Biology subjects

Smith, T. J.

Publications and source records attributed to Smith, T. J..

4 recordsLinked to original sources

bioRxiv2018

Coopting the Lap system of Pseudomonas fluorescens to reversibly customize bacterial cell surfaces

Initialattachment to a surface is a key and highly regulated step in biofilm formation. In this study, we present a platform for reversibly functionalizing bacterial cell surfaces, with an emphasis on designing biofilms. We engineered the Lap system of Pseudomonas fluorescens Pf0-1, which is normally used to regulate initial cell surface attachment, to display various protein cargo at the bacterial cell surface and control extracellular release of the cargo in response to changing levels of the second messenger cdi-GMP. To accomplish this goal, we fused the protein cargo between the N-terminal retention module and C-terminal secretion signal of LapA, and controlled surface localization of the cargo with natural signals known to stimulate or deplete c-di-GMP levels in P. fluorescens Pf0-1. We show this system can tolerate large cargo in excess of 500 amino acids, direct P. fluorescens Pf0-1 to surfaces it does not typically colonize, and program this microbe to sequester the toxic medal cadmium.

bioengineering↗

bioRxiv2017

Botulinum-neurotoxin-like sequences identified from an Enterococcus sp. genome assembly

Botulinum neurotoxins (BoNTs) are produced by diverse members of the Clostridia and result in a flaccid paralysis known as botulism. Exploring the diversity of BoNTs is important for the development of therapeutics and antitoxins. Here we describe a novel, bont-like gene cluster identified in a draft genome assembly for Enterococcus sp. 3G1_DIV0629 by querying publicly available genomic databases. The bont-like gene is found in a gene cluster similar to known bont gene clusters. Protease and binding motifs conserved in known BoNT proteins are present in the newly identified BoNT-like protein; however, it is currently unknown if the BoNT-like protein described here is capable of targeting neuronal cells resulting in botulism.

genomics↗

bioRxiv2017

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d{inverted exclamation}-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbCs calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.\n\nImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d{inverted exclamation}-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a \"cloud\" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

microbiology↗

bioRxiv2017

Cell surface associated LapA of Pseudomonas fluorescens is anchored inside its Type-1 Secretion TolC-like Pore

The type-1 secretion system (T1SS) of gram-negative bacteria enables a one-step translocation strategy known to move functionally diverse proteins from the cytoplasm into the extracellular environment without a periplasmic intermediate. LapA of Pseudomonas fluorescens Pf0-1 is a giant type-1 secreted (T1S) adhesin that facilitates biofilm formation only when displayed at the cell surface. A LapA-targeting periplasmic protease, LapG, connects intracellular cyclic diguanylate (c-di-GMP) levels with cell surface-associated LapA by cleaving and absolving LapA from the cell surface under conditions unsuitable for biofilm formation. Here, we demonstrate that LapA contains a novel N-terminal element, called the retention module (RM), which prohibits classical one-step T1S of LapA. We provide evidence that the RM of LapA tethers LapA at the cell surface through its outer membrane TolC-like pore, LapE, where LapA is accessible to the periplasmic protease LapG. We also demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins and represents a new subclass of T1SS ABC transporters exclusively involved in transporting LapA-like adhesins.\n\nSignificance StatementBacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is often key for initiating contact with a preferred substrate to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence these adhesins are secreted by a new subclass of T1SS ABC transporters.

microbiology↗