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Cationic Host Defense Peptides (HDPs) are endowed with a broad variety of activities, including direct antimicrobial properties and modulatory roles in the innate immune response. Even if it has been widely demonstrated that bacterial membrane represents the main target of peptide antimicrobial activity, the molecular mechanisms underlying membrane perturbation by HDPs have not been fully clarified yet. Recently, two cryptic HDPs have been identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, and with anti-biofilm, wound healing and immunomodulatory properties. Moreover, ApoB derived HDPs are able to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, by using a multidisciplinary approach, including time killing curves, Zeta potential measurements, membrane permeabilization assays, electron microscopy analyses, and isothermal titration calorimetry studies, the antimicrobial effects of ApoB cryptides have been analysed on bacterial strains either susceptible or resistant to peptide toxicity. Intriguingly, it emerged that even if electrostatic interactions between negatively charged bacterial membranes and positively charged HDPs play a key role in mediating peptide toxicity, they are strongly influenced by the composition of negatively charged bacterial surfaces and by defined extracellular microenvironments.

Colwellia psychrerythraea 34H is a Gram-negative cold-adapted microorganism that adopts many strategies to cope with the limitations due to the low temperatures of its habitat. In this study, we reported the complete characterization of the lipid A moiety from the lipopolysaccharide of Colwellia, to find out if structural features could be linked to the cold-adaptation strategy. The lipid A and its partially deacylated derivative were completely characterized by means of high resolution mass spectrometry, NMR spectroscopy, and chemical analysis. An unusual structure, consisting of 3-hydroxy unsaturated tetradecenoic acid as component of the primary acylation pattern, was identified. In addition, the presence of a partially acylated phosphoglycerol moiety on the secondary acylation site at 3-position of the reducing GlcN caused a tremendous natural heterogeneity of the lipid A structure. Biological activity assays indicated that Colwellia psychrerythraea 34H lipid A did not show any either agonistic or antagonistic effect when tested in human macrophages.

Microrganisms from sea ice, glacial and subglacial environments are currently under investigation due to their relevant ecological functions in these habitats, and to their potential biotechnological applications. The cold-adapted Colwellia psychrerythraea 34H produces extracellular polysaccharides with cryoprotection activity. We here describe the purification and detailed molecular primary and secondary structure of the exopolysaccharide (EPS) secreted by C. psychrerythraea 34H cells grown at 4 °C. The structure was determined by chemical analysis and NMR. The trisaccharide repeating unit of the EPS is constituted by a N-acetyl quinovosamine unit and two residues of galacturonic acid both decorated with alanine. In addition, the EPS was tested in vitro showing a significant inhibitory effect on ice recrystallization. In-depth NMR and computational analysis suggest a pseudohelicoidal structure which seems to prevent the local tetrahedral order of the water molecules in the first hydration shell, and could be responsible of the inhibition of ice recrystallization.\ud As cell cryopreservation is an essential tool in modern biotechnology and medicine, the observations reported in this paper could pave the way for a biotechnological application of Colwellia EPS.

Staphylococcus epidermidis, a harmless human skin colonizer, is a significant nosocomial pathogen in predisposed hosts because of its capability to form a biofilm on indwelling medical devices. In a recent paper, the purification and identification of the pentadecanal produced by the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, able to impair S. epidermidis biofilm formation, were reported. Here the authors report on the chemical synthesis of pentadecanal derivatives, their anti-biofilm activity on S. epidermidis, and their action in combination with antibiotics. The results clearly indicate that the pentadecanal derivatives were able to prevent, to a different extent, biofilm formation and that pentadecanoic acid positively modulated the antimicrobial activity of the vancomycin. The cytotoxicity of these new anti-biofilm molecules was tested on two different immortalized eukaryotic cell lines in view of their potential applications.

