Bai, F.-W. | bioRxiv Science

bioRxiv2024

Previously uncharacterized aliphatic amino acid positions modulate the apparent catalytic activity of the EAL domain of ZMO_1055 and other cyclic di-GMP specific EAL phosphodiesterases

The ubiquitous second messenger cyclic di-GMP signaling system decides bacterial life style transition between sessility and motility. GGDEF diguanylate cyclase and EAL phosphodiesterase domains conventionally direct the turnover of this signaling molecule being subject to micro- and macroevolution. While the highly conserved signature amino acids involved in divalent ion binding and catalysis have readily been identified, recognition of amino acid substitutions that modulate the catalytic activity is rare. Associated with development towards cellulose-mediated self-flocculation in Zymomonas mobilis ZM401, the A526V substitution, previously not been recognized to affect the functionality of the EAL domain, downregulates the apparent catalytic activity of the PAS-GGDEF-EAL ZMO1055 phosphodiesterase compared to parental Z. mobilis ZM4 and equally in homologous protein domains independently of the genetic background. Substitution of A526, which is conserved among homologs, with amino acids with longer aliphatic side chains than valine have an even more pronounced effect. Thus single amino acid substitutions that lead to alterations in the catalytic activity of cyclic di-GMP turnover domains amplify the signaling output and thus significantly contribute to the flexibility and adaptability of the cyclic di-GMP signaling network. In this context, ZMO1055 seems to be a current evolutionary target.

microbiology↗

bioRxiv2021

Exploration of molecular mechanism underlying the self-flocculation of Zymomonas mobilis through comparative omics analyses and experimental validations for developing robust production systems

Zymomonas mobilis metabolizes sugar through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation and more substrate to product formation with improved yield, since ATP is dissipated predominately through growth for intracellular energy homeostasis, making it a platform to be engineered as microbial cell factories, particularly for producing bulk commodities with major cost from feedstock consumption. ZM401, a self-flocculating mutant, presents advantages for production including cost-effective biomass recovery through gravity sedimentation, self-immobilization within bioreactors for high cell density to improve productivity and enhanced tolerance to environmental stresses for high product titers, but molecular mechanism underlying this phenotype is largely unknown. In this work, we sequenced and assembled the genome of ZM401 to explore genetic basis for the self-flocculation of the bacterial cells through comparative genomic and transcriptomic analyses, molecular docking simulations for enzymes encoded by functional genes and their substrates/activators, and experimental validations. Our results demonstrated that the single nucleotide deletion in ZMO1082 disrupted its stop codon for the putative gene being fused with ZMO1083, which created an exciting gene encoding the subunit A of the bacterial cellulose synthase with unique function for synthesizing cellulose microfibrils to flocculate the bacterial cells, and the single nucleotide mutation in ZMO1055 compromised the function of bifunctional diguanylate cyclase/phosphodiesterase encoded by the gene on the degradation of c-di-GMP for its intracellular accumulation to activate the cellulose biosynthesis. These discoveries are significant not only for optimizing the self-flocculation of Z. mobilis, but also engineering other bacteria with the self-flocculating phenotype for robust production.

microbiology↗