Genome analysis of the salt-resistant Paludifilum halophilum DSM 102817T reveals genes involved in flux-tuning of ectoines and unexplored bioactive secondary metabolites.

Paludifilum halophilum DSM 102817T is the first member of the genus Paludifilum in the Thermoactinomycetaceae family. The thermohalophilic bacterium was isolated from the solar saltern of Sfax, Tunisia and was shown to be able to produce ectoines with a relatively high-yield and to cope with salt stress conditions. In this study, the whole genome of P. halophilum was sequenced and analysed. Analysis revealed 3,789,765 base pairs with an average GC% content of 51.5%. A total of 3775 genes were predicted of which 3616 were protein-coding genes and 73 were RNA genes. The genes encoding key enzymes for ectoines (ectoine and hydroxyectoine) synthesis (ectABCD) were identified from the bacterial genome next to a gene cluster (ehuABCD) encoding a binding-protein-dependent ABC transport system responsible for ectoines mobility through the cell membrane. With the aid of KEGG analysis, we found that the central catabolic network of P. halophilum comprises the pathways of glycolysis, tricarboxylic acid cycle, and pentose phosphate. In addition, anaplerotic pathways replenishing oxaloacetate and glutamate synthesis from central metabolism needed for high ectoines biosynthetic fluxes were identified through several key enzymes. Furthermore, a total of 18 antiSMASH-predicted putative biosynthetic gene clusters for secondary metabolites with high novelty and diversity were identified in P. halophilum genome, including biosynthesis of colabomycine-A, fusaricidin-E, zwittermycin A, streptomycin, mycosubtilin and meilingmycin. Based on these data, P. halophilum emerged as a promising source for ectoines and antimicrobials with the potential to be scaled up for industrial production, which could benefit the pharmaceutical and cosmetic industries.

The saltern-derived Paludifilum halophilum DSM 102817T is a new high-yield ectoines producer in minimal medium and under salt stress conditions.

In the present study, the growth conditions and accumulation of ectoines (ectoine and hydroxyectoine) by Paludifilum halophilum DSM 102817T under salt stress conditions have been investigated. The productivity assay of this strain for ectoines revealed that the highest cellular content was reached in the minimal glucose sea water medium (SW-15) within 15% salinity. The addition of 0.1% (w/v) aspartic acid to the medium allowed an average of four times higher biomass production, and a dry mycelial biomass of 1.76 g L-1 was obtained after 6 days of growth in shake flasks at 40 °C and 200 rpm. Among the inorganic cations supplemented to the glucose SW-15 medium, the addition of 1 mM Fe2+ yielded the highest amount of mycelial biomass (3.45 g L-1) and total ectoines content (119 mg g-1), resulting in about 410 mg L-1 of products at the end of exponential growth phase. After 1 h of incubation in an osmotic downshock solution containing 2% NaCl, 70% of this content was released by the mycelium, and recovering cells maintained a high survival, with a maximal growth rate (µ max) of about 93% of the control population exposed to 15% NaCl. During growth at optimal salinity and temperature (15% NaCl and 40 °C), P. halophilum developed a compact and circular pellets that were easy to separate by simple decantation from both fermentation media and after hypoosmotic shock. Overall, the ectoines excreting P. halophilum could be a promising resource for ectoines production in a commercially valuable culture medium and at a large-scale fermentation process.

Enhancement of Antibacterial Activity of Paludifilum halophilum and Identification of N-(1-Carboxy-ethyl)-phthalamic Acid as the Main Bioactive Compound.

The aim of this study was to determine the combined effect of fermentation parameters and enhance the production of cellular biomass and antibacterial compounds from Paludifilum halophilum SMBg3 using the response surface methodology (RSM). Eight variables were screened to assess the effects of fermentation parameters on growth and metabolite production by Taguchi experimental design. Among these, the initial pH, temperature, and the percentage of MgSO4·7H2O in the medium were found to be most influential. The Box-Behnken design was applied to derive a statistical model for the optimization of these three fermentation parameters. The optimal parameters were initial pH: 8.3, temperature growth: 44°C, and MgSO4·7H2O: 1.6%, respectively. The maximum yield of biomass and metabolite production were, respectively, 11 mg/mL dry weight and 15.5 mm inhibition zone diameter against Salmonella enterica, which were in agreement with predicted values. The bioactive compounds were separated by the thick-layer chromatography technique and analyzed by GC/MS, NMR (1D and 2D), and Fourier-transform infrared spectroscopy (FT-IR). In addition to several fatty acids, N-(1-carboxy-ethyl)-phthalamic acid was identified as the main antibacterial compound. This element exhibited a potent activity against the ciprofloxacin-resistant Salmonella enterica CIP 8039 and Pseudomonas aeruginosa ATCC 9027 with a minimum inhibitory concentration (MIC) value range of 12.5-25 µg/mL. Results demonstrated that P. halophilum strain SMBg3 is a promising resource for novel antibacterial production due to its high-level yield potential and the capacity for large-scale fermentation.

