Contrasting ability to take up leucine and thymidine among freshwater bacterial groups: implications for bacterial production measurements

We examined the ability of different freshwater bacterial groups to take up leucine and thymidine in two lakes. Utilization of both substrates by freshwater bacteria was examined at the community level by looking at bulk incorporation rates and at the single-cell level by combining fluorescent in situ hybridization and signal amplification by catalysed reporter deposition with microautoradiography. Our results showed that leucine was taken up by 70–80% of Bacteria-positive cells, whereas only 15–43% of Bacteria-positive cells were able to take up thymidine. When a saturating substrate concentration in combination with a short incubation was used, 80–90% of Betaproteobacteria and 67–79% of Actinobacteria were positive for leucine uptake, whereas thymidine was taken up by < 10% of Betaproteobacteria and by < 1% of the R-BT subgroup that dominated this bacterial group. Bacterial abundance was a good predictor of the relative contribution of bacterial groups to leucine uptake, whereas when thymidine was used Actinobacteria represented the large majority (> 80%) of the cells taking up this substrate. Increasing the substrate concentration to 100 nM did not affect the percentage of R-BT cells taking up leucine (> 90% even at low concentrations), but moderately increased the fraction of thymidine-positive R-BT cells to a maximum of 35% of the hybridized cells. Our results show that even at very high concentrations, thymidine is not taken up by all, otherwise active, bacterial cells

Spatio-temporal niche separation of planktonic Betaproteobacteria in an oligo-mesotrophic lake

We investigated the diversity of planktonic Betaproteobacteria and the seasonal population changes of betaproteobacterial taxa in an oligo-mesotrophic lake (Piburger See, Austria). Focus was put on the vertical distribution of the investigated populations and on differences between their respective cell fractions with apparent amino acid incorporation. On average, 66% of betaproteobacterial cells and 73% of their diversity could be attributed to four clades within three lineages that were further analysed by fluorescence in situ hybridization. The numbers of bacteria from the R-BT subclade of the beta I lineage and from the PnecB subgroup of the beta II lineage were rather constant throughout the water column. In contrast, members of another subgroup of beta II (PnecC) and bacteria related to Methylophilus (beta IV) were particularly numerous in the oxygen-depleted zone. In general, only moderate seasonal changes in abundance were observed in the upper water layers, whereas there was a clear relationship between decreasing oxygen levels and the rise of bacteria from the PnecC and beta IV clades in deeper strata. On average, almost 80% of beta I bacteria, but < 15% of cells from the beta IV clade, showed amino acid incorporation. Our results suggest that the studied populations occupy distinct vertical and ecophysiological niches in Piburger See

Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements

In the non-heterocyst. marine cyanobacterium Trichodesmium nitrogen fixation is con fined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems. changing the effective cross-section of PSI!. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption. fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method. we used them to deconvolute the in vivo fluorescence spectra of richodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle. and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium

Acclimation of Trichodesmium erythraeum ISM101 to high and low irradiance analysed on the physiological, biophysical and biochemical level

As the nonheterocystous diazotrophic cyanobacterium Trichodesmium lives both at the ocean surface and deep in the water column, it has to acclimate to vastly different irradiances. Here, we investigate its strategy of light acclimation in several ways.In this study, we used spectrally resolved fluorescence kinetic microscopy to investigate the biophysics of photosynthesis in individual cells, analysed cell extracts for pigment and phycobiliprotein composition, measured nitrogenase activity and the abundance of key proteins, and assayed protein synthesis/degradation by radioactive labelling.After acclimation to high light, Trichodesmium grew faster at 1000 μmol m-2 than at 100 μmol m-2 S-1. This acclimation was associated with decreasing cell diameter, faster protein turnover, the down-regulation of light-harvesting pigments and the outer part of the phycobiliprotein antenna, the up-regUlation of light-protective carotenoids, changes in the coupling of phycobilisomes to thereaction centres and in the coupling of individual phycobiliproteins to the phycobilisomes. The latter was particularly interesting, as it represents an as yet unreported light acclimation strategy.Only in the low light-acclimated culture and only after the onset of actinic light did phycourobilin and phycoerythrin contribute to photochemical fluorescence quenching, showing that these phycobiliproteins may become quickly (in seconds) very closely coupled to photosystem 11. This fast reversible coupling also became visible in the nonphotochemical changes of the fluorescence quantum yield