Light is a driving force behind the synthesis of biomass in photoautotrophs. In pelagic ecosystems, highly variable light strongly affects microalgae at an ecological (success and succession), molecular (acclimation, carbon allocation), photosynthetic and growth levels. Therefore, manipulation of light appears to be an attractive tool for enhancing growth of microalgae in order to extend the use of these organisms in biotechnological field. In this study, we investigate the responses of the diatom alga Skeletenoma marinoi to different growth light regimes, that induced composite acclimative patterns manifesting in strong alterations in the cell biology. Although the growth rate is dependent upon the integrated daily light dose received by the algae, we demonstrate that physiological and biochemical acclimation responds to additional light stimuli. The application of fluctuating red light on a sinusoidal blue light spectral distribution induces an increase in non-photochemical quenching and the de-epoxidation state of the xanthophyll cycle along with modifications of the metabolic state of the cells, e.g. an increase in carbohydrates, glycolipids and saturated fatty acids. Providing an unnatural square-wave light course induces drastic changes in photosynthesis, pigment and macromolecular composition of the cells.

The marine environment is the largest aquatic ecosystem on Earth and it harbours microorganisms responsible for more than 50% of total biomass of prokaryotes in the world. All these microorganisms produce extracellular polymers that constitute a substantial part of the dissolved organic carbon, often in the form of exopolysaccharides (EPS). In addition, the production of these polymers is often correlated to the establishment of the biofilm growth mode, during which they are important matrix components. Their functions include adhesion and colonization of surfaces, protection of the bacterial cells and support for biochemical interactions between the bacteria and the surrounding environment. The aim of this review is to present a summary of the status of the research about the structures of exopolysaccharides from marine bacteria, including capsular, medium released and biofilm embedded polysaccharides. Moreover, ecological roles of these polymers, especially for those isolated from extreme ecological niches (deep-sea hydrothermal vents, polar regions, hypersaline ponds, etc.), are reported. Finally, relationships between the structure and the function of the exopolysaccharides are discussed.

Background Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. Results We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small sweet protein derived from monellin, with potential for food industry applications as a high intensity sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. Conclusions Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions. Electronic supplementary material The online version of this article (10.1186/s12934-018-0974-z) contains supplementary material, which is available to authorized users.

Background The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. Results Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. Conclusions Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle. Electronic supplementary material The online version of this article (10.1186/s12866-018-1360-z) contains supplementary material, which is available to authorized users.

In this work, four exopolysaccharide-producing lactic acid bacteria (LAB) strains, newly isolated from Tunisian spontaneously fermented foods and beverages, namely bovine and turkey meat sausages (BMS and TMS), date palm sap (DPS) and cow milk (CM), were identified as Leuconostoc citreum-BMS, Leuconostoc mesenteroides-TMS, Pediococcus pentosaceus-DPS and Leuconostoc pseudomesenteroides-CM, respectively. The isolated strains showed the ability to withstand simulated human gastrointestinal (GI) tract conditions (low pH, lysozyme, bile salts, pepsin and pancreatin) and showed high surface hydrophobicity (79-90%), besides their ability to act against Escherichia coli and Listeria monocytogenes and to produce exopolysaccharides (EPS). Therefore, these isolates can be served as potential probiotics. The produced EPS were growth-associated suggesting that they are primary metabolites. The molecular weights were higher than 106Da using HPLC-SEC. 2D-NMR results indicated that all the samples were mixtures of dextran and levan, except for EPS-CM which was a levan-type EPS. Furthermore, the EPS samples showed an abitlity to inhibit and to disrupt pathogenic biofilms and showed high thermostability studied via differential scanning calorimetry (DSC) with melting points higher than 224°C making them promising to be used in thermal processed foods.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.

Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: -> 4)-beta-d-GlcpNAcA-(1 -> 3)-beta-d-QuipNAc4NAc-(1 -> 3)-beta-d-GalpNAc-(1 ->. The 3D model, generated in accordance with H-1,H-1-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 A degrees C, this molecule displays only a low ice recrystallization inhibition activity. Open Access 2019-09-01

Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of itsstructural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core.

