Biology subjects

Kot, W. P.

Publications and source records attributed to Kot, W. P..

5 recordsLinked to original sources

bioRxiv2024

A single strand-based library preparation method for unbiased virome characterization

The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent limitations and biases associated with current sequencing library preparation methods. To overcome these problems, we repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA+Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.

microbiology↗

bioRxiv2022

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice

Many prokaryotes harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, very few of these systems have been experimentally confirmed to be active in gut bacteria. Here, we experimentally demonstrate that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage-assays in vitro but also in vivo using gnotobiotic (GB) mice. An increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Interestingly, much less new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

microbiology↗

bioRxiv2020

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets

The direct use of medical zinc oxide (ZnO) in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders. This study assessed whether rapeseed meal added two brown macroalagae species (Saccharina latissima and Ascophylum nodosum) and fermented using lactic acid bacteria (FRS) could improve piglet performance and gut health. The weaned piglets were fed one of three different feeding regimens (n = 230 each): basal diet, 2.5% and 5% FRS from day 28 of life to day 85. The piglets fed with 2.5% FRS presented superior phenotype with alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier as well as numerically improvements of final body weight. Colon microbiota composition was determined using amplicon sequencing of the V3 and V1 - V8 region of the 16S rRNA gene using Illumina Nextseq and Oxford Nanopore MinION sequencing, respectively. The two amplicon sequencing strategies showed high consistence between the detected bacteria. Both sequencing technologies showed that the FRS fed piglets had a distinctly different microbial composition relative to the basal diet. Compared with piglets fed the basal diet, Prevotella stercorea was verified by both technologies to be more abundant in the FRS piglets, and positively correlated with colon mucosa thickness and negatively correlated with blood levels of leucocytes and IgG. In conclusion, FRS supplementation improved gut health of weaner piglets, and altered their gut microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not cause further improvements.

microbiology↗

bioRxiv2020

Genomic characterization of three novel Pseudomonas phages within the subfamily of Autographivirinae isolated from organic waste

Three phages targeting Pseudomonas syringae GAW0113 have been isolated from organic waste samples: Pseudomonas phage Bertil, Misse and Strit. The phages have double-stranded DNA genomes ranging from 41342 to 41374 bp in size comprising 50 to 51 open reading frames. The three phages genomes are highly similar and genomic comparison analyses shows that they all belong to the Autographivirinae subfamily of the family Podoviridae. The phages are however only distantly related to other members of this family, and have limited gene synteny with type-phages of other genera within Autographivirinae, suggesting that the newly isolated phages could represent a new genus.

microbiology↗

bioRxiv2019

A novel and direct mobilome approach improves the detection of larger-sized circular elements across kingdoms

Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate immense genetic diversity. MGEs include DNA elements such as plasmids, transposons and Insertion Sequences (IS-elements), as well as bacteriophages (phages), and they serve as a vast communal gene pool. These mobile DNA elements represent a human health risk as they can add new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting circular MGEs, referred to as mobilomes, allows the expansion of our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomes from bacterial communities are not studied to the same extent as metagenomics, partly because of methodological biases arising from multiple displacement amplification (MDA), often used in previous metamobilome publications. In this study, we show that MDA is detrimental to the detection of larger-sized plasmids if small plasmids are present by comparing the abundances of reads mapping to plasmids in a wastewater sample spiked with a mock community of selected plasmids with and without MDA. Furthermore, we show that it is possible to produce samples consisting almost exclusively of circular MGEs and obtain a catalog of larger, complete, circular MGEs from complex samples without the use of MDA.\n\nImportanceMobile genetic elements (MGEs) can transport genetic information between genomes in different bacterial species, adding new traits, potentially generating dangerous multidrug-resistant pathogens. In fact, plasmids and circular MGEs can encode bacterial genetic specializations such as virulence, resistance to metals, antimicrobial compounds, and bacteriophages, as well as the degradation of xenobiotics. For this reason, circular MGEs are crucial to investigate, but they are often missed in metagenomics and ecological studies. In this study, we present, for the first time, an improved method, which reduces the bias towards small MGEs and we demonstrate that this method can unveil larger, complete circular MGEs from complex samples without the use of multiple displacement amplification. This method may result in the detection of larger-sized plasmids that have hitherto remained unnoticed and therefore has the potential to reveal novel accessory genes, acting as possible targets in the development of preventive strategies directed at pathogens.

microbiology↗