Text|Explore the secret history of the tomb
Editor|Exploring the Secret History of the Tomb
preface
The fixation of atmospheric diazepine organisms as ammonia has a significant impact on the extent of marine primary production. Only a few bacteria are known to contribute to open up nitrogen fixation in the marine environment, and they typically make up only a small portion (< 0.1%) of the overall microbial community.
Among them, it is estimated that the single-celled nitrogen-fixing bacterium Crocosphaera watsonii is an important contributor to marine nitrogen fixation. Most of the primary productivity in tropical and subtropical marine regions is expected to be limited by nitrogen, while nitrogen sources for diazo organisms are replenished by large atmospheric reservoirs and are essentially unlimited.
Conversely, iron is considered a key micronutrient for marine nitrogen-fixing bacteria because they use an iron nitrogenase protein complex containing homodimeric ferritin with a 4Fe:4S metal cluster (NifH) and a heterotetrameric molybdenum ferritin with an 8Fe:7S P cluster and 7Fe and 1 Mo MoFe cofactors (NifDK, α, and β subunits).
Both field experiments and models predict that the distribution of nitrogen fixation in the ocean is mainly limited by iron availability. Although iron is so important for marine nitrogen fixation, there is limited understanding of how marine nitrogen-fixing bacteria adapt to this low-iron environment.
The coexistence of oxygenated photosynthesis and nitrogen-fixing metabolism presents unique challenges for nitrogen-fixing bacteria due to the high demand for iron and the chemical incompatibility of molecular oxygen and nitrogenase protein complexes, and previous elemental studies measuring iron content in crocodiles whole cells have found increased iron content during dark periods of nitrogen fixation.
Although this finding is consistent with theoretical studies that predict that marine nitrogen-fixing bacteria require large amounts of iron, it has been somewhat controversial due to its effects on the dynamics of intracellular iron but poorly understood diurnal cycles.
Several single-celled nitrogen-fixing bacteria, including Crocosphaera watsonii, have been observed to fix nitrogen during dark periods, which is widely thought to be an adaptation to the temporal separation of photosynthesis and nitrogen fixation to avoid oxygen disruption of the nitrogenase complex.
Global transcription studies have also observed large-scale changes in the transcriptome of the single-celled nitrogen-fixing bacteria Crocosphaera watsonii, Cyanothece, Gloeothece, and Synechococcus during the diurnal cycle, consistent with temporal separation of photosynthesis and nitrogen fixation.
However, the possibility that the circadian cycle of the transcriptome or proteome may affect iron demand has not been previously discussed, possibly because transcriptional studies provide information about gene expression rather than actual enzyme banks and immunological protein studies to date.
The simultaneous detection of NifH subunits in Crocosphaera and Gloeothece is limited to a few selected proteins, and no global or absolute quantitative proteome studies on nitrogen-fixing bacteria have been published.
Therefore, it remains uncertain whether the observed circadian transcriptome cycle requires the maintenance of a relatively consistent proteome, or indeed results in dramatic changes in global proteome composition during each diurnal cycle.
Recent advances in proteomics technology promise to elucidate the mechanistic links between the biochemistry of important microorganisms and the global biogeochemical cycle, and there are two broad approaches to mass spectrometry-based proteomics: the global shotgun proteomics approach.
The relative abundance of hundreds of proteins can be investigated simultaneously and semi-quantitatively using spectral counting, as well as peptide standards for absolute quantification of proteins of interest using selective reaction monitoring (SRM) mass spectrometry and isotope labeling methods.
Although there have been several culture-based marine microbial studies and field assessments of protein-rich, the combined application of global and targeted proteomics approaches to biogeochemistry-related issues, such as iron restriction of marine diazo nutrition, has not been reported.
Results and discussion
The diurnal cycle and stock of ferrometalloenzymes in Crocosphaera watsonii were investigated from three different culture experiments using global and targeted liquid chromatography mass spectrometry (LC-MS) proteomics techniques.
These experiments were all grown in a 14:10 light:dark cycle: a day and night experiment sampled during the light and dark photoperiods (sampling for 10 hours from the light on and 7 h from the light on) used 1D and 2D LC-MS methods to analyze the global proteome.
The time course study (here DIEL) is sampled every 2-3 h for 30 h (4 h from the light on) and the absolute number of global proteome and target protein is analyzed using 1D LC-MS.
and a group of biological triplicate cultures (sampled at 6.5 h in the light period, 3 h in the dark period, and 6 h) to analyze the protein abundance of interest by mass spectrometry.
