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A series of Tn9l 71ac insertions define the comnG region of the Bacillus subtUis chromosome. comG mutants are deficient in competence and specifically in the binding of exogenous DNA. The genes included in the comG region are first expressed during the transition from the exponential to the stationary growth phase. From nucleotide sequence information, it was concluded that the comG locus contains seven open reading frames (ORFs), several of which overlap at their termini. High-resolution Si nuclease mapping and primer extension were used to identify the 5' terminus of the comG mRNA. The sequence upstream from the comG start site closely resembled the consensus recognition sequence for the major B. subtilis vegetative RNA polymerase holoenzyme. Complementation analysis confirmed that the comG ORF1 protein is required for the ability of competent cultures to resolve into two populations with different cell densities on Renografin (E. R. Squibb & Sons, Princeton, N.J.) gradients, as well as for full expression of comE, another late competence locus. The predicted comG ORF1 protein showed significant similarity to the virB ORF11 protein from Agrobacterium tumefaciens, which is probably involved in T-DNA transfer. The N-terminal sequences of comG ORF3 and, to a lesser extent, the comG ORF4 and ORF5 proteins were similar to a class of pilin proteins from members of the genera Bacteroides, Pseudomonas, Neisseria, and MoraxeeUa. All of the comG proteins except comG ORF1 possessed hydrophobic domains that were potentially capable of spanning the bacterial membrane. It is likely that these proteins are membrane associated, and they may comprise part of the DNA transport machinery. When present in multiple copies, a DNA fragment carrying the comG promoter was capable of inhibiting the development of competence as well as the expression of several late com genes, suggesting a role for a transcriptional activator in the expression of those genes.

Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

Biotechnological production of recombinant proteins for human therapy requires a cultivation of the host organism in a nutrient medium free of animal substances. Therefore, various nutrient media for the new expression system Leishmania tarentolae were developed and examined according to their cultivation conditions as static suspension culture and agitated culture. Investigations resulted in the development of a serum-free but hemin containing medium, based on yeast extract and buffer salts. Here we report that a high and stable specific growth rate of 0.103 h(-1) and a maximal cell density of 1 x 10(9) cells ml(-1) is obtained in an alternative medium, the YE-medium. For the newly developed medium, the successful expression of enhanced green fluorescent protein and the adaptation of the cultivation from the agitated culture to the bioreactor could be shown. Furthermore, an analytical method for detection of the essential, organic iron source hemin was established. The consumption of hemin was monitored because hemin is a potentially important process parameter for bioprocess control. With knowledge of these results, an improved expression system is available as an alternative to commonly used cell cultures for the production of recombinant proteins.