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Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA.

ABSTRACT: The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

SUBMITTER: Clark IC

PROVIDER: S-EPMC8000134 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Soil microbial community assembly is crucial for understanding the mechanisms of microbial communities that regulate ecosystem-level functioning. The relative contributions of stochastic and deterministic processes to microbial community assembly remain poorly defined, and major questions exist concerning the soil organic carbon (SOC) dynamics of microbial community assembly in deep soil. Here, the bacterial community assembly processes were explored across five soil profile depths (up to 80 cm) during a 15-year field experiment involving four fertilization regimes. We found that the bacterial community assembly was initially governed by deterministic selection in topsoil but was progressively structured by increasing stochastic dispersal with depth. The migration rate (m) and β-null deviation pattern supported the hypothesis of a relatively greater influence of dispersal in deep soil, which was correlated with bacterial community assembly by stochastic processes. These changes in the entire community assembly reflected consistent assembly processes of the two most dominant phyla, Acidobacteria and Chloroflexi Structural equation modeling showed that soil features (pH and total phosphorus) and bacterial interactions (competition and network complexity) were significantly related to bacterial community assembly in the 0-to-10-cm and 10-to-20-cm layers. Partial Mantel tests, structural equation modeling, and random forest modeling consistently indicated a strong and significant correlation between bacterial community assemblages and SOC dynamics, implying that bacterial assembly processes would potentially suppress SOC metabolism and mineralization when the contributions of stochastic dispersal to communities increased in deeper layers. Our results have important implications for integrating bacterial community assembly processes into the predictions of SOC dynamics.IMPORTANCE We have provided a framework to better understand the mechanisms governing the balance between stochastic and deterministic processes and to integrate the shifts in community assembly processes with microbial carbon metabolism. Our study reinforced that environmental filtering and bacterial cooccurrence patterns influence the stochastic/deterministic continuum of soil bacterial community assembly and that stochasticity may act through deeper soil layers to influence carbon metabolism. Delineating theoretically the potential linkages between community assembly and SOC dynamics across a broad range of microbial systems represents an interesting topic for future research.

Project description:BackgroundThe evolution of Next-Generation Sequencing (NGS) has considerably reduced the cost per sequenced-base, allowing a significant rise of sequencing projects, mainly in prokaryotes. However, the range of available NGS platforms requires different strategies and software to correctly assemble genomes. Different strategies are necessary to properly complete an assembly project, in addition to the installation or modification of various software. This requires users to have significant expertise in these software and command line scripting experience on Unix platforms, besides possessing the basic expertise on methodologies and techniques for genome assembly. These difficulties often delay the complete genome assembly projects.ResultsIn order to overcome this, we developed SIMBA (SImple Manager for Bacterial Assemblies), a freely available web tool that integrates several component tools for assembling and finishing bacterial genomes. SIMBA provides a friendly and intuitive user interface so bioinformaticians, even with low computational expertise, can work under a centralized administrative control system of assemblies managed by the assembly center head. SIMBA guides the users to execute assembly process through simple and interactive pages. SIMBA workflow was divided in three modules: (i) projects: allows a general vision of genome sequencing projects, in addition to data quality analysis and data format conversions; (ii) assemblies: allows de novo assemblies with the software Mira, Minia, Newbler and SPAdes, also assembly quality validations using QUAST software; and (iii) curation: presents methods to finishing assemblies through tools for scaffolding contigs and close gaps. We also presented a case study that validated the efficacy of SIMBA to manage bacterial assemblies projects sequenced using Ion Torrent PGM.ConclusionBesides to be a web tool for genome assembly, SIMBA is a complete genome assemblies project management system, which can be useful for managing of several projects in laboratories. SIMBA source code is available to download and install in local webservers at http://ufmg-simba.sourceforge.net .

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Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA.

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