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A semisynthetic strategy to obtain monophosphoryl lipid A derivatives equipped with clickable (azide, alkyne, double bond, or thiol precursor) moieties, starting from the native lipid A isolated from Escherichia coli, is presented. These lipid A derivatives can be conjugated with other interesting biomolecules, such as tumor-associated carbohydrate antigens (TACAs). In this way, the immunostimulant activity of monophosphoryl lipid A can significantly improve the immunogenicity of TACAs, thus opening access to potential self-adjuvant anticancer vaccine candidates. A monophosphoryl lipid A−Thomson-Friedenreich (TF) antigen conjugate was obtained to demonstrate the feasibility of this methodology, which stands as a valuable, rapid, and scalable alternative to the highly complex approaches of total synthesis recently reported to the same aim. A preliminary evaluation of the immunological activity of this conjugate as well as of other semisynthetic lipid A derivatives was also reported.

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.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea. Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricus. IMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

Shewanella sp. HM13 is a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel. It produces a large amount of outer membrane vesicles (OMVs), which are particles released in the medium where the bacterium is cultured. This strain biosynthesizes a single major cargo protein in the OMVs, a fact that makes Shewanella sp. HM13 a good candidate for the production of extracellular recombinant proteins. Therefore, the structural characterization of the components of the vesicles, such as lipopolysaccharides, takes on a fundamental role for understanding the mechanism of biogenesis of the OMVs and their applications. The aim of this study was to investigate the structure of the oligosaccharide (OS) isolated from Shewanella sp. HM13 cells as the first step for a comparison with that from the vesicles. The lipooligosaccharide (LOS) was isolated from dry cells, purified, and hydrolyzed by alkaline treatment. The obtained OS was analyzed completely, and the composition of fatty acids was obtained by chemical methods. In particular, the OS was investigated in detail by 1H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry. The oligosaccharide was characterized by the presence of a residue of 8-amino-3, 8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) and of a d, d-heptose, with both residues being identified in other oligosaccharides from Shewanella species., This article belongs to the Special Issue Marine Bacteria as Sources of Bioactive Compounds.

The probiotic features of strain GM newly isolated from Tunisian spontaneously fermented goat milk and identified as Bacillus tequilensis-GM were assessed. Strain GM showed high resistance to saliva (90.64%), gastric juice (88.55%), intestinal juice (72.83%) and resistance to bile salts (65.22%), was able to act against Listeria monocytogenes ATCC 15313, Escherichia coli ATCC 25922 and Enterococcus feacalis ATCC 25912, showed high surface hydrophobicity (77.3%) and was sensitive to most of the studied antibiotics. Strain GM did not exhibit any hemolytic activity whereas it was able to produce protease, amylase and β-galactosidase. Moreover, results showed that strain GM produced high molecular weight β-(2 → 6)-levan with high ability to inhibit and to disrupt pathogenic biofilms and with high ability to reduce syneresis of sucrose-supplemented skimmed milk. B. tequilensis-GM can therefore be suitable to be used as starter culture in fermented dairy products, since it possesses desirable probiotic properties in addition to its ability to produce levan.

Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.

Colwellia psychrerythraea 34H is a psychrophilic Gram-negative bacterium, able to survive at subzero temperatures by producing a unique capsular polysaccharide (CPS) with anti-freeze properties similar to those of the well-known anti-freeze (glyco)proteins. The tetrasaccharide repeating unit of the CPS - constituted of alternating amino sugars and uronic acid moieties in a glycosaminoglycan-like fashion with an amide-linked threonine (Thr) decoration - was synthesized as an O-n-propyl glycoside. The synthesis faced some challenging features such as building up a crowded [→2)α-d-Galp(1→] moiety as well as differentiating the two uronic units for the regioselective insertion of the Thr amide only on one of them. NMR data for the obtained tetrasaccharide confirmed the structure proposed for the C. psychrerythraea polysaccharide.

In the present work, an exopolysaccharide (EPS) was produced by a newly isolated Bacillus tequilensis-GM ropy strain. Results showed that EPS-GM was produced simultaneously with the growth phase and reached its maximum (2.9 g/L) when sucrose was used as carbon source. According to GC-MS and HPLC-SEC results, the recovered EPS-GM was composed by fructose, glucose, mannose, galactose, galactosamine and N-acetyl-glucosamine, with high molecular weight ranging between 1.8 and 2.5 × 106 Da. Different properties of EPS-GM were tested. Results showed that crude EPS-GM has several potential features, including high thermostability evaluated by DSC (with a melting point of 222 °C), good emulsifying properties and stability at both neutral and acidic pH, and high capacities to adsorb heavy metal ions (94.96% of Fe2+, 97.66% of Zn2+ and 98.24% of Cu2+) making it promising to be used in different industrial fields.

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.

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

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.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea. Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

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)-β-D-GlcpNAcA-(1 →3)-β-D-QuipNAc4NAc-(1 →3)-β-D-GalpNAc-(1 →. The 3D model, generated in accordance with

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.

The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

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.