus, a novel archaeal member of the MarR transcription factor family.

The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators abundantly distributed throughout the bacterial and archaeal domains. Here we describe the identification and characterization of BldR2, as a new member of this family, in the archaeon Sulfolobus solfataricus and report physiological, biochemical, and biophysical investigation of its stability and DNA binding ability. Transcriptional analysis revealed the upregulation of BldR2 expression by aromatic compounds in the late-logarithmic growth phase and allowed the identification of cis-acting sequences. Our results suggest that BldR2 possesses in solution a dimeric structure and a high stability against both temperature and chemical denaturing agents; the protein binds site specifically to its own promoter, Sso1082, with a micromolar binding affinity at two palindromic sites overlapping TATA-BRE and the transcription start site. Benzaldehyde and salicylate, ligands of MarR members, are antagonists of binding of DNA by BldR2. Moreover, two single-point mutants of BldR2, R19A and A65S, properly designed for obtaining information about the dimerization and the DNA binding sites of the protein, have been produced and characterized. The results point out an involvement of BldR2 in the regulation of the stress response to aromatic compounds and point to arginine 19 as a key amino acid involved in protein dimerization, while the introduction of serine 65 increases the DNA affinity of the protein, making it comparable with those of other members of the MarR family.

Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels.

The unicellular green microalga Chlamydomonas reinhardtii is a powerful photosynthetic model organism which is capable of heterotrophic growth on acetate as a sole carbon source. This capacity has enabled its use for investigations of perturbations in photosynthetic machinery as mutants can be recovered heterotrophically. Fixation of acetate into cellular carbon metabolism occurs first by its conversion into acetyl-CoA by a respective synthase and the generation of succinate by the glyoxylate cycle. These metabolic steps have been recently determined to largely occur in the peroxisomes of this alga; however, little is known about the trafficking and import of acetate or its subcellular compartmentalization. Recently, the genes of five proteins belonging to the GPR1/FUN34/YaaH (GFY) superfamily were observed to exhibit increased expression in C. reinhardtii upon acetate addition, however, no further characterization has been reported. Here, we provide several lines of evidence to implicate Cr GFY1-5 as channels which share structural homology with bacterial succinate-acetate channels and specifically localize to microbodies, which are surprisingly distinct from the glyoxylate cycle-containing peroxisomes. We demonstrate structural models, gene expression profiling, and in vivo fluorescence localization of all five isoforms in the algal cell to further support this role.

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.

Combined elicitation of methyl-jasmonate and red light on stilbene and anthocyanin biosynthesis.

Vitis vinifera cell suspensions are a suitable system to study the metabolic regulation of a large range of polyphenols, including flavonoids and stilbenes that play important roles in plant development. Grape cv. Barbera petioles cell cultures were treated with red light and 10 µM methyl-jasmonate (MeJA), alone or in combination, to investigate their influence and/or induction effect on the production of anthocyanins, catechins and free and mono-glucosylated stilbenes. The synthesis of total anthocyanins was slightly decreased by red light alone, while MeJA and MeJA plus red light increased the levels of these metabolites. When compared to the relative controls, the red light treatment decreased the amount of catechins and increased their release in the culture medium, while MeJA alone or in combination with red light increased their production. Red light treatment generally enhanced the amount of free and mono-glucosylated stilbenes during the entire observation period, as well as the percentage of their release in the media. Treatment with MeJA strongly promoted the production of total stilbenes, which was further elicited by the MeJA plus red light treatment. During the combined treatment, the presence of the light stimulus improved the effect of MeJA by anticipating the maximum increase of stilbenes which were also largely released (up to 90%). These results demonstrate that, in grapevine, as in other plant systems, the change of conditions in which the MeJA stimulus is perceived (e.g. going from total white to red light) drastically modifies the plant response to this hormone. The present paper confirms that the jasmonate transduction pathway is integrated into an elaborate signaling network that also comprehends the red light signaling pathway.

Thermal stabilization of psychrophilic enzymes: a case study of the cold-active hormone-sensitive lipase from Psychrobacter sp. TA144.

Cold-adapted enzymes possess high specific activity at low and moderate temperatures with respect to their mesophilic and thermophilic homologs; it is accepted that they have a less rigid and more flexible structure in the region surrounding the active site. However, the low stability of such molecules could represent the main barrier for their application in some industrial bioprocesses. The aim of this article was to investigate the ability of the naturally occurring osmolytes to increase the thermal stability and the specific activity of the cold-active lipase from Psychrobacter sp. TA144 (PsyHSL), which belongs to the hormone-sensitive lipase group. The effect of trimethylamine N-oxide (TMAO), betaine, and L-proline addition on the activity and thermal stability of PsyHSL was investigated by means of biochemical and biophysical techniques. It turned out that in the presence of 3 M TMAO, the enzyme specific activity enhanced up to 250% at 50°C, while the addition of 1 M TMAO increased the thermostability fivefold at 45°C. Our experiments demonstrated that, even in the case of a psychrophilic enzyme, osmoprotectants, particularly TMAO, addition may be considered an efficient strategy to improve the protein thermal stability and specific activity at higher temperatures.