Biology subjects

Swarts, D. C.

Publications and source records attributed to Swarts, D. C..

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↗

bioRxiv2023

RNA-guided RNA silencing by an Asgard archaeal Argonaute

Eukaryotic Argonaute proteins achieve gene repression and defense against viruses and transposons by RNA-guided RNA silencing. By contrast, known prokaryotic Argonautes adopt single-stranded DNA as guides and/or targets, leaving the evolutionary origin of RNA-guided RNA silencing elusive. Here, we show an evolutionary expansion of Asgard archaeal Argonautes (asAgos), including the discovery of HrAgo1 from the Lokiarchaeon Candidatus Harpocratesius repetitus that shares a common origin with eukaryotic PIWI proteins. HrAgo1 exhibits RNA-guided RNA cleavage in vitro and RNA silencing in human cells. The cryo-EM structure of HrAgo1 combined with quantitative single-molecule experiments reveals that HrAgo1 possesses hybrid structural features and target binding modes bridging those of the eukaryotic AGO and PIWI clades. Finally, genomic evidence suggests that eukaryotic Dicer-like processing of double-stranded RNA likely emerged as a mechanism of generating guide RNA for asAgos prior to eukaryogenesis. Our study provides new insights into the evolutionary origin and plasticity of Argonaute-based RNA silencing.

microbiology↗

bioRxiv2023

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, suppressing SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation. KEY POINTSO_LIInactive monomeric SPARTA is autoinhibited by the C-terminal tail of TIR-APAZ C_LIO_LITarget DNA recognition causes C-terminal tail release, pAgo restructuring, and dimerization C_LIO_LITIR domain rotation enables catalytic activation and cooperative tetramer formation C_LI

biochemistry↗

bioRxiv2019

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures ([≥]65{degrees}C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5-end and thymidines in the sub-seed segment (siDNA nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37{degrees}C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

molecular biology↗