No abstract

To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA-rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS-disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.

A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

Background: The aetiology of infraorbital dark circles is complex and multi-factorial. The aim of this research was to measure and characterize dark circles and to determine the physiological changes associated with the occurrence of this aesthetically unpleasing issue. Materials and methods: Clinical, photographic and instrumental assessments were performed on Caucasian skin to determine the most appropriate methodologies to measure dark circles, comparing different zones of the infraorbital region in subjects with and without dark circles. Exploratory studies were also carried out on African and Far East Asian skin, as well as on tracking the natural variation of dark circles over seven days in Caucasian subjects. Results: Under-eye dark circles in Caucasian subjects are characterized by significantly darker coloured skin and higher colour deviations between the dark circle region and surrounding areas of skin. Multispectral image analysis produced a higher melanin index in subjects with dark circles, suggesting hyperpigmentation in the affected area, in addition to a higher haemoglobin index. The higher haemoglobin index, combined with preliminary assessments of the vascular network by videocapillaroscopy, suggests there may be more dilated, thicker or increased numbers of capillaries in subjects with dark circles. Ultrasound analysis showed a significant difference in skin thickness between the two groups of subjects linking the appearance of dark circles to thinner skin under the eye. Many of these trends were also observed in African and Far East Asian skin. The results also showed intra-individual, intra-day and inter-day variability of dark circle intensity in subjects with Caucasian skin. Conclusion: Three key physiological factors associated with the occurrence of infraorbital dark circles are hyperpigmentation, a tendency for more dilated, thicker or increased number of capillaries and thinner skin in the under-eye area. The combination of these three factors provides a robust indication of the presence of infraorbital dark circles.