Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.     

Strategies for maximizing metallothionein promoter regulated recombinant protein production in mammalian cell cultures

A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn⁺² or Cd⁺²) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd⁺² dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd⁺² although cell-associated Cd⁺² levels remain high. Further addition of Cd⁺² to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd⁺² is sequestered into a form which does not stimulate the MT promoter. Cd⁺² dosing regimes which maintained cell associated Cd⁺² concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd⁺² loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line.     

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.     

Unraveling regulatory networks in plant defense using microarrays

DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.     

Is there an inner nose?

Although behavioral and neuropsychological data regarding the existence of images for odors are inconclusive, reconsideration of earlier EEG work provides reasonably clear evidence for an inner nose. However, further EEG studies and neuroimaging data seem essential for conclusive demonstration of an inner nose.      

Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains

Glycosylated cellulases from Cellulomonas fimi were compared with their non-glycosylated counterparts synthesized in Escherichia coli from recombinant DNA. Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH. However, the glycosylated enzymes are protected from attack by a C. fimi protease when bound to cellulose, while the non-glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose.     

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.     

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.