Short and ultrashort antimicrobial peptides anchored onto soft commercial contact lenses inhibit bacterial adhesion.

Commercial soft contact lenses were chemically modified to incorporate antibacterial properties. Contact lenses and especially soft contact lenses present a risk of eye microbial infection that eventually may lead to vision loss. This is a significant health issue given the large population of contact lenses wearers worldwide. In order to introduce bactericidal activity in hydrogel contact lenses, one short and one ultrashort antimicrobial peptides, LKKLLKLLKKLLKL (LK) and IRIRIRIR (IR), were selected. These peptides were anchored on the surface of contact lenses using a linker (1,4-butanediol diglycidyl ether) under mild conditions (room temperature, pH = 7.4). Physical and chemical properties of peptide-functionalized contact lenses were investigated through several analytical techniques including wettability, Raman confocal microscopy, fluorescence studies, refractometry and spectrophotometry. These studies demonstrated that contact lens modification occurred at the nanolevel (ng/lens). Bacterial cultures showed that peptide-functionalized contact lenses can drastically reduce bacterial adhesion and viability when exposed to Pseudomonas aeruginosa and Staphylococcus aureus. These systems offer the potential to minimise corneal bacterial infection and represent a suitable platform for future ophthalmic devices.

Marinobacter maroccanus sp. nov., a moderately halophilic bacterium isolated from a saline soil.

During the taxonomic investigation of exopolymer-producing halophilic bacteria, a rod-shaped, motile, Gram-stain-negative, halophilic bacterium, designated strain N4T, was isolated from a saline soil located in northern Morocco. Optimal growth of the isolate was at 30-37 ºC and at pH 7.0-8.0, in the presence of 5-7 % (w/v) NaCl. Useful characteristics for the phenotypic differentiation of strain N4T from other Marinobacter species included α-chymotrypsin and α-glucosidase activities and the carbohydrate assimilation profile. The major fatty acids detected in strain N4T were C16:0 and C18:1ω9c and the predominant respiratory quinone was ubiquinone-9. Sequence analysis of the 16S rRNA gene indicated that strain N4T belonged to the genus Marinobacter and was closely related to the type strains of Marinobacter adhaerens (99.04 % similarity), Marinobacter salsuginis (98.97 %) and Marinobacter flavimaris (98.36 %). Phylogenetic analysis of the rpoD gene sequence also showed that the nearest neighbours of strain N4T were M. salsuginis (91.49 % similarity), M. adhaerens and M. flavimaris (90.63 %). Strain N4T showed 87.98 % average nucleotide identity with M. flavimaris and M. salsuginis, and 87.47 % with M. adhaerens. Regarding in-silico genome-to-genome distance, strain N4T showed DNA-DNA hybridization values of 33.30 % with M. adhaerens, 34.60 % with M. flavimaris and 34.70 % with M. salsuginis. The DNA G+C content of strain N4T was 57.3 mol%. Based on the results of phenotypic characterization, phylogenetic analysis and genome comparison, strain N4T represents a novel species of the genus Marinobacter, for which the name Marinobacter maroccanus sp. nov. is proposed. The type strain is N4T (=CECT 9525T=LMG 30466T).

Complete Genome Sequence of Marinobacter flavimaris LMG 23834T, Which Is Potentially Useful in Bioremediation.

The complete genome sequence of the halophilic strain Marinobacter flavimaris LMG 23834T is presented here. The genomic information of this type strain will be useful for taxonomic purposes and for its potential use in bioremediation studies.

Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity.

Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty acids, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.

Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa.

Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa_LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N-terminal region of Pa_LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn-1) and 14/16 (sn-2) carbon atoms in length. Carbon atoms from the sn-1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa_LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. aeruginosa with host cells.

Production of 10(S)-hydroxy-8(E)-octadecenoic and 7,10(S,S)-hydroxy-8(E)-octadecenoic ethyl esters by Novozym 435 in solvent-free media.

