Biology subjects

Boleij, A.

Publications and source records attributed to Boleij, A..

4 recordsLinked to original sources

bioRxiv2020

Nutrition or nature: disentangling the complex forces shaping prokaryote pan-genomes

Microbial pan-genomes are shaped by a complex combination of stochastic and deterministic forces. Even closely related genomes often exhibit extensive variation in their gene content. Understanding what drives this variation requires exploring the interactions of genes with each other and with their external environments. However, to date, conceptual models of pan-genome dynamics often represent genes as independent units and provide limited information about their mechanistic interactions. Here, we use pan-reactomes as proxies for pan-genomes since they can explicitly represent the interactions between the genes that code for metabolic reactions and simulate complex phenotypes that interact with the metabolic environment. We interpreted pan-reactomes as dynamic pools of metabolic reactions that are potentially gained or lost and simulated the routes along which different lineages lose reactions in alternative environments. We performed these simulations on the pan-reactomes of 46 bacterial and archaeal families covering a broad taxonomic range. These simulations allowed us to disentangle metabolic reactions whose presence does, and does not depend on the metabolite composition of the external environment, allowing us to identify reactions constrained "by nutrition" and "by nature", respectively. By comparing the frequency of reactions from the first group with their observed frequencies in bacterial and archaeal families, we predicted the metabolic niches that shaped the genomic composition of these lineages in their evolutionary past. Moreover, we found that the lineages that were shaped by a more diverse metabolic niche also occur in more diverse biomes as assessed by global environmental sequencing datasets. Together, we introduce a computational framework for analyzing and interpreting pan-reactomes that provides new insights into the ecological and evolutionary drivers of pan-genome composition.

evolutionary biology↗

bioRxiv2020

Optimized DNA isolation method for microbiome analysis of human tissues

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies ([~]2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, {gamma}-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

microbiology↗

bioRxiv2019

Growth rate alterations of human colorectal cancer cells by 157 gut bacteria

Several bacteria in the human gut microbiome have been associated with colorectal cancer (CRC) by high-throughput screens. In some cases, molecular mechanisms have been elucidated that drive tumorigenesis, including bacterial membrane proteins or secreted molecules that interact with the human cancer cells. For most gut bacteria, however, it remains unknown if they enhance or inhibit cancer cell growth. Here, we screened bacteria-free supernatants (secretomes) and inactivated cells of over 150 cultured bacterial strains for their effect on CRC cell growth. We observed family-level and strain-level effects that often differed between bacterial cells and secretomes, suggesting that different molecular mechanisms are at play. Secretomes of Bacteroidaceae, Enterobacteriaceae, and Erysipelotrichaceae bacteria enhanced CRC cell growth, while most Fusobacteriaceae cells and secretomes inhibited growth, contrasting prior findings. In some bacteria, the presence of specific functional genes was associated with CRC cell growth rates, including the virulence genes TcdA in Clostridiales and FadA in Fusobacteriaceae, which both inhibited growth. Bacteroidaceae cells that enhanced growth were enriched for genes of the cobalamin synthesis pathway, while Fusobacteriaceae cells that inhibit growth were enriched for genes of the ethanolamine utilization pathway. Together, our results reveal how different gut bacteria have wide-ranging effects on cancer cells, contribute a better understanding of the effects of the gut microbiome on the human host, and provide a valuable resource for identifying candidate target genes for potential microbiome-based diagnostics and treatment strategies.

microbiology↗

bioRxiv2018

Bacterial Zombies And Ghosts: Production Of Inactivated Gram-Positive And Gram-Negative Species With Preserved Cellular Morphology And Cytoplasmic Content

There are many approaches available to inactivate bacteria, each with a different efficacy, impact on cell integrity, and potential for application in high-throughput. The aim of this study was to compare these approaches and develop a standardized protocol for generation of intact Gram-positive as well as on Gram-negative \"bacterial zombies\", i.e. cells that are metabolically dead with retained cellular integrity. Here, we introduce the term \"bacterial zombies\" in addition to \"bacterial ghosts\" to differentiate inactivated bacteria with preserved cellular integrity from those with perforated membranes, where DNA and cytoplasmic contents have been released. This differentiation of inactivated bacteria is important if the cell content is the subject of study, or if cell contents in the media may cause unwanted effects in downstream applications. We inactivated eight different bacterial species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA and visualization by scanning electron microscopy. Based on these results, we discuss the choice of bacterial inactivation methods, and conclude that beta-propiolactone and ethanol are the most promising approaches for standardized generation of bacterial zombies.\n\nIMPORTANCEFor applications such as vaccination or analyses that are sensitive to bacterial growth, inactivated bacteria are preferred because they simplify the analyses and the interpretation of results. This study compared various bacterial inactivation treatments that maintain cell integrity and may be used in high-throughput. Our results demonstrated that beta-propiolactone and 70% ethanol were the best techniques to achieve these goals.

microbiology↗