Biology subjects

Hartono, S.

Publications and source records attributed to Hartono, S..

4 recordsLinked to original sources

bioRxiv2026

Stressostat cultivation of Lactococcus lactis improves lactate stress resistance through mutations in RNA polymerase

Adaptive laboratory evolution is used to improve the phenotypes of microorganisms and to characterise the mechanisms underlying resistance against complex growth inhibition. Here we focused on lactic acid bacteria (LAB) as starter cultures for food fermentations. Production of LAB starter cultures is challenging due to growth inhibition by organic acids, mainly lactate, produced during fermentation. By utilising stressostat cultivation we generated Lactococcus lactis isolates with enhanced lactate resistance. Using a combination of (meta)genomics, proteomics and pH-controlled batch fermentations, we deciphered the lactate resistance mechanisms of these L. lactis isolates. Proteome responses of L. lactis, combined with similar growth inhibition at high salt, suggest that high lactate mainly causes osmotic stress. We identified RNA polymerase (RNAP) mutations in subunits {beta} (rpoB) and {beta} (rpoC) as key mutations, causing pleiotropic effects in the proteome. These proteome adaptations are linked to enhanced lactate resistance, particularly the resistance to hyperosmotic stress without glycine-betaine supplementation, likely by altering cross-linking of the peptidoglycan in the cell envelope via downregulation of MurE. The proteome changes indicate that lactate might (indirectly) cause oxidative stress. Combined, our study shows that RNAP mutations enhanced lactate resistance through pleotropic effects in the proteome that changed L. lactis responses against multiple stresses. HighlightsO_LILactate resistant variants were isolated from stressostat cultivations C_LIO_LIMetagenomics revealed evolutionary trajectory during stressostat cultivation C_LIO_LIRNAP mutations were identified as key drivers for improved lactate resistance C_LIO_LIRNAP mutations altered the proteome, enhancing the osmotic stress resistance C_LI

microbiology↗

bioRxiv2025

Protein-mediated stabilization and nicking of the non-template DNA strand dramatically affect R-loop formation in vitro

R-loops are an important class of non-B DNA structures that form co-transcriptionally. Using in vitro transcription and unbiased quantitative sequencing readouts, we show that the addition of single-strand DNA binding proteins co-transcriptionally can drive a 3- to 5-fold increase of R-loop frequency without significant changes to R-loop distribution. We propose that this is caused by stabilizing and preventing the collapse of short nascent R-loops. This suggests that R-loop formation is highly dynamic and highlights single strand binding proteins as players in cellular R-loop regulation. We further show that non-template strand DNA nicks are powerful initiators of R-loop formation, increasing R-loop frequencies by up to two orders of magnitude. Atomic force microscopy (AFM) revealed that the non-template strand in nick-initiated structures is often flayed away from the RNA:DNA hybrid and engaged in self-pairing, creating unique forked R-loop features. DNA nicks, one of the most frequent DNA lesions in cells, are therefore potential hotspots for opportunistic R-loop initiation and may cause the formation of a novel class of R-loops. Overall, this work highlights the importance of the displaced single-strand on R-loop initiation and dynamics. SIGNIFICANCE STATEMENTR-loops are three-stranded DNA:RNA hybrid structures that form during transcription and play critical roles in both gene regulation and genome stability. This study reveals that single-stranded DNA-binding protein (SSB) increases R-loop frequency by stabilizing nascent R-loops, revealing a role for this protein in R-loop dynamics. Additionally, single strand DNA breaks, common cellular lesions, are identified as potent initiators of R-loops, increasing their rate of formation by up to 100-fold. Using atomic force microscopy, the study uncovers unique forked structures in nick-initiated R-loops, representing a novel class of secondary R-loop structures. These findings provide new insights into how cellular factors and DNA damage influence R-loop formation, with implications for understanding genome instability in health and disease.

molecular biology↗

bioRxiv2024

The R-loop Grammar predicts R-loop formation under different topological constraints

R-loops are transient three-stranded nucleic acids that form during transcription when the nascent RNA hybridizes with the template DNA, freeing the DNA non-template strand. There is growing evidence that R-loops play important roles in physiological processes such as control of gene expression, and that they contribute to chromosomal instability and disease. It is known that R-loop formation is influenced by both the sequence and the topology of the DNA substrate, but many questions remain about how R-loops form and the 3-dimensional structures that they adopt. Here we represent an R-loop as a word in a formal grammar called the R-loop grammar and predict R-loop formation. We train the R-loop grammar on experimental data obtained by single-molecule R-loop footprinting and sequencing (SMRF-seq). Despite not containing explicit topological information, the R-loop grammar accurately predicts R-loop formation on plasmids with varying starting topologies and outperforms previous methods in R-loop prediction. Author summaryR-loops are prevalent triple helices that play regulatory roles in gene expression and are involved in various diseases. Our work improves the understanding of the relationship between the nucleotide sequence and DNA topology in R-loop formation. We use a mathematical approach from formal language theory to define an R-loop language and a set of rules to model R-loops as words in that language. We train the resulting R-loop grammar on experimental data of co-transcriptional R-loops formed on different DNA plasmids of varying topology. The model accurately predicts R-loop formation and outperforms prior methods. The R-loop grammar distills the effect of topology versus sequence, thus advancing our understanding of R-loop structure and formation.

genomics↗

bioRxiv2020

Vascular Endothelial Growth Factor as an Immediate-Early Activator of UV-induced Skin Injury

The negative health consequences of acute ultraviolet (UV) exposure are evident, with reports of 30,000 emergency room visits annually to treat the effects of sunburn in the United States alone. Acute effects of sunburn include erythema, edema, and severe pain, and chronic overexposure to UV radiation can lead to skin cancer. While the pain associated with the acute effects of sunburn may be relieved by current interventions, existing post-sunburn treatments are not capable of reversing the cumulative and long-term pathological effects of UV exposure, an unmet clinical need. Here we show that activation of the vascular endothelial growth factor (VEGF) pathway is a direct and immediate consequence of acute UV exposure, and activation of VEGF signaling is necessary for the initiating the acute pathological effects of sunburn. In UV-exposed human subjects, VEGF signaling is activated within hours. Topical delivery of VEGF pathway inhibitors, targeted against the ligand VEGF-A (gold nanoparticles conjugated with anti-VEGF antibodies) and small molecule antagonists of VEGF receptor signaling, prevent the development of erythema and edema in UV-exposed mice. Collectively, these findings suggest targeting VEGF signaling may reduce the subsequent inflammation and pathology associated with UV-induced skin damage, which reveals a new post-exposure therapeutic window to potentially inhibit the known detrimental effects of UV on human skin.

molecular biology↗