Novozym 435, lipase B from Candida antarctica, was used in this study for the production of ethyl esters. For the first time, trans-hydroxy-fatty acid ethyl esters were synthesized in vitro in solvent-free media. We studied the effects of the substrate-ethanol molar ratio and enzyme synthetic stability of the biocatalyst. To determine the structure of the formed compounds, Fourier transformed infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry were used, three less time-consuming structural techniques. trans-Hydroxy-fatty acid ethyl esters were synthesized with a reaction yield of 90 % or higher with optimal reaction conditions.

Mixed monolayer of DPPC and lysine-based cationic surfactants: an investigation into the antimicrobial activity.

In this paper, we report studies which aim to elucidate the mechanisms involved in the antimicrobial activity of three cationic lysine-based surfactants: LLM, LALM, and C6 (LL)2. To this end, a simple membrane model (i.e., 1,2-dipalmitoyl-sn-phosphatidylcholine, DPPC) was used to explore the monolayer properties at the air/liquid interface. Compression π-A isotherms of mixtures of DPPC/lysine surfactants at different pH showed an expansion of the DPPC monolayer, suggesting cationic lysine surfactant/DPPC interactions, which strongly depend on surfactant structure and hydrophobic interactions. Antimicrobial activity of the three surfactants has also been assessed with transmission electron microscopy, observing the effects on Staphylococcus aureus and Escherichia coli . The three surfactants caused various kinds of damage to the bacteria tested, such as structural alterations, leakage of internal material, and cell destruction.

Sphingobacterium detergens sp. nov., a surfactant-producing bacterium isolated from soil.

A novel Gram-negative-staining strain, designated 6.2S(T), was isolated from a soil sample and identified as a biosurfactant producer. Its taxonomic position was investigated using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 °C, with 0-3% (w/v) NaCl, and at pH 7.0. Based on 16S rRNA gene sequence analysis, strain 6.2S(T) was found to be a member of the genus Sphingobacterium and was most closely related to four type species of the genus, showing sequence similarities of 96.8-98.9%. Partial chaperonin 60 (cpn60) gene sequence analysis was useful in resolving the phylogenetic relationships between strain 6.2S(T) and closely related taxa, with similarities ranging from 85.5% (with Sphingobacterium thalpophilum DSM 11723(T)) to 90.3% (with Sphingobacterium canadense CR11(T) and Sphingobacterium multivorum JCM 21156(T)). The results of DNA-DNA hybridization experiments between the novel strain and its closest relatives gave a DNA-DNA relatedness value of less than 70%, and consequently confirmed that this new strain did not belong to a previously described species of the genus Sphingobacterium. The major fatty acids were summed feature 3 (iso-C(15:0) 2 OH and/or C(16:1)ω7c); iso-C(15:0); iso-C(17:0) 3-OH and C(16:0). The G+C content of the genomic DNA was 40.0 mol%. According to its phenotypic and genotypic characteristics and the phylogenetic data, strain 6.2S(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium detergens sp. nov. is proposed. The type strain is 6.2S(T) ( = CECT 7938(T) = LMG 26465(T)).

Rhamnolipid surfactants: alternative substrates, new strategies.

This chapter concentrates on the various possibilities of using alternative substrates and new strategies. Such strategies include an integrated production system to reduce the environmental impact and an attempt to minimize residues, which reinforces socio-economic and region-structural development. Additionally, we offer an overview of the physicochemical and biological properties of rhamnolipid surfactants associated with the applications of these molecules in different circumstances.

Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.

