Genome Mining and Characterization of Biosynthetic Gene Clusters in Two Cave Strains of Paenibacillus sp.

The genome sequencing and mining of microorganisms from unexplored and extreme environments has become important in the process of identifying novel biosynthetic pathways. In the present study, the biosynthetic potential of Paenibacillus sp. strains 23TSA30-6 and 28ISP30-2 was investigated. Both strains were isolated from the deep oligotrophic Krubera-Voronja Cave and were found to be highly active against both Gram-positive and Gram-negative bacteria. Genome mining revealed a high number of biosynthetic gene clusters in the cave strains: 21 for strain 23TSA30-6 and 19 for strain 28ISP30-2. Single clusters encoding the biosynthesis of phosphonate, terpene, and siderophore, as well as a single trans-AT polyketide synthase/non-ribosomal peptide synthetase, were identified in both genomes. The most numerous clusters were assigned to the biosynthetic pathways of non-ribosomal peptides and ribosomally synthesized and post-translationally modified peptides. Although four non-ribosomal peptide synthetase gene clusters were predicted to be involved in the biosynthesis of known compounds (fusaricidin, polymyxin B, colistin A, and tridecaptin) of the genus Paenibacillus, discrepancies in the structural organization of the clusters, as well as in the substrate specificity of some adenylation domains, were detected between the reference pathways and the clusters in our study. Among the clusters involved in the biosynthesis of ribosomally synthesized peptides, only one was predicted to be involved in the biosynthesis of a known compound: paenicidin B. Most biosynthetic gene clusters in the genomes of the cave strains showed a low similarity with the reference pathways and were predicted to represent novel biosynthetic pathways. In addition, the cave strains differed in their potential to encode the biosynthesis of a few unique, previously unknown compounds (class II lanthipeptides and three non-ribosomal peptides). The phenotypic characterization of proteinaceous and volatile compounds produced by strains 23TSA30-6 and 28ISP30-2 was also performed, and the results were compared with those of genome mining.

Speciation of methyltins by dispersive liquid-liquid microextraction and gas chromatography with mass spectrometry.

Dispersive liquid-liquid microextraction in combination with an in situ derivatization is suggested for methyltin compound sampling and preconcentration from water solutions. The derivatization was carried out with sodium tetraethylborate at pH 3. The effects of extraction and disperser solvents type, volume, and extraction time on the extraction efficiency were investigated. 1,2-Dichlorobenzene was used as an extraction solvent and ethanol was used as a disperser solvent. The calibration graphs for all the analytes were linear up to 2 µg (Sn) L(-1), correlation coefficients were 0.998-0.999, LODs were 0.13, 0.05, and 0.06 ng (Sn) L(-1) for trimethyltin, DMT, and monomethyltin, respectively. Repeatabilities of the results were acceptable with RSDs up to 12.1%. A possibility to apply the proposed method for methyltin compound determination in water samples was demonstrated.

Retention behaviour of imidazolium ionic liquid cations on 1.7 µm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases.

The potential of 1.7 µm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.

Dispersion-solidification liquid-liquid microextraction for volatile aromatic hydrocarbons determination: comparison with liquid phase microextraction based on the solidification of a floating drop.

Two microextraction techniques--liquid phase microextraction based on solidification of a floating organic drop (LPME-SFO) and dispersive liquid-liquid microextraction combined with a solidification of a floating organic drop (DLLME-SFO)--are explored for benzene, toluene, ethylbenzene and o-xylene sampling and preconcentration. The investigation covers the effects of extraction solvent type, extraction and disperser solvents' volume, and the extraction time. For both techniques 1-undecanol containing n-heptane as internal standard was used as an extracting solvent. For DLLME-SFO acetone was used as a disperser solvent. The calibration curves for both techniques and for all the analytes were linear up to 10 microg/mL, correlation coefficients were in the range 0.997-0.998, enrichment factors were from 87 for benzene to 290 for o-xylene, detection limits were from 0.31 and 0.35 microg/L for benzene to 0.15 and 0.10 microg/L for o-xylene for LPME-SFO and DLLME-SFO, respectively. Repeatabilities of the results were acceptable with RSDs up to 12%. Being comparable with LPME-SFO in the analytical characteristics, DLLME-SFO is superior to LPME-SFO in the extraction time. A possibility to apply the proposed techniques for volatile aromatic hydrocarbons determination in tap water and snow was demonstrated.

Silicone glue coated stainless steel wire for solid phase microextraction.

A new solid phase microextraction (SPME) fiber based on high-temperature silicone glue coated on a stainless steel wire is presented. The fiber coating can be prepared easily in a few minutes, it is mechanically stable and exhibits relatively high thermal stability (up to 260 degrees C). The extraction properties of the fiber to benzene, toluene, ethylbenzene, and xylenes (BTEX) were examined using both direct and headspace SPME modes coupled to gas chromatography-flame ionization detection. The effects of the extraction and desorption parameters including extraction and desorption time, sampling and desorption temperature, and ionic strength on the extraction/desorption efficiency have been studied. For both headspace and direct SPME the calibration graphs were linear in the concentration range from 0.5 microg L(-1) to 10 mg L(-1) (R2>0.996) and detection limits ranged from 0.07 to 0.24 microg L(-1). Single fiber repeatability and fiber-to-fiber reproducibility were less than 6.8 and 21.5%, respectively. Finally, headspace SPME was applied to determine BTEX in petrol station waste waters with spiked recoveries in the range of 89.7-105.2%.