An Efficient Transformation Method for the Bioplastic-Producing "Knallgas" Bacterium Ralstonia eutropha H16.

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic ß-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis, and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism. To overcome this limitation, a systematic study is performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells are made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieves a transformation efficiency of (3.86 ± 0.29) × 105 cfu µg-1 DNA, a 103 -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism.

A novel P0 glycoprotein transgene activates expression of lacZ in myelin-forming Schwann cells.

P0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P0 promoter-based transgenes has been inconsistent. As much as 9 kb of 5' flanking sequence fused to lacZ never yielded detectable levels of beta-galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P0 gene, including 6 kb of 5' flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin-forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2-kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P0 mRNA. These data suggest that intragenic or 3' flanking sequences are necessary to generate the remarkable levels of endogenous P0 gene expression.

Cloning and sequence of the cDNA encoding the beta 4 integrin subunit in rat peripheral nerve.

beta 4 and alpha 6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. beta 4 is also expressed in Schwann cell (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for beta 4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding beta 4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in beta 4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of beta 4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived beta 4 integrin are highly similar to those cloned from epithelia.