7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Utilization of the key regulatory protein CP12 to re-direct reductive power for production in cyanobacteria

Regulation & small proteins

Cyanobacteria as phototrophic microorganisms bear great potential to produce chemicals and fuels from sustainable resources such as light, CO2, and water. The recently gained knowledge on internal electron and carbon fluxes and the regulation of cellular metabolism brings about opportunities to channel the energy towards product formation to overcome low efficiencies in product formation that hinder economical application to date. For instance, intrinsic regulators might pose interesting engineering targets to tune metabolic fluxes. In case of reductive processes like hydrogen production or redox catalysis, the partition of redox equivalents determines the efficiency. Here, the small regulatory protein CP12 controlling the major competitor for electrons – the Calvin-Benson-Bassham (CBB) cycle – was utilized to minimize electron drain in the model cyanobacterium Synechocystis sp. PCC 6803. In particular, we made use of phage-based CP12 variants to downregulate the CBB cycle. Unlike the native CP12 of Synechocystis, which only inhibits the CBB cycle in the dark and gets deactivated by a reduced cell status1,2, CP12 homologs from cyanophages appear to be active when the infected host cell is exposed to light. This, together with other metabolic interventions, lead to an enhanced electron availability for phage reproduction3. To simulate this circumstance, we constructed Synechocystis strains that encode those CP12 homologs. In addition, we used rational protein design targeting the elimination of redox-control from the CP12 variant native to Synechocystis. In total, 6 different CP12 variants were inducibly expressed in Synechocystis. Intriguingly, impairment of growth upon induction of CP12 expression could be detected in different strengths for all variants. For promising variants, titratability of this effect could be demonstrated and their impact on the metabolism and hydrogen production was analysed. With this, we demonstrated that small proteins can be used to re-direct metabolic capacities in cyanobacteria.1 McFarlane, et al. 2019, PNAS 116(42): 20984-209902 Lucius et al. 2022, Front. Plant Sci. 13: 10287943 Thompson et al. 2011, PNAS 108: E757-E764

Oral Presentation

Prof. Dr.

Mapping prokaryotic translatomes with ribosome profiling and its derivative approaches

Ribosome profiling (Ribo-seq) is a powerful technique that has been widely used to study and globally annotate translatomes, including small open reading frames (sORFs). The method has enabled the detection of novel translated sORFs in the model organism Escherichia coli and in several other prokaryotes. However, for many bacterial and archaeal species this information is still lacking. To gain insights into the translatomes and hidden world of small proteins (≤ 50-100 amino acids), we have been adapting and optimizing Ribo-seq approaches for diverse prokaryotes. For this purpose, we have established conventional as well as optimized new Ribo-seq derivates based on specific translation inhibitors to detect translation initiation and translation termination sites (TIS and TTS). Furthermore, we have developed several computational tools to aid in the analysis of our datasets. Among these tools are the HRIBO pipeline, which processes raw data and performs metagene profiling analysis, ORF prediction, differential expression analysis, and ORFBounder, which automatically identifies peaks for TIS and TTS libraries.We have applied such Ribo-seq profiling approaches and bioinformatics analyses to several species, including Haloferax volcanii, the plant symbiont Sinorhizobium meliloti, [CS1] and the human pathogens Campylobacter jejuni and Helicobacter pylori. The approaches, together with independent mass spectrometry and western blot validation, expanded the number of small proteins in the different organisms. Additionally, to gain insights into potential sORF functions, we have also generated Ribo-seq datasets under different growth and stress conditions. Moreover, future aims and challenges will involve applying Ribo-seq to anaerobic bacteria from the microbiota or mixtures of organisms, such as during host interaction. Additionally, we created RIBOBASE, an online repository of prokaryotic transcriptomic and translatomic datasets that facilitates the visualization, sharing of data online and direct access to the data. Altogether, our experimental and computational approaches will be beneficial for both current and future research on small proteins in prokaryotes.

CemR atypical response regulator impacts energy conversion in Campylobacteria

Campylobacter jejuni and Arcobacter butzleri are microaerophilic food-borne human gastrointestinal pathogens of the Campylobacteria class. According to WHO, campylobacteriosis is the leading cause of diarrhoeal diseases, while A. butzleri is an emerging pathogen associated with diarrhoea, rapidly increasing in incidence worldwide. These species face different atmospheric conditions during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Thus, they need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonisation sites. Interestingly, no regulators redirecting energy conversion pathways in response to oxygen as an electron acceptor have been found in Campylobacteria (doi.org/10.1016/bs.ampbs.2019.02.003).Our previous research on gastric human pathogen Helicobacter pylori, relative to C. jejuni and A. butzleri, revealed that the orphan response regulator HP1021 is a redox switch protein controlling the transcription of approx. 30% of genes (doi.org/10.1093/nar/gkab440, doi.org/10.1038/s41467-023-42364-6). The HP1021 regulon includes the tricarboxylic acid cycle (TCA) genes/proteins.Here, we studied the regulons of HP1021 homologues in C. jejuni and A. butzleri, Cj1608 and Abu0127, respectively. We used RNA-seq and LC-MS/MS to identify genes/proteins regulated by Cj1608 and Abu0127 and define the Cj1608/Abu0127 dependent pathways used by C. jejuni and A. butzleri to respond to oxidative stress. We found that these regulators are involved in the control of more than 30% of genes in each species. The genes/proteins affected most in both species are related to metabolism and energy conservation processes, mostly TCA and electron transport chain. Using phenotypic analyses, we confirmed that energy conversion processes in Cj1608/Abu0127 deletion mutants are disturbed, which causes their delayed growth. Moreover, our combined data indicate that these regulators respond to oxygen levels and redirect metabolism towards optimal energy conservation. Thus, we named the proteins Campylobacteria energy and metabolism regulators – CemR.

Raum 13

Oral Session

Regulation & Small Proteins

Oral Sessions

Oral Session
Regulation & Small Proteins

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Oral SessionRegulation & Small Proteins

Appointment

Chair

Topic

Programme

Oral Session
Regulation & Small Proteins