Biology subjects

Georg, J.

Publications and source records attributed to Georg, J..

10 recordsLinked to original sources

bioRxiv2026

Functional decoding reveals a hidden regulatory layer of the Salmonella transcriptome during infection

Bacterial transcriptomes contain extensive, largely unexplored regulatory information beyond annotated genes, including small regulatory RNAs (sRNAs), yet distinguishing functionally active transcripts from the broader non-coding transcriptome remains a fundamental challenge, particularly in the context of host-pathogen interactions. Here, we develop an unbiased highthroughput functional screening strategy to decode the regulatory potential of the Salmonella enterica transcriptome during macrophage infection. A pooled expression library comprising 875 RNA fragments derived from infection-relevant conditions was screened for effects on bacterial invasion and intracellular survival, revealing distinct and stage-specific regulatory activities. Functional characterization uncovered non-canonical sRNAs originating from 5' untranslated regions that differentially modulate virulence-associated programs spanning the SPI-1, SPI-4 and SPI-2 pathogenicity islands. One attenuates the SPI-1 and SPI-4 secretion systems, limiting bacterial adhesion and invasion, whereas the other promotes invasion-associated programs while repressing pathways supporting intracellular survival. Rather than acting solely on individual virulence determinants, these regulatory activities reveal a broader layer of posttranscriptional control that coordinates bacterial adaptation across the dynamic host environment. Our study establishes a scalable strategy for extracting functional regulatory information from condition-specific bacterial transcriptomes and provides a framework for uncovering context-dependent RNA-mediated control of complex host-associated phenotypes.

microbiology↗

bioRxiv2024

An unprecedented small RNA-riboswitch interaction controls expression of a bifunctional pump that is essential for Staphylococcus aureus infection

Maintaining manganese and iron homeostasis is critical for the human pathogen Staphylococcus aureus. To counteract metal-based host defense strategies (e.g., nutritional immunity, metal poisoning), S. aureus uses a combination of metal-sensing transcription factors and regulatory RNAs to maintain metal homeostasis. In this study, we uncovered an unprecedented interaction between a cis- and a trans-acting regulatory RNA controlling a conditionally essential gene, mntY, encoding a Mn efflux pump. This broadly conserved RNA-RNA interaction between a Fe-responsive sRNA and a Mn-sensing riboswitch allows the integration of Feand Mn-related stresses, notably encountered at the infection site, to fine-tune mntY expression. Remarkably, deletion of the mntY gene is strongly pleomorphic, causing growth defects, altering virulence factor expression, immune evasion, and survival during infection. We demonstrated that MntY is critical for the adaptation of S. aureus to both low and high Mn environments, due to its dual role in metalation of Mn-dependent exoenzymes and Mn detoxification. These findings point to MntY as a promising new therapeutic target to combat multidrug-resistant staphylococcal infections.

microbiology↗

bioRxiv2024

A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and an oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.

synthetic biology↗

bioRxiv2024

Characterization of YlxR/Ssr1238, a conserved RNA binding protein in a model cyanobacterium

Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Structural prediction suggests an RNA-binding interface for the protein YlxR/Ssr1238 in the cyanobacterium Synechocystis 6803. Two pairs of cysteine residues are arranged as possibly coordinating an Fe-S cluster and appear widely conserved in the homologous proteins of other cyanobacteria. Overexpression of Ssr1238 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript derived from gene ssl3177, which encodes a central enzyme in cell wall remodeling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and participates in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the B. subtilis YlxR protein functions as an RNase P modulator (RnpM), but suggest additional functionalities and extend its proposed role to the phylum cyanobacteria.

microbiology↗

bioRxiv2023

Balanced cell division is secured by two different regulatory sites in OxyS RNA

By interfering with cell division, the Escherichia coli oxidative stress-induced OxyS small RNA brings about cell cycle arrest thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division OxyS inhibits mepS encoding an essential peptidoglycan endopeptidase responsible for cell elongation. Furthermore, a phylogenetic evolutionary target conservation analysis of OxyS homologs to identify OxyS most common function revealed that the majority of OxyS targets belong to the category of "cell cycle" followed by "peptidoglycan metabolism", especially cell elongation. mepS is the most frequent target of this category, predicted to be regulated by OxyS in 64 % of the 146 organisms studied. We suggest that cell cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress induced sRNA OxyS.

