Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 µm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.

Multiplex gene transfer by genetic transformation between isolated S. pneumoniae cells confined in microfluidic droplets.

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell-cell interactions and overcome the limitations of current methods involving bulk mixed cultures. To determine the relevance and reliability of this new method for study of bacterial genetic transformation, we compared recombination events occurring in 44 recombinants recovered after competence-mediated gene exchange between pairs of cells confined in femtoliter-scale droplets vs. those occurring in exchanges in parallel bulk culture mixtures. The pattern of recombination events in both contexts exhibited the hallmarks of the macro-recombination exchanges previously observed within the more complex natural contexts of biofilms and long-term evolution in the human host.

A Chemically Patterned Microfluidic Paper-based Analytical Device (C-µPAD) for Point-of-Care Diagnostics.

A chemically patterned microfluidic paper-based analytical device (C-µPAD) is developed to create fluidic networks by forming hydrophobic barriers using chemical vapor deposition (CVD) of trichlorosilane (TCS) on a chromatography paper. By controlling temperature, pattern size, and CVD duration, optimal conditions were determined by characterizing hydrophobicity, spreading patterns, and flow behavior on various sized fluidic patterns. With these optimal conditions, we demonstrated glucose assay, immunoassay, and heavy metal detection on well-spot C-µPAD and lateral flow C-µPAD. For these assays, standard curves showing correlation between target concentration and gray intensity were obtained to determine a limit of detection (LOD) of each assay. For the glucose assays on both well-spot C-µPAD and lateral flow C-µPAD, we achieved LOD of 13 mg/dL, which is equivalent to that of a commercial glucose sensor. Similar results were obtained from tumor necrosis factor alpha (TNFα) detection with 3 ng/mL of LOD. For Ni detection, a colorimetric agent was immobilized to obtain a stationary and uniform reaction by using thermal condensation coupling method. During the immobilization, we successfully functionalized amine for coupling the colorimetric agent on the C-µPAD and detected as low as 150 µg/L of Ni. These C-µPADs enable simple, rapid, and cost-effective bioassays and environmental monitoring, which provide practically relevant LODs with high expandability and adaptability.

Fast and green microwave-assisted conversion of essential oil allylbenzenes into the corresponding aldehydes via alkene isomerization and subsequent potassium permanganate promoted oxidative alkene group cleavage.

Essential oil allylbenzenes from have been converted quickly and efficiently into the corresponding benzaldehydes in good yields by a two-step "green" reaction pathway based on a solventless alkene group isomerization by KF/Al(2)O(3) to form the corresponding 1-arylpropene and a subsequent solventless oxidation of the latter to the corresponding benzaldehyde by KMnO(4)/CuSO(4).5H(2)O. The assistance by microwave irradiation results in very short reaction times (<15 minutes). The green conversion of eugenol (4-allyl-2-methoxyphenol) into vanillin (4-hydroxy-3-methoxybenzaldehyde) has been carried out in a similar way, requiring however two additional microwave-assisted synthetic steps for acetylation of the hydroxy group prior to the oxidation reaction, and for the final deacetylation of vanillin acetate (4-acetoxy-3-methoxybenzaldehyde) by KF/Al(2)O(3) under solvent-free conditions, respectively.