[0.65% false-positive rate (FPR)] among 477 unique proteins identified in global proteomic one-dimensional analysis of day and night experiments, 37 proteins were found to vary in abundance by a factor of 2 or greater between day and night.
These results were confirmed in the deeper 2D chromatography proteome, where 160 of the 1,108 identified proteins (0.11% FPR) showed greater than 2-fold changes. The time course of the circadian cycle clearly shows the cycle of protein abundance.
Lighting effects
Among them, 100 proteins were found to be above the threshold spectral count signal (≥10) and showed diurnal variation in amplitude (max-min ≥14) Cluster analysis revealed two broad categories, which include key photosystems or nitrogen-fixing proteins, which increase abundance during the day and night, respectively.
These experiments show that the diurnal cycle observed in the transcriptome of single-celled nitrogen-fixing bacteria exhibits large-scale changes in the global proteome, with more than 20% of the measured proteins in the diurnal experiment showing diurnal variation.
In diurnal and diurnal experiments, metalloenzymes involved in nitrogen fixation were those with the most significant abundances in Crocosphaera watsonii, largely absent during the day and appearing at night.
Based on these global experiments, the absolute abundance of nine protein targets in the DIEL experiment was determined by triple quadrupole SRM mass spectrometry using isotope-labeled internal standards.
The abundance of the three nitrogenase-fixing metalloproteins ranges from undetectable levels during photoperiods to one of the most abundant proteins in the proteome during dark periods.
At its peak abundance, the ratio of ferritin to molybdenum ferritin subunit (α used here) is 3.5:1, which is consistent with the previously observed 3:1 cell ratio and the stoichiometry of 2:1 within nitrogenase.
Together, these global and targeted proteome measurements provide evidence for nocturnal synthesis of metalloenzymes in nitrogenase-fixing enzymes and subsequent complete diurnal degradation.
An increase in the abundance of flavo-oxedoxin during dark periods was observed, similar to nitrogenase-fixing enzyme metalloenzymes, and in contrast to the increased abundance of flavin oxorin observed in most phytoplankton under iron-deficient conditions.
This suggests that ferroxyprotein-flavin replacement of electron transport in photosynthesis does not occur significantly in crocodiles, with global proteomes sampled from short-term iron deprivation experiments during illumination to distinguish iron effects from dark expression.
Protein effects
An increase in flavanin in response to iron stress was not observed 24 h after exogenous siderophore addition, consistent with the hypothesis that flavo-oxedoxin can act as an electron carrier for nitrogenases rather than ferroxenoxins.
Even under iron-plentiful conditions, the use of flavin oxederin at night appears to be an adaptation to the overall minimization of iron requirements in this microbial cell.
In the mirror image of nitrogenase-fixing metalloenzymes and flavin oxedoxin, many proteins are more abundant during photoperiods, in particular, several proteins from iron-rich photosystem I decrease during dark periods and increase during light periods.
Includes PsaA, PsaB, PsaD, and PsaF. Iron-containing cytochromes b6 and c550 are among the most abundant cytochromes detected by spectral counting (f, P450 and b559 and c oxidases are also detected),
And measured by targeted mass spectrometry, abundance decreases substantially during dark periods. Degradation of large amounts of cytochrome B6 and C550 releases large amounts of heme during dark periods. Two heme oxygenases capable of releasing iron from heme were identified in the proteome,
They show diurnal variation and may be important in the iron cycle. Iron-storing bacterial ferritin has also been identified in the proteome, one of which shows a certain circadian periodicity. Given that iron is thought to be added and removed from ferritin cages without degradation, there is not necessarily a circadian cycle for bacterial ferritin.
Transcription and immunology
These results depict a dynamic Crocosphaera proteome that alternates its metalloenzyme stocks to accommodate alternating time demands for nitrogen fixation and photosynthetic biochemical activity. Although Crocosphaera's unique nocturnal nitrogenase activity is one of its defining features in laboratory and field studies,
However, the fate of nitrogenase-fixing protein complexes during illumination remains controversial due to technical limitations of transcriptional and immunological methods, where transcripts do not measure protein abundance and immunological methods may not detect post-translationally modified proteins.
