Table_3_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.XLSX

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

<i>isp2Δ</i> spores show a delay in germination.

<p>(A) <i>isp2Δ</i> spores have a germination defect on solid YPD medium. Colonies of wild type and <i>isp2Δ</i> strains after growth at room temperature for 63h (germination) and 51h (vegetative growth). Scale bars, 100μm (5x magnification). (B) Quantification of colony sizes using ImageJ. The average colony size of <i>isp2Δ</i> spores was only 20.5% of wild type spores, whereas the average colony size of <i>isp2Δ</i> yeast was 45.7% of wild type yeast. The difference in size between colonies from yeast growth and spore germination for <i>isp2Δ</i> strains was significant (p = 7.1x10^-48) but not for the wild type strain (p = 7.8x10^-1). Data represent number (n) of independent experiments and are shown as a mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (C) Germination delay for <i>isp2Δ</i> spores in liquid YPD media. Optical density at a wavelength of 600nm (OD₆₀₀) was measured every 3min over 50h. The y-axis shows OD₆₀₀ and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (D) Average time taken to double initial OD₆₀₀. Quantified doubling times were nearly identical for wild type and <i>isp2Δ</i> yeast (p = 0.38); however, <i>isp2Δ</i> spores took significantly longer than wild type to double the population (p = 1.2×10⁻¹⁰). Data represent number (n) of independent experiments and are shown as mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (E) Morphological changes during germination of <i>isp2Δ</i> and wild type spores. Spores were exposed to YPD liquid media to trigger germination at room temperature and photographed at 0h and 12h. Scale bars, 5μm (1000× magnification).</p

Summary of spore-enriched protein mutant phenotypes.

<p><i>C</i>. <i>neoformans</i> spores are produced through sexual development (above the gray bar). Sexual development occurs between haploid yeast of opposite mating types (<b>a</b> and α) and includes mate detection and cell fusion, dikaryotic filamentation, basidium formation and production of spores in chains. Spores can disperse and germinate into yeast (below the gray bar) to complete the life cycle. Gene names in red text are placed at the process in which deletion mutants show phenotypes. Six genes, <i>ISP3-7</i> and <i>SFH5</i>, did not result in detectable phenotypes in any assays.</p

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in <i>Cryptococcus</i>

<div><p>Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus <i>Cryptococcus neoformans</i> to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in <i>C</i>. <i>neoformans</i>. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.</p></div

Image_1_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.TIFF

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

Table_4_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.XLSX

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

Assessment of the proteomic data.

<p>(A) Venn diagram of spore proteins and yeast proteins identified. 2232 and 2192 proteins were identified from spores and yeast, respectively, with a majority of 1858 existing in both cell types. (B) Virtual two-dimensional gel diagrams of the predicted <i>C</i>. <i>neoformans</i> proteome (upper panel) and identified proteins in either yeast (middle panel) or spores (lower panel). Each dot represents a protein, with the x-axis showing isoelectric point (pI) and the y-axis as molecular weight (MW, Dalton).</p

Table_1_Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.XLSX

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.</p

Spores derived from wild type by <i>isp2Δ</i> crosses show wild type rates of germination.

<p>(A) Spores purified from WT × <i>isp2Δ</i> strains were grown in YPD and measured via optical density (OD₆₀₀) every 3min over 50h. The y-axis shows OD₆₀₀ and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (B) Model of Isp2 activity during germination. Germination encompasses two stages: a morphological transition and growth initiation before the active replication of vegetative growth. Isp2 protein (black triangles) is present in mature spores from WT × WT crosses and persists during germination through the morphological transition to contribute to optimal growth initiation. In contrast, there is no Isp2 in spores from <i>isp2Δ</i> × <i>isp2Δ</i> crosses, and thus, a delay of ~2h during germination occurs, specifically during the growth initiation phase. Notably, spores from WT × <i>isp2Δ</i> crosses do not show a delay in germination and therefore contain Isp2 protein similar to wild type spores, regardless of genotype. Spores are shown as ovals with stalks, whereas yeast are shown as spheres. Large and small spheres together represent budding yeast. Isp2 protein is represented by black triangles.</p