Cyanobacteria in motion

This work was supported by a grant from the DFG (WI 2014/7-1) to A.W

Surface characterisation reveals substrate suitability for Cyanobacterial phototaxis

Cyanobacteria respond to light stimulation, activating localized assembly of type IV pili for motility. The resulting phototactic response is highly dependent on the nature of the incoming light stimulus, and the final motility parameters depend on the surface properties. Conventionally, phototaxis studies are carried out on hydrogel surfaces, such as agarose, with surface properties, that vary in time due to experimental conditions. This study considers five substrates, widely utilized in microfluidic technology, to identify the most suitable alternative for performing reliable and repeatable phototaxis assays. The surfaces are characterized via a contact angle goniometer to determine the surface energy, white light interferometry for roughness, zeta-potentials and AFM force distance curves for charge patterns, and XPS for surface composition. Cell motility assays showed 1.25 times increment on surfaces with a water contact angle of 80 compared to a reference glass surface. To prove that motility can be enhanced, polydimethylsiloxane (PDMS) surfaces were plasma treated to alter their surface wettability. The motility on the plasma-treated PDMS showed similar performance as for glass surfaces. In contrast, untreated PDMS surfaces displayed close to zero motility. We also describe the force interctions of cells with the test surfaces using DLVO (Derjaguin-Landau-Verwey-Overbeek) and XDLVO (extended DLVO) theories. The computed DLVO/XDLVO force-distance curves are compared with those obtained using atomic force microscopy. Our findings show that twitching motility on tested surfaces can be described mainly from adhesive forces and hydrophobicity/hydrophilicity surface properties

Appendages of the Cyanobacterial Cell

Extracellular non-flagellar appendages, called pili or fimbriae, are widespread in gram-negative bacteria. They are involved in many different functions, including motility, adhesion, biofilm formation, and uptake of DNA. Sequencing data for a large number of cyanobacterial genomes revealed that most of them contain genes for pili synthesis. However, only for a very few cyanobacteria structure and function of these appendages have been analyzed. Here, we review the structure and function of type IV pili in Synechocystis sp. PCC 6803 and analyze the distribution of type IV pili associated genes in other cyanobacteria. Further, we discuss the role of the RNA-chaperone Hfq in pilus function and the presence of genes for the chaperone-usher pathway of pilus assembly in cyanobacteria

Data_Sheet_1_Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803.PDF

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.</p

Mediatorless, Reversible Optical Nanosensor Enabled through Enzymatic Pocket Doping

This study presents a reversible, mediatorless, near-infrared glucose sensor based on glucose oxidase-wrapped SWCNTs. A combination of fluorescence, absorption, and Raman spectroscopy measurements suggest a fluorescence enhancement mechanism based on localized enzymatic doping of SWCNT defect sites that does not rely on added mediators. The cyclic addition and removal of glucose is shown to enhance and recover fluorescence, demonstrating reversibility

Xeno Nucleic Acid Nanosensors for Enhanced Stability Against Ion-Induced Perturbations

The omnipresence of salts in biofluids creates a pervasive challenge in designing sensors suitable for in vivo applications. Fluctuations in ion concentrations have been shown to affect the sensitivity and selectivity of optical sensors based on single-walled carbon nanotubes wrapped with single-stranded DNA (ssDNA–SWCNTs). We herein observe fluorescence wavelength shifting for ssDNA–SWCNT-based optical sensors in the presence of divalent cations at concentrations above 3.5 mM. In contrast, no shifting was observed for concentrations up to 350 mM for sensors bioengineered with increased rigidity using xeno nucleic acids (XNAs). Transient fluorescence measurements reveal distinct optical transitions for ssDNA- and XNA-based wrappings during ion-induced conformation changes, with XNA-based sensors showing increased permanence in conformational and signal stability. This demonstration introduces synthetic biology as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nanobioengineered sensors with augmented capabilities

Site-Specific Protein Conjugation onto Fluorescent Single-Walled Carbon Nanotubes

Semiconducting single-walled carbon nanotubes (SWCNTs) are among the few photostable optical emitters that are ideal for sensing, imaging, drug delivery, and monitoring of protein activity. These applications often require strategies for immobilizing proteins onto the nanotube while preserving the optical properties of the SWCNTs. Site-specific and oriented immobilization strategies, in particular, offer advantages for improving sensor and optical signaling responses. In this study, we demonstrate site-specific protein immobilization of a model of enhanced yellow fluorescent protein with a single engineered cysteine residue, using either single-stranded DNA or a pyrene-containing linker to interact with the SWCNT surface. Protein expression and bioconjugation were characterized using a combination of gel electrophoresis, absorbance, fluorescence, mass spectrometry, and circular dichroism measurements. The results confirm successful protein immobilization onto SWCNTs, which retain their near-infrared fluorescence following conjugation. The successful demonstration of these bioconjugation strategies serves as a basis for more cost-effective, site-specific immobilization strategies that can help preserve protein folding and functionality

Directed evolution of the optoelectronic properties of synthetic nanomaterials

Directed evolution is a powerful approach to tailor protein properties toward new or enhanced functions. Herein, we use directed evolution to engineer the optoelectronic properties of DNA-wrapped single-walled carbon nanotube sensors through DNA mutation. This approach leads to an improvement in the fluorescence intensity of 56% following two evolution cycles