Biology subjects

Wiegard, A.

Publications and source records attributed to Wiegard, A..

5 recordsLinked to original sources

bioRxiv2026

Easy-to-use whole-genome sequencing workflows and standardized practices to uncover hidden genetic variation in Synechocystis PCC 6803 wild-type and knock-out strains

Knock-out mutants are often used to study gene function by disrupting a specific gene and comparing the mutant to a wild-type strain. Reliable interpretation, however, requires that the two strains differ only by the intended mutation and that the observed phenotype is caused specifically by the deleted gene. In the highly polyploid cyanobacterium Synechocystis sp. PCC 6803, this is particularly challenging because incomplete segregation can mask genetic heterogeneity or secondary suppressor mutations. The genetic variation among laboratory wild-type lines can further confound phenotypic analyses. We show that these challenges can be addressed by routine strain validation via whole-genome sequencing (WGS). To this end, we developed and tested user friendly workflows for short-read (Illumina), long-read (Oxford Nanopore Technologies; ONT), and hybrid data, providing standardized quality control, variant calling, and structural variant detection. We benchmarked their performance in detecting single-nucleotide polymorphisms (SNPs), small indels, and structural variants using simulated datasets across different coverages and mixed populations. Applying the workflows to three Synechocystis sp. PCC 6803 wild-type lines revealed multiple sequence and structural differences relative to the reference genome, including previously undescribed genetic variants, underscoring the importance of documenting the strain background and the value of long-read sequencing. Characterization of two independent 6-phosphogluconate dehydrogenase (gnd) knock-out mutants and their complemented strains highlighted how a failed rescue can reveal a phenotype unrelated to the intended knock-out. An automated literature analysis revealed that only a minority of the investigated Synechocystis studies that used knock-out mutants included complementation as a control (39%), whereas this practice is more common in studies involving Escherichia coli (63%) and Saccharomyces cerevisiae (55%). Based on these results, we propose a practical guide for standardizing knock-out phenotyping in Synechocystis PCC 6803. Combined with accessible workflows for routine whole-genome validation, this framework aims to support more robust and reproducible knock-out studies in the future.

microbiology↗

bioRxiv2026

Live-cell imaging enables reporter-free monitoring of the circadian rhythm in individual Synechocystis cells

In vivo monitoring of circadian rhythms depends on reliable and non-invasive detection methods. This is often achieved by expressing reporter genes heterologously under the control of a circadian promoter. The activity or fluorescence of the gene product is then used as a readout. To avoid the need for generation of such reporter strains, we recently established a reporter-free detection method for cyanobacterial batch cultures. To determine whether these rhythms are driven at the level of individual cells or result from population-based effects, such as gating of cell division, we analyzed individual Synechocystis sp. PCC 6803 cells by combining a microfluidic cultivation technique with multipoint time-lapse microscopy imaging at the single-cell resolution. Hundreds of time-lapse image sequences, acquired over a period of up to ten days, were processed using our deep learning cell segmentation workflow. Although the cells had been entrained by a 12-hour light-dark cycle, neither cell size nor cell division displayed circadian rhythms. This indicates the absence of circadian gating of cell division in Synechocystis. Instead, we observed circadian oscillation in the average brightness of the phase contrast of individual Synechocystis cells. To demonstrate how phase-contrast analysis of single cells can be complemented by backscatter analysis of batch cultures, we investigated the wildtype, a deletion mutant known to affect circadian rhythms ({Delta}kaiC3) and complementation strains at both, the single-cell and batch levels. We concluded that phase contrast and backscatter likely measured the same rhythmic changes in the refractive index of the cells. The method presented here will advance circadian research by enabling the analysis of circadian rhythms in individual cells without the need for expression of reporter molecules.

microbiology↗

bioRxiv2023

Self-sustained rhythmic behavior of Synechocystis PCC 6803 under continuous light conditions in the absence of light-dark entrainment

Circadian clocks regulate biological activities, providing organisms a fitness advantage under diurnal changing conditions by allowing them to anticipate and adapt to recurring external changes. In recent years attention was drawn to the entrainment by intracellular cycles. Photosynthetic Cyanobacteria coordinate their gene expression, metabolism, and other activities in a circadian fashion. Solely, three proteins, KaiA, KaiB, and KaiC, constitute the well-studied circadian clock of the cyanobacterial model, Synechococcus elongatus PCC 7942. It remained inconclusive for a long time whether Synechocystis sp. PCC 6803, an important organism for biotechnological applications, can also maintain circadian rhythms under continuous illumination. Using an approach, which does not require genetic modification, we investigated the growth behavior of Synechocystis via non-invasive online backscattering measurement and verified all three criteria for true circadian oscillators: temperature compensation, entrainment by external stimuli, and a self-sustained freerunning period of about 24 hours. Since manipulation of the circadian clock (Synechocystis {Delta}kaiA1B1C1) led to a significant reduction in glycogen content, disruption of glycogen synthesis (Synechocystis {Delta}glgC) entirely inhibited glycogen formation and both mutants lost oscillations, we hypothesize that the oscillations reflect glycogen metabolism. Significance StatementMonitoring circadian rhythms in cyanobacteria usually requires genetically modified reporter strains or intensive sampling for downstream analysis. Even for the main cyanobacterial model Synechocystis sp. PCC 6803 it was debated for years to which extent undamped circadian oscillations are really present until a suitable reporter strain was developed. We applied online backscatter measurements as an alternative readout to monitor circadian oscillations in cyanobacteria. In Synechocystis the temperature-compensated kaiA1B1C1-driven 24 h metabolic oscillations did not require light-dark entrainment, highlighting the relevance of the clock for the carbon metabolism even under continuous light, an aspect which should be considered for industrial set-ups. Our method opens the possibility to extend circadian analysis to non-GMO and monitor metabolic rhythmicity during high-density cultivation.

systems biology↗

bioRxiv2023

Co-inhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription

Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive transcription also occurs during replication, perturbing replisome progression and leading to DNA damage. The synergistic effect of TOP1 and BRD4 inhibition is specific for cancer cells leaving normal cells unharmed, highlighting the sensitivity of the tumor to these transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of high transcription and replication. One Sentence SummaryTOP1 and BRD4 inhibitors synergize to selectively kill pancreatic cancer in vivo via readthrough transcription without emergence of drug resistance

cancer biology↗

bioRxiv2019

Synechocystis KaiC3 displays temperature and KaiB dependent ATPase activity and is important for viability in darkness

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of{Delta} kaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a crosstalk between the KaiC1 and KaiC3 based systems.

microbiology↗