Characterisation of the Prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment research biomarker

Sediment cores from Lake Suigetsu, Japan are recognised as a key record of past climate reconstruction because of the finely laminated sediments that provide precise event stratigraphy. Here, we study the relationship between the microbial communities in the lake sediments of Lake Suigetsu during validated episodes of environmental change. We use fossil DNA from the lake sediment and utilising the PCR-DGGE technique, we detected the presence of several taxa. Among the investigated sediment cores, the Acidobacteria community was found to be the most abundant while the Actinobacteria community was the least. The results showed that the overall bacterial community structure and their diversity were significantly affected by sediment depths, rather than the availability of nutrient (i.e. TOC and TN). The first event was the introduction of saline water in Lake Suigetsu. Historical records have described this event occurring during 1664 AD, which equates to the sediment depth of approximately 81.64 cm. A metagenomics study based on selected sediment depths has exhibited a shift in the bacterial taxa, consistent with the transition of lake salinity from freshwater to brackish. Bacillaceae and Clostridiaceae were found to be more predominant in the brackish sediments relative to the freshwater sediments. Evidence of the seawater incursion was found in the sediment depths between 82.16 and 83.16 cm. The second event was a climate event dated back to the Late Quaternary period. The results presented here show that bacterial diversity and species richness become increased when climate changed from a cold to warmer conditions. The metagenomics analysis on the sediment deposits has demonstrated distinctive differentiations in bacterial taxa during the climate transition from the colder to warmer episodes. This observation could be related to the rapid adaptation/tolerance of bacteria to environmental changes, or simply the effect of depth. Although the temperature-dependent δ15N isotope can be strongly correlated to the bacterial communities, the weak selectivity of the δ15N isotope could result in false correlation between the δ15N isotope and the diversity of the bacterial communities. The application of molecular and culture-dependent techniques was used to characterise bacterial diversity in the sedimentary records of Lake Suigetsu. The culture-based techniques showed a better representation of high GC Actinobacteria while molecular techniques revealed a better profile of Gram negative bacteria. Furthermore, based on a polyphasic approach, several putatively new species have been identified, notably Actinobacteria strains that belong to the genera Dermacoccus, Dietzia, Leifsonia and Rhodococcus. Among the tested strains, a novel Rhodococcus isolate that was recovered from the freshwater sediment, merits recognition of new species status and the name Rhodococcus meromictica sp. nov is proposed

Thermostable in vitro transcription-translation compatible with microfluidic droplets

Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughputThis work has received funding from the European Union\u2019s Research and Innovation Framework programs FP7 and Horizon 2020 under Grant Agreement numbers 324439, 635595, 685474, 695669 and 10100560 and from the Spanish Ministry of Economy and Competitiveness under grant number BIO-2013-44963-R. The CBM is funded by \u201CCentre of Excellence Severo Ochoa\u201D Grant CEX2021-001154-S from MICIU/AEI / https://doi.org/10.13039/501100011033 and receives institutional support by Fundaci\u00F3n Ram\u00F3n Arece

Culture Dependent Determination of the Prokaryotic Community of Lake Suigetsu, Japan: towards a novel palaeoenvironment research biomarker

Lake Suigetsu is a small tectonic lake in central Japan containing annually laminated (varved) sediment. An ongoing international project is combining over 600 radiocarbon ages from terrestrial leaf macrofossils with varve ages from the core (SG06) to generate a terrestrial radiocarbon calibration model stretching back to the limit of radiocarbon dating (c. 50, 000 BP). This core chronology provides an ideal basis for quantitative studies of climate change and palaeoenvironmental reconstruction. The present study aimed to identify and elucidate the diversity of the microbial communities down the sediment core with the intention to develop novel biomarkers for past climate change using a culture-dependent approach. Lake sediment samples taken at 932.44, 1363.76 and 2434 cm composite depths, representing c. 6860, 10911 and 23600 years before present were subjected to dispersion and differential centrifugation (DDC) coupled with selective isolation. A total of 204 representative isolates were further identified using 16S rRNA gene sequencing, with 139 found to reside within 18 genera belonging to the class Actinobacteria. Five genera were recovered solely from the 932.44 cm sample, with eight genera residing within the 1363.76 cm sample. Only two genera were recovered from all three samples. This may suggest that the development of the prokaryotic community during and post-deposition could be influenced by climatic conditions at the time of deposition and/or community development during burial. Results support the use of prokaryotic communities as potential palaeoenvironmental indicators

