Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

ARCHAEOLOGICAL, SOIL AND MICROBIOLOGICAL STUDIES OF THE MOSKVA RIVER FLOODPLAIN DEPOSITS AT THE ZVENIGOROD BIOLOGICAL PLANT

В статье приводятся результаты применения методов почвенно-микробиологического исследования культурных слоев в пойменных отложениях Москвы-реки. Выявлена корреляция пиков численности сапротрофных и термофильных бактерий с горизонтами погребенных почв в толще аллювия, к которым приурочены археологические находки. Показано, что с помощью микробиологических анализов можно выявить следы хозяйственной активности, которая датируется ранним бронзовым веком. The paper describes results of soil and microbiological studies of the occupation layers in the Moskva River floodplain deposits. The study identified a correlation between peaks in the count of the saprotrophic and thermophilic bacteria with the horizons of buried soils in the stream sediments where archaeological finds were made. The paper shows that micro bioassays can help identify traces of economic activity dating to the early Bronze Age.

Theoretical and Experimental Investigation of the Thermal Inactivation of Thermoanaerobacterium Thermosaccharolyticum and Geobacillus Stearothermophilus in Different Canned Food Matrices

In the canning industry, thermal preservation processes typically are designed based on Clostridium botulinum thermal destruction kinetics. However, some bacteria can still survive, necessitating implementation of stricter timetemperature regimen for sterilization process. The aim of this study was to compare processing effectiveness at F0 (sterilization value) 8 ±1 min from the perspective of the vegetable-based product canning facility, while analyzing the inactivation, viability, and recovery of thermophilic bacteria. Four commercial products [tomato soup and rassolnik soup - acidified food (AF), and mushroom soup and pea porridge - low-acid food (LACF)] with different heat transfer characteristics (convection and conduction) were inoculated with 6.6 log10 spores/ml Geobacillus stearothermophilus LMKK 244 (reported as DSM 6790 and ATCC 10149 in other collections) and 4.810 log spores/ml Thermoanaerobacterium thermosaccharolyticum DSM 571 spore suspensions. Food samples contaminated with bacterial spores were processed in a steam-air retort at 118 °C for 75 min. G. stearothermophilus and T. thermosaccharolyticum growth was not detected in AF samples (pH = 4.4 and 4.5), but was observed in LACF samples (pH = 5.1 and 5.8). Practical evaluation showed that T. thermosaccharolyticum did not survive thermal processing, which was verified using a presence/absence test after incubation at 55 °C. G. stearothermophilus did not survive thermal processing, but recovered in pea porridge (pH = 5.8) during incubation. Our observations showed that food pH is a crucial factor determining microorganism survival during heat treatment and may be used by the vegetable-based product canning facilities to improve the food sterilization conditions.

Bacterial Diversity and CAZyme Potential Revealed in Pandanus Rich Thermal Spring Cluster of India: A Non-cultivable 16S rRNA Sequencing Approach

In the present study, we explored four different geothermal spots of the Deulajhari spring cluster at a proximity of 10–20 meters with temperatures of 43 to 65°C to unravel their genesis, bacterial diversity and CAZyme potential. However, minor variations in physicochemical properties; TOC, sodium, chloride, zinc and nitrate were observed, including the pH of the spring openings. Illumina based amplicon sequencing revealed Firmicutes, Proteobacteria and Chloroflexi as the major bacterial phylum with higher abundance in the DJ04 sample. The alpha diversity of all the springs was almost same, whereas beta diversity revealed variations in the degree of uniqueness of OTUs at different temperatures. Statistical analysis established a positive correlation between sulfur content with Heliobacterium, Thermodesulfovibrio, Thermodesulfobacterium and Herpetosipho as well as TOC and HCO3 with Thermoanaerobacter, Desulfovibrio, Candidatus solibacter and Dehalogenimona. The major hydrocarbon family genes and Carbohydrate Active Enzyme pathways were predicted to be highest in DJ04 with elevated concentrations of HCO3 and TOC. Higher homogeneity in geo-physicochemical and microbial features direct the possibility of the common origin of these springs through plumbing systems. However, the minor variations in diversity and functionality were due to variations in temperature in spring openings through the mixing of subsurface water contaminated with carbohydrates from leaf biomass litter. Functional characterization of the thermophilic bacteria of this spring provides essential scope for further industrial applications. The biogeochemical reasons hypothesized for the genesis of unique multiple openings in the cluster are also of interest to conservation scientists for taking measures toward necessary laws and regulations to protect and preserve these springs.