Ligand-Binding PAS Domains in a Genomic, Cellular, and Structural Context

A major roadblock to understanding how microbes in the gastrointestinal tract colonize and influence the physiology of their hosts is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. We describe the application of population-based genomic sequencing after chemical mutagenesis to map bacterial genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility. Surprisingly, less than half of these genes were annotated as motility-related based on sequence homologies. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. In the process, we reannotated genes belonging to a broad family of diguanylate cyclases and phosphodiesterases to highlight their specific role in motility and assigned functions to uncharacterized genes. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host. These results indicate that genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains to define the role of genes in complex environments can be accomplished in bacteria without the development of species-specific molecular genetic tools. motility | mutagenesis | comparative genomics | gene annotation

Section: Significancesupporting

confidence: 64%

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

Bae

Mueller

Wong

et al. 2016

“…Indeed, PakD, PakB, and PakA all possess multiple PAS and/or GAF domains. Because of low sequence identity among these domain classes, it is generally not possible to predict the actual signal sensed by PAS or GAF domains (33)(34)(35), and the presence of multiple domains complicates the picture. Another kinase, PakG, does not harbor any domains other than the DHp and CA domains of the catalytic core, suggesting that it might be the protein level of PakG that is regulated in response to stress or that its activity might be regulated by an accessory protein.…”

Section: Discussionmentioning

confidence: 99%

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Kaczmarczyk¹,

Hochstrasser²,

Vorholt³

et al. 2014

117

Significance Bacteria possess many regulatory systems to monitor their environment and adapt their physiology accordingly. Whereas most systems sense one specific signal, the general stress response (GSR) is activated by many signals and protects cells against a wide range of adverse conditions. In Alphaproteobacteria, the GSR is controlled by the response regulator PhyR, but little is known about the upstream pathways. Here, we establish the GSR as a complex regulatory network composed of a particular family of partially redundant sensor kinases and of additional response regulators that modulate PhyR activity in Sphingomonas melonis . Given the broad conservation of this kinase family, it is possible that it plays a general role in the GSR in Alphaproteobacteria.

“…1B). PAS domains function to sense environmental signals, such as light, oxygen, and redox state (30,31), and control regulatory processes in diverse organisms. Although the presence of GGDEF and EAL domains suggests that PA14_07500 could harbor both DGC and PDE activity, the early-onset and enhanced wrinkling of ΔPA14_07500 mutant colonies indicate that PA14_07500 functions as a PDE under the tested conditions.…”

Section: Colony Matrix Distribution Phenotypes Suggest That Phenazinementioning

confidence: 99%

Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP

Okegbe

Fields

Cole

et al. 2017

101

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttlemediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3′,5′)-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain-containing regulatory proteins found in complex eukaryotes.W hen microbial cells cannot import or are physically separated from metabolic electron donors or acceptors, diffusible compounds can act as electron carriers and support survival on these substrates (1, 2). These conditions arise in the presence of poised-potential electrodes or insoluble minerals, such as iron oxides (3-5), and in multicellular communities (biofilms) where the formation of chemical gradients leads to oxidant limitation for cells at depth (6-9). Diverse microbes secrete redoxactive compounds with the capacity to function as electron shuttles (10-12). In the pathogenic bacterium Pseudomonas aeruginosa PA14, electron-shuttling antibiotics called phenazines support survival on poised-potential electrodes and balance the intracellular redox state of cells in anoxic biofilm subzones (1, 9).Similar to those formed by many species of microbes, colonies of PA14 develop intricate wrinkle structures on agar-solidified growth media (13). Phenazines profoundly alter PA14 colony morphogenesis, inhibiting the onset of wrinkle formation and changing the organization of wrinkles (12) (Fig. 1A). Modeling of resource availability within colonies suggests that the earlier increase in the colony surface area-to-volume ratio in phenazine-null (Δphz) mutants maximizes access to oxygen for cells that would otherwise become limited for oxidant (14). Measurements of...