By mass spectrometry analysis of multiple trypsin peptides for each protein, this study showed almost complete degradation of metalloenzymes in nitrogenase-fixing enzyme complexes and partial degradation of certain iron-containing components of photosynthesis. Considering the potentially significant energy costs,
The extent of proteome cycling we observed in Crocosphaera watsonii was surprising. As mentioned earlier, this proteome cycle may have the dual function of preventing oxygen from destroying the nitrogenase-fixing complex, as well as providing a mechanism that significantly reduces the need for metabolic iron.
We believe the latter to be correct, based on stoichiometry calculations for quantitative metalloenzyme data in A-H and the associated iron stoichiometry for each metalloenzyme. By summarizing iron associated with major metalloenzymes, we calculated the amount of iron involved in nitrogen fixation and photosynthesis throughout the diurnal cycle.
By degrading these metalloenzymes when not in use, Crocosphaera reduced peak night, peak day, and minimum ferrometalease stocks by 38%, 40%, and 75%, respectively, at any given time, compared to the hypothetical scenario of these metalloenzymes not degraded.
Nitrogen fixation and photosynthesis
Another experiment that performed targeted protein analysis on organisms in triplicate was consistent with these estimates of iron conservation in diurnal experiments, with a 45 ± 19% reduction in iron use during the photoperiod (6.5 h after turning on the lights, n = 3), and 38 ± 14% ±less iron during dark periods (sampled 3 h and 6 h after lights out, n = 3 per section).
If iron-free flavoxedrin is observed during dark periods, these iron savings are replaced by iron-required ferroraxin. The iron released by the degradation of these metalloenzymes can have several fates,
These include storage in bacterial ferritin or chaperone proteins, loss from cells, and reuse in other metalloenzymes. Given the severe iron restriction conditions in the upper ocean, it appears that some of the released iron stocks are involved in nitrogen fixation and photosynthesis metabolism.
This iron protection strategy is similar to Hotbunking's maritime practice in that ships sail with more sailors (metalloenzyme requirements) than berths (iron atoms), while sailors in shifts share the same bunk—keeping the bunks hot (or atoms in iron use).
While it is methodologically difficult to record the sharing of individual iron atoms between metabolisms, our data clearly demonstrate the reduction of ferrometase banks and the simultaneous reduction of metabolic iron requirements through circadian proteome cycling.
Iron preservation by reducing metalloenzyme stocks may be a key component of the low-iron marine niche of crocodiles. Mucormyces is another dominant oxygenated marine nitrogen-fixing bacterium that differs from Crococodile in that it fixes both nitrogen and carbon during photoperiod.
The result was Trichodesmium sp. This iron protection strategy may not be employed as Crocosphaera did. This is consistent with Trichodesmium and Crocosphaera (> 35 and 16± 11 μ mol Fe mol C-1, respectively) and estimates of most cellular iron (22–50%) required for nitrogen fixation in Trichodesmium.
Previous studies of Crocosphaera watsonii cell iron observed an increase in iron concentration per cell during dark periods. Our calculated iron content per cell is attributed to nitrogen fixation and photosynthesis in line with the measured values,
Higher iron levels at night may be the result of circadian proteome cycling, plus daytime ferrous absorption and accumulation resulting from photochemical reduction and photoperiodic cell division.
Stocks of metalloenzymes
By reducing nitrogen-fixing and photosynthetic metalloenzyme banks when not in use, Crocosphaera watsonii has evolved to have 40% less iron than maintaining these enzymes throughout the circadian cycle. Although difficult to quantify, this process of resynthesizing enzymes on a daily basis is sure to consume a lot of effort.
Based on our quantitative measurements, the circadian cycle of only three nitrogenase-fixing metalloproteins contributed ~2.3% of the total protein, and all eight target proteins in Figure 3 contributed 5.0% (using the difference between maximum and minimum expression levels during the circadian cycle).
Many lower abundance proteins also undergo circadian cycles, and proteins contribute about 40% of cellular biomass, these calculations provide some rough energy costs to increase the energy costs required for protein synthesis,
This reduces the need for iron metabolism (referred to here as "hotbunking"). If Crocosphaera watsonii evolved hotbunking from an ancestor who maintained its metalloenzymes day and night, how would the trade-off between lower iron requirements and higher energy costs affect Crocosphaera's competitiveness relative to its ancestor and other nitrogen-fixing bacteria?
A numerical global ocean circulation and ecosystem model is a useful tool to explore the impact of hotbunking on Crocosphera, habitat and nitrogen fixation rates in the global oceans.
epilogue
Through this study, we can find that after reducing the metalloenzymes in nitrogen-fixing bacteria, the rate of iron corrosion is reduced, and this method can be used to protect iron.
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