The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.

Fertilization of Xenopus laevis eggs triggers a wave of increased [Ca2+]i. The exact signal transduction pathway culminating in this Ca2+ wave remains unknown. To determine whether increases in tyrosine kinase activity are part of this pathway, we microinjected tyrosine kinase inhibitors into unfertilized eggs. Upon fertilization, signs of activation were monitored, such as fertilization envelope liftoff and the Ca2+ wave (for eggs microinjected with lavendustin A). Various concentrations of lavendustin A and tyrphostin B46 were microinjected, as well as inactive forms of these compounds (lavendustin B and tyrphostin A1) to provide negative controls. Peptide A, a 20-amino-acid peptide derived from the SH2 region of pp60(v-src) tyrosine kinase, was also microinjected. Peptide A inhibits tyrosine kinase activity but not PKA or PKG activity. Dose-response curves for lavendustin A, tyrphostin B46, and peptide A show clear inhibition of vitelline envelope liftoff by these three compounds. Confocal imaging of eggs coinjected with lavendustin A and Oregon Green-dextran showed that the Ca2+ wave was inhibited under normal insemination conditions but that the block of the Ca2+ wave could be overcome with very high sperm densities. A phenomenon of small local Ca2+ increases termed "hot spots" seen in lavendustin A containing eggs is also described. Since this inhibition of egg activation by tyrosine kinase inhibitors can be overcome by Ca2+ microinjection, the inhibitors must act on a step in the signal transduction cascade that is upstream of the Ca2+ wave.

No abstract

The production of pigments during industrial fermentations is undesirable, and expensive purification steps are often required to remove colored compounds from anticipated commercial products. We observed that a recombinant Bacillus subtilis strain synthesizing the heterologous polysaccharide hyaluronic acid (HA) produced copious amounts of a red pigment with biochemical properties characteristic of pulcherrimin, a previously characterized iron-binding pyrazine compound. An apigmented B. subtilis mutant was isolated following chemical mutagenesis and compared to its parent strain using DNA microarray transcriptome analysis. Among the genes whose transcription was significantly (p < 0.05) altered in the apigmented mutant, yvmC, and cypX were selected as likely pulcherrimin biosynthetic genes on the basis of in silico bioinformatics examination which suggested that the yvmC gene product contains a putative phosphopantetheine domain characteristic of some cyclic peptide synthases, and the cypX gene encodes a cytochrome P450-like enzyme.Pulcherrimin biosynthesis was previously proposed to occur via cyclization of two leucine molecules followed by a redox reaction involving molecular oxygen. Additionally, yvmC and cypX genes are juxtaposed on the B. subtilis chromosome and appear to be coordinately expressed based on hierarchical cluster analysis of microarray data. Disruption of the yvmC or cypX genes yielded strains that did not produce red pigment. This study illustrates that the combination of transcriptional profiling and bioinformatics is an effective approach that can be employed to solve industrial fermentation problems.