Biology subjects

Hochberg, G.

Publications and source records attributed to Hochberg, G..

3 recordsLinked to original sources

bioRxiv2021

Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

synthetic biology↗

bioRxiv2021

A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading. SignificancePhenotypic heterogeneity benefits the proliferation of microbial populations in changing environments. Such heterogeneity can be created by recruitment of enzymatic activity to specific cellular compartments, e.g., the cell pole. Here we show how a GGDEF domain of a multidomain phosphodiesterase has adopted the function as a spatial on-switch that is specifically activated upon direct interaction with a polar landmark protein.

microbiology↗

bioRxiv2021

Structure and mechanistic features of the prokaryotic minimal RNase P

Endonucleolytic removal of 5-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

biochemistry↗