Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.     

A new occurrence of ambient inclusion trails from the ~1900‐million‐year‐old Gunflint Formation,Ontario: nanocharacterization and testing of potential formation mechanisms

Ambient inclusion trails (AITs) are tubular microstructures thought to form when a microscopic mineral crystal is propelled through a fine‐grained rock matrix. Here, we report a new occurrence of AITs from a fossilized microbial mat within the 1878‐Ma Gunflint Formation, at Current River, Ontario. The AITs are 1–15 μm in diameter, have pyrite as the propelled crystal, are infilled with chlorite and have been propelled through a microquartz (chert) or chlorite matrix. AITs most commonly originate at the boundary between pyrite‐ and chlorite‐rich laminae and chert‐filled fenestrae, with pyrite crystals propelled into the fenestrae. A subset of AITs originate within the fenestrae, rooted either within the chert or within patches of chlorite. Sulphur isotope data (³⁴S/³²S) obtained in situ from AIT pyrite have a δ³⁴S of ?8.5 to +8.0 ‰, indicating a maximum of ~30 ‰ fractionation from Palaeoproterozoic seawater sulphate (δ³⁴S ≈ +20 ‰). Organic carbon is common both at the outer margins of the fenestrae and in patches of chlorite where most AITs originate, and can be found in smaller quantities further along some AITs towards the terminal pyrite grain. We infer that pyrite crystals now found within the AITs formed via the action of heterotrophic sulphate‐reducing bacteria during early diagenesis within the microbial mat, as pore waters were becoming depleted in seawater sulphate. Gases derived from this process such as CO₂ and H₂S were partially trapped within the microbial mat, helping produce birds‐eye fenestrae, while rapid microquartz precipitation closed porosity. We propose that propulsion of the pyrite crystals to form AITs was driven by two complementary mechanisms during burial and low‐grade metamorphism: firstly, thermal decomposition of residual organic material providing CO₂, and potentially CH₄, as propulsive gases, plus organic acids to locally dissolve the microquartz matrix; and secondly, reactions involving clay minerals that potentially led to enhanced quartz solubility, plus increases in fluid and/or gas pressure during chlorite formation, with chlorite then infilling the AITs. This latter mechanism is novel and represents a possible way to generate AITs in environments lacking organic material.     

Cellulose-binding domains: potential for purification of complex proteins.

The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively. The gene fragment encoding the CBD can be fused to the gene of a protein of interest. Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner. Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp. are dimeric proteins. The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners. Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA. CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.     

Self-Powered Wireless Carbohydrate/Oxygen Sensitive Biodevice Based on Radio Signal Transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.     

Modelling evolution on design-by-contract predicts an origin of Life through an abiotic double-stranded RNA world

Background  

It is generally believed that life first evolved from single-stranded RNA (ssRNA) that both stored genetic information and catalyzed the reactions required for self-replication.     

Visualising gold inside tumour cells following treatment with an antitumour gold(I) complex

Gold(I) phosphine complexes, such as [Au(d2pype)(2)]Cl, (1, where d2pype is 1,2-bis(di-2-pyridyl phosphinoethane)), belong to a class of promising chemotherapeutic candidates that have been shown to be selectively toxic to tumourigenic cells, and may act via uptake into tumour cell mitochondria. For a more holistic understanding of their mechanism of action, a deeper knowledge of their subcellular distribution is required, but to date this has been limited by a lack of suitable imaging techniques. In this study the subcellular distribution of gold was visualised in situ in human breast cancer cells treated with 1, using nano-scale secondary ion mass spectrometry. NanoSIMS ion maps of (12)C(14)N(-), (31)P(-), (34)S(-) and (197)Au(-) allowed, for the first time, visualisation of cellular morphology simultaneously with subcellular distribution of gold. Energy filtered transmission electron microscopy (EFTEM) element maps for gold were also obtained, allowing for observation of nuclear and mitochondrial morphology with excellent spatial resolution, and gold element maps comparable to the data obtained with NanoSIMS. Following 2 h treatment with 1, the subcellular distribution of gold was associated with sulfur-rich regions in the nucleus and cytoplasm, supporting the growing evidence for the the mechanism of action of Au(I) compounds based on inhibition of thiol-containing protein families, such as the thioredoxin system. The combination of NanoSIMS and EFTEM has broader applicability for studying the subcellular distribution of other types of metal-based drugs.     

Cooperative tertiary interaction network guides RNA folding

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have?a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming?a unique, stable fold.