Regulation of the Erythrobacter litoralis DSM 8509 general stress response by visible light.

Extracytoplasmic function (ECF) sigma factors are environmentally responsive transcriptional regulators. In Alphaproteobacteria, σEcfG activates general stress response (GSR) transcription and protects cells from multiple stressors. A phosphorylation-dependent protein partner switching mechanism, involving HWE/HisKA_2-family histidine kinases, underlies σEcfG activation. The identity of these sensor kinases and the signals that regulate them remain largely uncharacterized. We have developed the aerobic anoxygenic photoheterotroph (AAP), Erythrobacter litoralis DSM 8509, as a comparative genetic model to investigate GSR. Using this system, we sought to define the role of visible light and a photosensory HWE kinase, LovK, in regulation of GSR transcription. We identified three HWE kinase genes that collectively control GSR: gsrK and lovK are activators, while gsrP is a repressor. In wild-type cells, GSR transcription is activated in the dark and nearly off in the light, and the opposing activities of gsrK and gsrP are sufficient to modulate GSR transcription in response to illumination. In the absence of gsrK and gsrP, lovK alone is sufficient to activate GSR transcription. lovK is a more robust activator in the dark, and light-dependent regulation by LovK requires that its N-terminal LOV domain be photochemically active. Our studies establish a role for visible light and an ensemble of HWE kinases in light-dependent regulation of GSR transcription in E. litoralis.

Influence of light on carbon utilization in aerobic anoxygenic phototrophs.

Aerobic anoxygenic phototrophs contain photosynthetic reaction centers composed of bacteriochlorophyll. These organisms are photoheterotrophs, as they require organic carbon substrates for their growth whereas light-derived energy has only an auxiliary function. To establish the contribution of light energy to their metabolism, we grew the phototrophic strain Erythrobacter sp. NAP1 in a carbon-limited chemostat regimen on defined carbon sources (glutamate, pyruvate, acetate, and glucose) under conditions of different light intensities. When grown in a light-dark cycle, these bacteria accumulated 25% to 110% more biomass in terms of carbon than cultures grown in the dark. Cultures grown on glutamate accumulated the most biomass at moderate light intensities of 50 to 150 µmol m(-2) s(-1) but were inhibited at higher light intensities. In the case of pyruvate, we did not find any inhibition of growth by high irradiance. The extent of anaplerotic carbon fixation was detemined by radioactive bicarbonate incorporation assays. While the carboxylation activity provided 4% to 11% of the cellular carbon in the pyruvate-grown culture, in the glutamate-grown cells it provided only approximately 1% of the carbon. Additionally, we tested the effect of light on respiration and photosynthetic electron flow. With increasing light intensity, respiration decreased to approximately 25% of its dark value and was replaced by photophosphorylation. The additional energy from light allows the aerobic anoxygenic phototrophs to accumulate the supplied organic carbon which would otherwise be respired. The higher efficiency of organic carbon utilization may provide an important competitive advantage during growth under carbon-limited conditions.

Influence of growth temperature and starvation state on survival and DNA damage induction in the marine bacterium Sphingopyxis alaskensis exposed to UV radiation.

Despite the considerable volume of literature describing the individual effects of temperature or UV light on aquatic bacteria, little is known about their combined effects. The current study was conducted to learn about the effects of growth temperature and duration of starvation on the response of a marine bacterium, Sphingopyxis alaskensis to UV-B or simulated solar radiation. Cells grown at 12 degrees C or 24 degrees C, and harvested at early or late stationary phase, were exposed to UV-B or simulated solar radiation (>290 nm). The predominant forms of UV-induced DNA damage, namely cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PP s), were quantified using a HPLC-mass spectrometry. While the commonly accepted view that DNA damage induced by UV-B radiation is temperature-independent, we observed in S. alaskensis that the yield of photoproducts for 12 degrees C was generally lower than for cells grown at 24 degrees C. The relative distribution of DNA photoproducts also varied with growth temperature, with an increased formation of TC 6-4PP for late compared to early stationary phase cells. In contrast, with the exception of cultures grown at 12 degrees C exposed to simulated solar radiation, the duration of stationary phase had no effect on total photoproduct formation. Collectively, these data indicate that growth temperature has more effect than duration of starvation on the formation of photoproducts in S. alaskensis.

Remarkable resistance to UVB of the marine bacterium Photobacterium angustum explained by an unexpected role of photolyase.

DNA damage and cell survival was assessed in the marine bacteria, Photobacterium angustum (GC%=39.6) and Sphingopyxis alaskensis (GC%=65.5) following UVB irradiation and recovery in the presence or absence of visible light. The extent of bipyrimidine photoproduct formation was analyzed by HPLC-MS/MS. S. alaskensis was chosen as a reference species since it was previously shown to be photoresistant. Interestingly, P. angustum exhibited an even higher level of survival to UVB irradiation than S. alaskensis. This higher photoresistance was associated with a decrease in the rate of formation of cyclobutane pyrimidine dimers (CPDs) at high UVB doses. Despite different distributions in UVB-induced lesions, the survival difference between the two marine bacteria could not be accounted for by qualitative differences in either photoreactivation or the rate of nucleotide excision repair of the photoproducts arising from the different bipyrimidine doublets (TT, CT, TC and CC). Dark repair was found to be much more efficient for P. angustum than S. alaskensis but the corresponding rate of photoproduct removal was lower than that observed at high UVB doses. We propose that the increased resistance of P. angustum under high UVB doses results from a UVB-induction of CPD photolyase(s) that may directly repair DNA damage and/or act indirectly by enhancing the rate of nucleotide excision repair.

The response of the marine bacterium Sphingopyxis alaskensis to solar radiation assessed by quantitative proteomics.

The adaptive response of the marine bacterium Sphingopyxis alaskensis RB2256 to solar radiation (both visible and ultraviolet) was assessed by a quantitative proteomic approach using iTRAQ (isobaric tags for relative and absolute quantification). Both growth phase (mid-log and stationary phase) and duration (80 min or 8 h) of different light treatments (combinations of visible light, UV-A and UV-B) were assessed relative to cultures maintained in the dark. Rates of total protein synthesis and viability were also assessed. Integrating knowledge from the physiological experiments with quantitative proteomics of the 12 conditions tested provided unique insight into the adaptation biology of UV and visible light responses of S. alaskensis. High confidence identifications were obtained for 811 proteins (27% of the genome), 119 of which displayed significant quantitative differences. Mid-log-phase cultures produced twice as many proteomic changes as stationary-phase cultures, while extending the duration of irradiation exposure of stationary-phase cultures did not increase the total number of quantitative changes. Proteins with significant quantitative differences were identified that were characteristic of growth phase and light treatment, and cellular processes, pathways and interaction networks were determined. Key factors of the solar radiation adaptive response included DNA-binding proteins implicated in reducing DNA damage, detoxification of toxic compounds such as glyoxal and reactive oxygen species, iron sequestration to minimize oxidative stress, chaperones to control protein re/folding, alterations to nitrogen metabolism, and specific changes to transcriptional and translational processes.