Cold-adapted Gram-negative bacteria inhabit snow, ice water, and frozen grounds and are perfectly adapted to live at very low temperatures. They have evolved several adaptation strategies, some of which concern the cellular membrane. Bacterial outer membrane, that functions as a selective barrier allowing the influx of nutrients and confers protection to the cell, is also considered a primary sensor of the cold. It is well known that one of the main responses to cold concerning the cell membrane is the increment of fluidity and the upregulation of genes encoding for proteins and membrane transporters. In this context, also the lipopolysaccharides, macromolecules constituting approximately 75% of the outer surface, may be modified in their structure in response to the conditions prevailing in the environment. In this chapter, for the first time, the role played by lipopolysaccharides structures in response to cold adaptation is analyzed. Some structural features of the lipopolysaccharides are modified as a consequence of living at low temperatures, thus confirming that all the outer membrane components are involved in adaptation and survival molecular mechanisms.

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air–liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.

Halomonas pantelleriensis DSM9661Τ is a Gram-negative haloalkaliphilic bacterium isolated from the sand of the volcanic Venus mirror lake, closed to seashore in the Pantelleria island in the south of Italy. It is able to optimally grow in media containing 3-15% (w/v) total salt and at pH between 9-10. To survive in these harsh conditions the bacterium has developed several strategies that probably concern the bacteria outer membrane, a barrier regulating the exchange with the environment. In such a context the lipopolysaccharides (LPSs), which are among the major constituent of the Gram-negative outer membrane, are thought to contribute to the restrictive membrane permeability properties. The structure of the lipid A family derived from the lipopolysaccharide of Halomonas pantelleriensis DSM 9661T is reported herein. The lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different numbers of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of ESI FT-ICR mass spectrometry and chemical analysis. Preliminary immunological assays were performed, and a comparison with the lipid A structure of the phylogenetic proximal Halomonas magadiensis is also reported.

Producing antimicrobials is a common adaptive behavior shared by many microorganisms, including marine bacteria. We report that SF214, a marine-isolated strain of Bacillus pumilus, produces at least two different molecules with antibacterial activity: a molecule smaller than 3 kDa active against Staphylococcus aureus and a molecule larger than 10 kDa active against Listeria monocytogenes. We focused our attention on the anti-Staphylococcus molecule and found that it was active at a wide range of pH conditions and that its secretion was dependent on the growth phase, medium, and temperature. A mass spectrometry analysis of the size-fractionated supernatant of SF214 identified the small anti-Staphylococcus molecule as a pumilacidin, a nonribosomally synthesized biosurfactant composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. The analysis of the SF214 genome revealed the presence of a gene cluster similar to the srfA-sfp locus encoding the multimodular, nonribosomal peptide synthases found in other surfactant-producing bacilli. However, the srfA-sfp cluster of SF214 differed from that present in other surfactant-producing strains of B. pumilus by the presence of an insertion element previously found only in strains of B. safensis.

Waste frying oil (WFO) is a complex and heterogeneous waste collected mainly from restaurants and, to a lesser extent, from food processors and households. In the last decades, interest for this waste has deeply grown and a strong competition in grabbing this raw material has begun. About 90% of WFOs collected in Europe is recycled for biodiesel production, being the high free fatty acids (FFAs) concentration in WFOs one of the main drawback limiting the yield of the conversion process. In this study we proposed a pit stop in the WFO to biodiesel conversion race by introducing an upstream microbial fermentation step aimed at reducing the FFAs content through its conversion into biopolymers, i.e. Polyhydroxyalkanoates (PHA). A properly engineered Escherichia coli strain, able to produce PHA exclusively from FFAs, but not secreting lipases, was tested in this process. Recombinant production of a near P(HHx) homopolymer was achieved in a process requiring an aqueous pretreatment step aimed to reduce the content of non-lipid carbon sources, which compete with FFAs for microbial growth impairing the PHA production. A WFO with a halved FFAs content was recovered after fermentation, thus rendering it more attractive for further conversion in biodiesel. Despite the quite low production yield achieved at this stage, the idea of no-competitiveness behind the process was verified, leaving space to further strain improvement strategies to boost PHA yield.