Although Anoxybacillus and Geobacillus, two genera of thermophilic bacteria close to the genus Bacillus, have only been described recently, the number of species in these genera has increased rapidly. Four thermophilic, lipolytic strains (DR01, DR02, DR03 and DR04) isolated from a hot spring in Veracruz (Mexico), which could not be identified phenotypically, were subjected to 16S rRNA gene sequence analysis. Three strains were identified as belonging to the genus Anoxybacillus, but strain DR03 was identified as Geobacillus pallidus. This result led us to perform a phylogenetic analysis of the genera Anoxybacillus and Geobacillus based on 16S rRNA gene sequences from all the type strains of these genera. Phylogenetic trees showed three major clusters, Anoxybacillus-Geobacillus tepidamans, Geobacillus sensu stricto and Geobacillus pallidus, while the 16S rRNA gene sequences of G. pallidus (DR03 and the type strain) showed low similarity to sequences of Anoxybacillus (92.5-95.1 %) and Geobacillus (92.8-94.5 %) species, as well as to Bacillus subtilis (92.2-92.4 %). In addition, G. pallidus could be differentiated from Anoxybacillus and Geobacillus on the basis of DNA G+C content and fatty acid and polar lipid profiles. From these results, it is proposed that Geobacillus pallidus should be classified in a novel genus, for which we propose the name Aeribacillus, as Aeribacillus pallidus gen. nov., comb. nov. The type strain of Aeribacillus pallidus is H12(T) (=ATCC 51176(T) =DSM 3670(T) =LMG 19006(T)).

Effect of emulsified feeding of oily substrate via submerged ceramic membranes on surfactant production in Pseudomonas aeruginosa fermentation.

Aqueous two-phase systems often face mass transfer limitations due to very poor miscibility of the fluids, and to enhance the homogeneity (or emulsification) in the reaction volume, high energy inputs are required which result in high shear forces in the culture medium. For the purposes of emulsification, microporous systems have advantages over other conventional methods due to mild operating conditions and narrow droplet-size distribution. In this study, emulsification within the culture volume was achieved by feeding the oily substrate (dispersed phase) into the aqueous medium (dispersion phase) via ceramic membranes integrated in the bioreactor. The method was investigated for bioprocesses aimed at producing rhamnolipids and polyhydroxyalkanoates (PHA). Better homogenization of the mixed volume and hence improved consumption of oily substrate was successful. Surfactants are produced by various bacterial cultures, especially Pseudomonas aeruginosa species, when oil is present as the only carbon source. Are surfactants produced only as a result of bacteria feeding on the oily substrate, or as a requirement to feed on the oily substrate, owing to their surface-active characteristics? This paper also intends to draw some conclusions in this respect.

Chemoenzymatic synthesis and antimicrobial and haemolytic activities of amphiphilic bis(phenylacetylarginine) derivatives.

Novel bis(N(alpha)-phenylacetyl-L-arginine)-alpha,omega-alkanediamide dihydrochloride (bis(PhAcArg)) derivatives with antimicrobial activity were designed and synthesised by a chemoenzymatic strategy. The new structures consist of two N(alpha)-phenylacetyl-L-arginine moieties connected by an alkanediamine spacer chain of 6, 8, 10, 12, and 14 methylene units through amide bonds. The key step in the chemoenzymatic strategy is the double aminolysis of the N(alpha)-phenylacetyl-L-arginine methyl ester by the corresponding alpha,omega-alkanediamine catalyzed by papain in ethanolic media. The compounds synthesised were tested as antimicrobials against 15 bacterial and 8 fungal species. The antimicrobial activity and selectivity depend strongly on the spacer chain length. The bis(PhAcArg) derivative with the spacer chain of 12 methylene groups gave the lowest MIC values against Gram-positive bacteria, whereas that with 14 methylene units was the best against Gram-negative bacteria. Interestingly, these novel compounds showed enhanced antibacterial activity relative to the lead compound, bis(N(alpha)-caproyl-L-arginine)-1,3-propanediamide dihydrochloride (C(3)(CA)(2)), and moderate antifungal activity. Moreover, tests of haemolytic activity toward human erythrocytes revealed that haemolysis increases with spacer chain length. Importantly, the compounds were classified as not irritating to eyes, with the exception of the compound with the spacer chain of 14 methylene groups, which was a slight eye irritant.