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.
Light, oxygen, voltage (LOV) based fluorescent proteins (FPs) represent a promising alternative to fluorescent reporters of the green fluorescent protein family. For certain applications like multicolor imaging or the design of FRET-based biosensors, the generation of spectrally shifted LOV-based FPs would be required. In a recent theoretical study ( Khrenova J. Phys. Chem. B 2015 , 119 ( 16 ), pp 5176 - 5183 ), the photophysical properties of a variant of the LOV-based fluorescent protein iLOV were predicted using quantum mechanics/molecular mechanics (QM/MM) approaches. The variant contained a lysine residue at the position of a highly conserved glutamine residue (Q489K), which directly interacts with the O4 and N5 atom of the flavin mononucleotide (FMN) chromophore. On the basis of QM/MM calculations, iLOV-Q489K was suggested to possess substantially red-shifted absorption and fluorescence-emission maxima with respect to parental iLOV. Here, we describe the experimental characterization of this variant, which, surprisingly contrary to the theoretical prediction, shows blue-shifted absorption and fluorescence-emission maxima. Using molecular dynamics (MD) simulations and QM/MM calculations, the molecular basis for the contradictory theoretical and experimental results is presented. Essentially, our computational analysis suggests that, in the Q489K variant, two possible side-chain conformers exist: (i) a least populated conformer K489in forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489out with the side-chain amino group flipped away from the FMN chromophore forming a new hydrogen bond with the backbone oxygen of G487. QM/MM calculated spectra of the K489out conformer are blue-shifted compared to the calculated spectra of parental iLOV, which is in accordance with experimental data. This suggests that the change in the conformation of K489 from K498in to K489out accounts for the change in the direction of the spectral shift from red to blue, thus reconciling theory and experiment.
In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK ) encoding P II -like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P II -like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P II -like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.
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