microbiology↗

bioRxiv2022

Feedback regulation of RNase E during UV-stress response in the cyanobacterium Synechocystis sp. PCC 6803

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5'-untranslated region (5' UTR); however, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation via its own 5' UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

microbiology↗

bioRxiv2021

Expression of the F0F1 ATP synthase inhibitor Atp{Theta} depends on small basic DNA-binding proteins and differential mRNA stability

F0F1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. Atp{Theta} is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. Atp{Theta} inhibits the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, Atp{Theta} is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2 and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that basic transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCEF0F1 ATP synthases are protein complexes that produce ATP, the universal biological energy source in all kinds of organisms. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis but they cannot use the same mechanism as plants that inhibit chloroplast ATP synthases entirely during the night because respiratory and photosynthetic complexes are both located in the same membrane system. Atp{Theta} is a small peptide inhibitor of the reverse ATPase function in cyanobacteria encoded by the gene atpT. The production of Atp{Theta} is highly regulated to ensure that it is only synthetized when it is needed. In the here presented work we found that three transcription factors contribute to the regulation of atpT expression. However, we identified the control of mRNA stability as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the Atp{Theta}-based inhibitory mechanism within the context of the cellular redox and energy balance.

plant biology↗

bioRxiv2021

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome and proteome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

systems biology↗

bioRxiv2018

Global analysis of the RpaB regulon based on the positional distribution of HLR1 sequences and comparative differential RNA-Seq data

The transcription factor RpaB regulates the expression of genes encoding photosynthesis-associated proteins during light acclimation. The binding site of RpaB is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two random nucleotides. Here, we used high-resolution mapping data of transcriptional start sites (TSSs) in the model Synechocystis sp. PCC 6803 in conjunction with the positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The results demonstrate that RpaB regulates the expression of more than 150 promoters, driving the transcription of protein-coding and non-coding genes and antisense transcripts under low light and upon the shift to high light when DNA binding activity is lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. Selected examples were validated by multiple experimental approaches, including chromatin affinity purification, reporter gene, northern hybridization and electrophoretic mobility shift assays. We found that RpaB controls ssr2016/pgr5, which is involved in cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH proteases; gcvP/slr0293, encoding a crucial photorespiratory protein; and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA, respectively. In addition to photosynthetic gene functions, RpaB contributes to the control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the CRISPR system and the circadian clock, making it one of the most versatile regulators in cyanobacteria.\n\nSignificance StatementRpaB is a transcription factor in cyanobacteria and in the chloroplasts of several lineages of eukaryotic algae. Like other important transcription factors, the gene encoding RpaB cannot be deleted, making the study of deletion mutants impossible. Based on a bioinformatic approach, we increased the number of known genes controlled by RpaB by a factor of 5. Depending on the distance to the TSS, RpaB mediates transcriptional activation or repression. The high number and functional diversity among its target genes and co-regulation with other transcriptional regulators characterize RpaB as a regulatory hub.

microbiology↗

bioRxiv2017

6S RNA Supports Recovery From Nitrogen Depletion In Synechocystis Sp. PCC 6803

BackgroundThe 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles.\n\nResultsPhysiological characterization of a 6S RNA deletion strain ({Delta}ssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type.\n\nTranscriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in {Delta}ssaA. In vivo pull-down studies of the RNA polymerase complex indicated a promoting effect of 6S RNA on the recruitment of the cyanobacterial housekeeping {sigma} factor SigA, concurrently supporting dissociation of group 2 {sigma} factors during recovery from nitrogen starvation.\n\nConclusionsThis study reveals 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by regulating the switch from group 2 {sigma} factors SigB, SigC and SigE to SigA- dependent transcription.

molecular biology↗