Barcoded microbial system for high-resolution object provenance.

Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.

Cell cycle progression in Caulobacter requires a nucleoid-associated protein with high AT sequence recognition.

Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed in Caulobacter The Escherichia coli nucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously in Caulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

Novel Modeling Approach to Generate a Polymeric Nanofiber Scaffold for Salivary Gland Cells.

BACKGROUND: Electrospun nanofibers have been utilized in many biomedical applications as biomimetics of extracellular matrix proteins that promote self-organization of cells into 3D tissue constructs. As progress towards an artificial salivary gland tissue construct, we prepared nanofiber scaffolds using PLGA, a biodegradable and biocompatible material. METHOD OF APPROACH: We used electrospinning to prepare nanofiber scaffolds using PLGA with both DMF and HFIP as solvents. Using a design of experiment (DOE) approach, system and process parameters were optimized concurrently and their effects on the diameter of the resulting fibers were computed into a single model. A transfer function was used to reproducibly produce nanofibers of a defined diameter, which was confirmed by SEM. The mouse salivary gland epithelial cell line, SIMS, was seeded on the nanofiber scaffolds, and morphology, cell proliferation, and viability were assayed. RESULTS: Varying two or more parameters simultaneously yielded trends diverging from the linear response predicted by previous studies. Comparison of two solvents revealed that the diameter of PLGA nanofibers generated using HFIP is less sensitive to changes in the system and process parameters than are fibers generated using DMF. Inclusion of NaCl reduced morphological inconsistencies and minimized process variability. The resulting nanofiber scaffolds supported attachment, survival and cell proliferation of a mouse salivary gland epithelial cell line. In comparison with glass and flat PLGA films, the nanofibers promoted self-organization of the salivary gland cells into 3D cell clusters, or aggregates. CONCLUSIONS: These data indicate that nanofiber scaffolds promote salivary gland cell organization, and suggest that a nanofiber scaffold could provide a platform for engineering of an artificial salivary gland tissue construct. This study additionally provides a method for efficient production of nanofiber scaffolds for general application in tissue engineering.