Antagonistic Properties of Some Halophilic Thermoactinomycetes Isolated from Superficial Sediment of a Solar Saltern and Production of Cyclic Antimicrobial Peptides by the Novel Isolate Paludifilum halophilum.

This study has focused on the isolation of twenty-three halophilic actinomycetes from two ponds of different salinity and the evaluation of their ability to exert an antimicrobial activity against both their competitors and several other pathogens. From the 23 isolates, 18 strains showed antagonistic activity, while 19 showed activities against one or more of the seven pathogen strains tested. Six strains exhibited consistent antibacterial activity against Gram-negative and Gram-positive pathogens characterized at the physiological and molecular levels. These strains shared only 94-95% 16S rRNA sequence identity with the closely related species of the Thermoactinomycetaceae family. Among them, the potent strain SMBg3 was further characterized and assigned to a new genus in the family for which the name Paludifilum halophilum (DSM 102817T) is proposed. Sequential extraction of the antimicrobial compounds with ethyl acetate revealed that the crude extract from SMBg3 strain had inhibitory effect on the growth of the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa. Based on the HRESI-MS spectral data, the cyclic lipopeptide Gramicidin S and four cyclic dipeptides (CDPs) named cyclo(L-4-OH-Pro-L-Leu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(L-Leu-L-Pro) were detected in the fermentation broth of Paludifilum halophilum. To our knowledge, this is the first report on the isolation of these compounds from members of the Thermoactinomycetaceae family.

Paludifilum halophilum gen. nov., sp. nov., a thermoactinomycete isolated from superficial sediment of a solar saltern.

A novel filamentous, halophilic, thermotolerant bacterium, strain SMBg3T was isolated from superficial sediment of a solar saltern in Sfax, Tunisia. The isolate is Gram-staining-positive, aerobic, catalase- and oxidase-positive. Optimum growth occurred at 40-45 °C, with 10 % (w/v) NaCl and at pH 8.0-9.0. Long and well developed aerial and substrate mycelia, with long chains of fluorescent and circular spores, were observed on all tested media. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMBg3T belongs to an independent phylogenetic lineage of the family Thermoactinomycetaceae and shows a gene sequence similarity of 94 % with Desmospora activa DSM 45169T 94.2 % with Kroppenstedtia eburnea DSM 45196T, 94.3 % with Kroppenstedtia guangzhouensis KCTC 29149T, 94.3 % with Melghirimyces algeriensisDSM 45474T and 94.5 % with Salinithrix halophila CECT 8506T. The predominant menaquinone is MK-7, but MK-8 and some minor unidentified components are also present in trace amounts. The major cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0. In addition to four major polar lipids identified as phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmethylethanolamine, five minor unknown lipids were detected in cell membranes. The DNA G+C content of strain SMBg3T is 51.2 mol%. Strain SMBg3T is distinct from recognized genera of the family Thermoactinomycetaceae by morphological, biochemical and chemotaxonomic characteristics. On the basis of physiological and phylogenetic data, strain SMBg3T represents a novel species of a new genus in the family Thermoactinomycetaceae for which the name Paludifilum halophilum gen. nov., sp. nov. is proposed. The type strain of the type species is SMBg3T (=DSM 102817T=CCUG 68698T).

Hexane Extract of Echinops spinosissimus Turra subsp. spinosus from Tunisia: A Potential Source of Acetylated Sterols - Investigation of its Biological Activities.

The hexane extract of Echinops spinosissimus Turra subsp. spinosus flower heads was analyzed for its fatty acid and sterol composition. Its physicochemical characteristics were also studied. The saponification, iodine and peroxide values were determined as 255 mg KOH/g, 42.57 g I2 /100 g and 110 m equiv. O2 /kg of oil, respectively. The oleic (C18:1; 61.14%), palmitic (C16:0; 21.36%) and linoleic (C18:2; 10.45%) acids were the dominant fatty acids. This extract was also found to contain high levels of ß-sitosterol and stigmasterol (44.97% and 34.95% of total sterols, respectively). On the other hand, the identification of terpenoid compounds was investigated by using GC/MS, which revealed fourteen major terpenoids mainly taraxasterol, lupeol, pseudotaraxasterol, lup-22(29)-en-3-yl acetate, taraxasteryl acetate, α-amyrin, ß-amyrin, pseudotaraxasteryl acetate, hop-20(29)-en3-ß-ol, α-amirenone, along with ß-sitosterol and stigmasterol. Moreover, we have evaluated the in vitro antibacterial and antifungal activities of the unsaponifiable matter and a fraction isolated from this extract. These activities were conducted using the diffusion disc methods and broth microdilution assay. The resulted fraction from this extract showed the highest antibacterial activity with significant minimum inhibitory concentrations (MIC) values 125.0 µg/ml against Staphylococcus aureus, Micrococcus luteus and Bacillus cereus. However, it did exhibit no substantial antifungal activity.