Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

Dinoroseobacter shibae DFL12T , a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12T is able to synthesize the vitamins B 1 and B 12 for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B 12 are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B 12 was confirmed to be functional, and D. shibae DFL12T was shown to provide the growth-limiting vitamins B 1 and B 12 to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12 T is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12 T has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12 T shows the most complex viral defense system of all Rhodobacterales sequenced to date.

In this study photophysical characteristics of LOV-based fluorescent proteins which are essential for analytic methods as well as imaging approaches have been comparatively analyzed in detail.

We previously characterized a LOV protein PpSB2-LOV, present in the common soil bacterium Pseudomonas putida, that exhibits a plant phototropin LOV-like photochemistry [Krauss, U., Losi, A., Gartner, W., Jaeger, K. E., and Eggert, T. (2005) Phys. Chem. Chem. Phys. 7, 2804-2811]. Now, we have identified a second LOV homologue, PpSB1-LOV, found in the same organism with approximately 66% identical amino acids. Both proteins consist of a conserved LOV core flanked by short N- and C-terminal extensions but lack a fused effector domain. Although both proteins are highly similar in sequence, they display drastically different dark recovery kinetics. At 20 degrees C, PpSB2-LOV reverts with an average time constant of 137 s from the photoequilibrium to the dark state, whereas PpSB1-LOV exhibits an average dark recovery time constant of 1.48 x 10(5) s. Irrespective of the significant differences in their dark recovery behavior, both proteins showed nearly identical kinetics for the photochemically induced adduct formation. In order to elucidate the structural and mechanistic basis of these extremely different dark recovery time constants, we performed a mutational analysis. Six amino acids in a distance of up to 6 A from the flavin chromophore, which differ between the two proteins, were identified and interchanged by site-directed mutagenesis. The amino acid substitution R66I located near the FMN phosphate in LOV domains was identified in PpSB1-LOV to accelerate the dark recovery by 2 orders of magnitude. Vice versa, the corresponding substitution I66R slowed down the dark recovery in PpSB2-LOV by a factor of 10. Interestingly, the interchange of the C-terminal extensions between the two proteins also had a pronounced effect on the dark recovery time constants, thus highlighting a coupling of these protein regions to the chromophore binding pocket.

Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.