Biology subjects

Seemann, S. E.

Publications and source records attributed to Seemann, S. E..

5 recordsLinked to original sources

bioRxiv2022

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids the enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and a control in a time-series RNA-seq experiment. We observe transcription in 542 previously un-annotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. Via gene-set over-representation analysis of the differentially expressed genes, we identified putatively regulated biological processes; overall the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

microbiology↗

bioRxiv2022

CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis

Enhancing yield during bacterial enzyme production could have positive economic and environmental impacts. For cell factories, such improvements in yields could potentially be obtained by fine-tuning the metabolic processes and their regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two -amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Complex operons that are regulated by a wide variety of transcription factors, non-coding and structured RNAs add to the challenge of finding yield-affecting candidates. Surprisingly, we found that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation (75% of novel RNA predictions had absolute logFC > 2). Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expressed annotations, including both novel candidate and prior annotated ncRNAs, we selected 53 non-coding candidates. The targeting with CRISPRi knockdowns transcription in a genomic region on both the sense and the antisense strand. Thus, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

microbiology↗

bioRxiv2021

Development of the entorhinal cortex occurs via parallel lamination during neurogenesis.

The entorhinal cortex (EC) is the spatial processing center of the brain and structurally is an interface between the three layered paleocortex and six layered neocortex, known as the periarchicortex. Limited studies indicate peculiarities in the formation of the EC such as early emergence of cells in layers (L) II and late deposition of LIII, as well as divergence in the timing of maturation of cell types in the superficial layers. In this study, we examine developmental events in the entorhinal cortex using an understudied model in neuroanatomy and development, the pig and supplement the research with BrdU labeling in the developing mouse EC. We determine the pig serves as an excellent anatomical model for studying human neurogenesis, given its long gestational length, presence of a moderate sized outer subventricular zone and early cessation of neurogenesis during gestation. Immunohistochemistry identified prominent clusters of OLIG2+ oligoprogenitor-like cells in the superficial layers of the lateral EC (LEC) that are sparser in the medial EC (MEC). These are first detected in the subplate during the early second semester. MRI analyses reveal an acceleration of EC growth at the end of the second trimester. BrdU labeling of the developing MEC, shows the deeper layers form first and prior to the superficial layers, but the LV/VI emerges in parallel and the LII/III emerges later, but also in parallel. We coin this lamination pattern parallel lamination. The early-born Reln+ stellate cells in the superficial layers express the classic LV marker, Bcl11b (Ctip2) and arise from a common progenitor that forms the late deep layer LV neurons. In summary, we characterize the developing EC in a novel animal model and outline in detail the formation of the EC. We further provide insight into how the periarchicortex forms in the brain, which differs remarkably to the inside-out lamination of the neocortex.

developmental biology↗

bioRxiv2019

The BSGatlas: An enhanced annotation of genes and transcripts for the Bacillus subtilis genome with improved information access

The genome of Bacillus subtilis continues to provide exiting genomic insights. However, the growing collective genomic knowledge about this micro-organism is spread across multiple annotation resources. Thus, the full annotation is not directly accessible neither for specific genes nor for large-scale high-throughput analyses. Furthermore, access to annotation of non-coding RNA genes (ncRNAs) and polycistronic mRNAs is difficult. To address these challenges we introduce the Bacillus subtilis genome atlas, BSGatlas, in which we integrate and unify multiple existing annotation resources. Our integration provides twice as many ncRNAs than the individual resources, improves the positional annotation for 70% of the combined ncRNAs, and makes it possible to infer specific ncRNA types. Moreover, we unify known transcription start sites, termination, and transcriptional units (TUs) as a comprehensive transcript map. This transcript map implies 815 new TUs and 6, 164 untranslated regions (UTRs), which is a five-fold increase over existing resources. We furthermore, find 2, 309 operons covering the transcriptional annotation for 93% of all genes, corresponding to an improvement by 11%. The BSGatlas is available in multiple formats. A user can either download the entire annotation in the standardized GFF3 format, which is compatible with most bioinformatics tools for omics and high-throughput studies, or view the annotation in an online browser at http://rth.dk/resources/bsgatlas.\n\nImportanceThe Bacillus subtilis genome has been studied in numerous context and consequently multiple efforts have been made in providing a complete annotation. Unfortunately, a number of resources are no longer maintained, and (i) the collective annotation knowledge is dispersed over multiple resources, of which each has a different focus of what type of annotation information they provide. (ii) Thus, it is difficult to easily and at a large scale obtain information for a genomic region or genes of interest. (iii) Furthermore, all resources are essentially incomplete when it comes to annotating non-coding and structured RNA, and transcripts in general. Here, we address all three problems by first collecting existing annotations of genes and transcripts start and termination sites; afterwards resolving discrepancies in annotations and combining them, which doubled the number of ncRNAs; inferring full transcripts and 2,309 operons from the combined knowledge of known transcript boundaries and meta-information; and critically providing it all in a standardized UCSC browser. That interface and its powerful set of functionalities allow users to access all the information in a single resource as well as enables them to include own data on top the full annotation.

genomics↗

bioRxiv2019

Sandwich cortical lamination and single-cell analysis decodes the developing spatial processing system.

Classic studies investigating how and when the entorhinal cortex (component of the memory processing system of the brain) develops have been based on traditional thymidine autoradiography and histological techniques. In this study, we take advantage of modern technologies to trace at a high resolution, the cellular complexity of the developing porcine medial entorhinal cortex by using single-cell profiling. The postnatal medial entorhinal cortex comprises 4 interneuron, 3 pyramidal neuron and 2 stellate cell populations which emerge from intermediate progenitor and immature neuron populations. We discover four MGE-derived interneurons and one CGE-derived interneuron population as well as several IN progenitors. We also identify two oligodendrocyte progenitor populations and three populations of oligodendrocytes. We perform a proof-of-concept experiment demonstrating that porcine scRNA-seq data can be used to develop novel protocols for producing human entorhinal cells in-vitro. We identified six transcription factors (RUNX1A1, SOX5, FOXP1, MEF2C, TCF4, EYA2) important in neurodevelopment and differentiation from one RELN+ stellate cell population. Using a lentiviral vector approach, we reprogrammed human induced pluripotent stem cells into stellate cell-like cells which expressed RELN, SATB2, LEF1 and BCL11B. Our findings contribute to the understanding of the formation of the brains cognitive memory and spatial processing system and provides proof-of-concept for the production of entorhinal cells from human pluripotent stem cells in-vitro.

developmental biology↗