The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse phosphorus stress response.

Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem function in the ocean. Diatoms are an abundant class of marine phytoplankton that are responsible for significant amounts of primary production. With the control they exert on the oceanic carbon cycle, there have been a number of studies focused on how diatoms respond to limiting macro and micronutrients such as iron and nitrogen. However, diatom physiological responses to P deficiency are poorly understood. Here, we couple deep sequencing of transcript tags and quantitative proteomics to analyze the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. A total of 318 transcripts were differentially regulated with a false discovery rate of <0.05, and a total of 136 proteins were differentially abundant (p<0.05). Significant changes in the abundance of transcripts and proteins were observed and coordinated for multiple biochemical pathways, including glycolysis and translation. Patterns in transcript and protein abundance were also linked to physiological changes in cellular P distributions, and enzyme activities. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P through increased production of metalloenzymes, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to respond to variations in environmental P availability.

Examination of microbial proteome preservation techniques applicable to autonomous environmental sample collection.

Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC-MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although further study is warranted on additional marine microbes.

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii.

The marine nitrogen fixing microorganisms (diazotrophs) are a major source of nitrogen to open ocean ecosystems and are predicted to be limited by iron in most marine environments. Here we use global and targeted proteomic analyses on a key unicellular marine diazotroph Crocosphaera watsonii to reveal large scale diel changes in its proteome, including substantial variations in concentrations of iron metalloproteins involved in nitrogen fixation and photosynthesis, as well as nocturnal flavodoxin production. The daily synthesis and degradation of enzymes in coordination with their utilization results in a lowered cellular metalloenzyme inventory that requires ∼40% less iron than if these enzymes were maintained throughout the diel cycle. This strategy is energetically expensive, but appears to serve as an important adaptation for confronting the iron scarcity of the open oceans. A global numerical model of ocean circulation, biogeochemistry and ecosystems suggests that Crocosphaera's ability to reduce its iron-metalloenzyme inventory provides two advantages: It allows Crocosphaera to inhabit regions lower in iron and allows the same iron supply to support higher Crocosphaera biomass and nitrogen fixation than if they did not have this reduced iron requirement.

A bi-site mechanism for Escherichia coli F1-ATPase accounts for the observed positive catalytic cooperativity.

Nucleotide binding to nucleotide-depleted F(1)-ATPase from Escherichia coli (EcF(1)) during MgATP hydrolysis in the presence of excess epsilon subunit has been studied using a combination of centrifugal filtration and column-centrifugation methods. The results show that nucleotide-binding properties of catalytic sites on EcF(1) are affected by the state of occupancy of noncatalytic sites. The ATP-concentration dependence of catalytic-site occupancy during MgATP hydrolysis demonstrates that a bi-site mechanism is responsible for the positive catalytic cooperativity observed during multi-site catalysis by EcF(1). The results suggest that a bi-site mechanism is a general feature of F(1) catalysis.

Studies of nucleotide binding to the catalytic sites of Escherichia coli betaY331W-F1-ATPase using fluorescence quenching.

Most studies of nucleotide binding to catalytic sites of Escherichia coli betaY331W-F(1)-ATPase by the quenching of the betaY331W fluorescence have been conducted in the presence of approximately 20 mM sulfate. We find that, in the absence of sulfate, the nucleotide concentration dependence of fluorescence quenching induced by ADP, ATP, and MgADP is biphasic, revealing two classes of binding sites, each contributing about equally to the overall extent of quenching. For the high-affinity catalytic site, the K(d) values for MgADP, ADP, and ATP equal 10, 43, and 185 nM, respectively. For the second class of sites, the K(d) values for these ligands are approximately 1,000x larger at 8.1, 37, and 200 microM, respectively. The presence of sulfate or phosphate during assay results in a marked increase in the apparent K(d) values for the high-affinity catalytic site. The results show, contrary to earlier reports, that Mg(2+) is not required for expression of different affinities for a nucleotide by the three catalytic sites. In addition, they demonstrate that the fluorescence of the introduced tryptophans is nearly completely quenched when only two sites bind nucleotide. Binding of ADP to the third site with a K(d) near mM gives little fluorescence change. Many previous results of fluorescence quenching of introduced tryptophans appear to require reinterpretation. Our findings support a bi-site catalytic mechanism for F(1)-ATPase.

Rotor/Stator interactions of the epsilon subunit in Escherichia coli ATP synthase and implications for enzyme regulation.

The H(+)-translocating F(0)F(1)-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F(0) to the synthesis of ATP by F(1). Although the epsilon subunit appears to be tightly associated with the gamma subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of epsilon can undergo significant conformational change as part of a regulatory process. Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of epsilon with an F(0) component of the rotor (subunit c) and with an F(1) component of the stator (subunit beta). Oxidation of the engineered F(0)F(1) causes formation of two disulfide bonds, betaD380C-S108C epsilon and epsilonE31C-cQ42C, to give a beta-epsilon-c cross-linked product in high yield. The results demonstrate the ability of epsilon to span the central stalk region from the surface of the membrane (epsilon-c) to the bottom of F(1) (beta-epsilon) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated epsilon (Uhlin, U., Cox, G. B., and Guss, J. M. (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the gamma subunit (Rodgers, A. J., and Wilce, M. C. (2000) Nat. Struct. Biol. 7, 1051-1054). The kinetics of beta-epsilon and epsilon-c cross-linking were studied separately using F(0)F(1) containing one or the other matched cysteine pair. The rate of cross-linking at the epsilon/c (rotor/rotor) interface is not influenced by the type of nucleotide added. In contrast, the rate of beta-epsilon cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP. This is consistent with a regulatory role for a reversible beta/epsilon (stator/rotor) interaction that blocks rotation and inhibits catalysis. Furthermore, the rate of beta-epsilon cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.