An Efficient Synthesis of the Bicyclic Darunavir Side Chain Using Chemoenzymatic Catalysis

Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective dynamic kinetic resolution of a -ketolactone. Subsequent lactone reduction with diisobutylaluminium hydride and phase transfer cyclization affords the bicyclic acetal fragment in 39% yield over four steps

Analysis of the microbial community and geochemistry of a sediment core from Great Slave Lake, Canada

Sediment cores taken from Great SlaveLake, Canada, were analysed to investigate theirmetabolically active microbial populations and geochemistry. The amplification of cDNA detectedmetabolically active bacterial (50 separate bands)and archaeal (49 separate band) communities. The bacterial communities were further resolved indicating active actinobacterial and c-proteobacterial communities(36 and 43 individual bands respectively).Redundancy discriminate analysis and Monte Carlopermutation testing demonstrated the significantimpact of geochemical parameters on microbial community structures. Geochemical analyses suggest that the upper 0.4 m represents soil weathering and erosion in the lake catchment. An increase in organic carbon in the lower core suggests either more primary productivity, indicating warmer climate conditions, associated with Holocene Climatic Optimum conditions pre 5,000 years BP or change from a reducing environment in the lower core to an oxidizing environment during more recent deposition. Drivers for bacterial, archaeal and actinobacterial community structures were sediment particle size, and its mineral composition. Depth also significantly affected cproteobacterial community structure. In contrast the organic carbon content did not significantly shape the microbial community structures within the sediment. This study indicates that geochemical parameters significantly contribute to microbial community structure in these sediments

Characterization of the prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment

Lake Suigetsu is a small tectonic lake in central Japan containing annually laminated sediment. A recent study combined more than 300 radiocarbon ages on terrestrial leaf macrofossils with varve ages from the core generated a quasi-continuous radiocarbon calibration model to the limit of radiocarbon dating (c.50,000BP). This core chronology provides an ideal basis for quantitative studies of climate change and palaeoenvironmental reconstruction. The present study aimed to identify and elucidate the diversity of the microbial communities down the sediment core to develop novel biomarkers for past climate change, using both culture-dependent and independent (molecular) techniques. Here we report the analysis of eighteen samples of the lake sediment sampled at every 4m of the 73.5m core covering the past 150,000 years using PCr-DGGE for 16S rrNA genes from Eubacteria and Archaea. in addition, two of the eighteen sediment samples, indicative of a salinity shift between 10911BP (freshwater) to 6860BP (brackish) were subjected to a Dispersion and Differential Centrifugation (DDC) culture-based approach. A total of 183 taxa were isolated from freshwater sediment samples and 71 taxa from sediments after the saline infux, respectively. This suggests that environmental shifts impose an effect over the diversity of microbial communities, endorsing their potential as biomarkers

Rhodococcus jostii: a home for Rhodococcus strain RHA1

The taxonomic position of Rhodococcus strain RHA1, an effective degrader of polychlorinated biphenyls with a large linear chromosome, was established using a polyphasic approach. The morphological and chemotaxonomic properties of the strain were typical of members of the genus Rhodococcus. The strain shared a high level of 16S rRNA sequence similarity (99.9 %) with the type strain of Rhodococcus jostii, a member of the Rhodococcus erythropolis subclade. The two strains shared a DNA:DNA relatedness value well above the cut-off point recommended for the circumscription of genomic species and had a broad range of phenotypic properties in common. The combination of genomic and phenotypic data show strain RHA1 to be a bona fide member of the species Rhodococcus jostii

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput