Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe oryzae.

A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO2) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.

Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.

The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

DNA Microarray-Based Identification of Genes Regulated by NtrC in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum NtrBC two-component system is a critical regulator of cellular nitrogen metabolism, including the acquisition and catabolism of nitrogenous compounds. To better define the roles of this system, genome-wide transcriptional profiling was performed to identify the NtrC regulon during the response to nitrogen limitation. Upon cells perceiving low intracellular nitrogen, they stimulate the phosphorylation of NtrC, which induces genes responsible for alteration of the core glutamine synthetase/glutamate synthase nitrogen assimilation pathway, including the genes for the glutamine synthetases and PII proteins. In addition, genes responsible for the import and utilization of multiple nitrogen sources, specifically nitrate and nitrite, were upregulated by NtrC activation. Mutational analysis of a candidate nitrite reductase revealed a role for NtrC in regulating the assimilation of nitrite, since mutations in both ntrC and the gene encoding the candidate nitrite reductase abolished the ability to grow on nitrite as a sole nitrogen source.

Soybean ureases, but not that of Bradyrhizobium japonicum, are involved in the process of soybean root nodulation.

Ureases are abundant in plants, bacteria, and in the soil, but their role in signaling between soybean and soil microorganisms has not been investigated. The bacterium Bradyrhizobium japonicum forms nitrogen-fixing nodules on soybean roots. Here, we evaluated the role(s) of ureases in the process of soybean nodulation. Chemotaxis assays demonstrated that soybean and jack bean ureases were more chemotactic toward bacterial cells than the corresponding plant lectins. The eu1-a,eu4 soybean, deficient in urease isoforms, formed fewer but larger nodules than the wild-type, regardless of the bacterial urease phenotype. Leghemoglobin production in wild-type plants was higher and peaked earlier than in urease-deficient plants. Inhibition of urease activity in wild-type plants did not result in the alterations seen in mutated plants. We conclude that soybean urease(s) play(s) a role in the soybean-B. japonicum symbiosis, which is independent of its ureolytic activity. Bacterial urease does not play a role in nodulation.

Temporal analysis of the magnaporthe oryzae proteome during conidial germination and cyclic AMP (cAMP)-mediated appressorium formation.

Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and highlight biological processes important for pathogenicity.

In-depth analysis of the Magnaporthe oryzae conidial proteome.

The filamentous fungus Magnaporthe oryzae (M. oryzae) is the causative agent of rice blast disease and presents a significant threat to worldwide rice production. To establish the groundwork for future research on the pathogenic development of M. oryzae, a global proteomic study of conidia was performed. The filter aided sample preparation method (FASP) and anion StageTip fractionation combined with long, optimized shallow 210 min nanoLC gradients prior to mass spectrometry analysis on an Orbitrap XL was applied, which resulted in a doubling of protein identifications in comparison to our previous GeLC analysis. Herein, we report the identification of 2912 conidial proteins at a 1% protein false discovery rate (FDR) and we present the most extensive study performed on M. oryzae conidia to date. A similar distribution between identified proteins and the predicted proteome was observed when subcellular localization analysis was performed, suggesting the detected proteins build a representative portion of the predicted proteome. A higher percentage of cytoplasmic proteins (associated with translation, energy, and metabolism) were observed in the conidial proteome relative to the whole predicted proteome. Conversely, nuclear and extracellular proteins were less well represented in the conidial proteome. Further analysis by gene ontology revealed biological insights into identified proteins important for central metabolic processes and the physiology of conidia.

Polyubiquitin is required for growth, development and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.

Evaluation of normalization methods on GeLC-MS/MS label-free spectral counting data to correct for variation during proteomic workflows.

Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with higher SpCs.

Bacterial outer membrane channel for divalent metal ion acquisition.

The prevailing model of bacterial membrane function predicts that the outer membrane is permeable to most small solutes because of pores with limited selectivity based primarily on size. Here, we identified mnoP in the Gram-negative bacterium Bradyrhizobium japonicum as a gene coregulated with the inner membrane Mn(2+) transporter gene mntH. MnoP is an outer membrane protein expressed specifically under manganese limitation. MnoP acts as a channel to facilitate the tranlocation of Mn(2+), but not Co(2+) or Cu(2+), into reconstituted proteoliposomes. An mnoP mutant is defective in high-affinity Mn(2+) transport into cells and has a severe growth phenotype under manganese limitation. We suggest that the outer membrane is a barrier to divalent metal ions that requires a selective channel to meet the nutritional needs of the cell.

Direct comparison of stable isotope labeling by amino acids in cell culture and spectral counting for quantitative proteomics.

Numerous experimental strategies exist for relative protein quantification, one of the primary objectives of mass spectrometry based proteomics analysis. These strategies mostly involve the incorporation of a stable isotope label via either metabolic incorporation in cell or tissue culture (¹5N/¹4N metabolic labeling, stable isotope labeling by amino acids in cell culture (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), or enzymatically catalyzed incorporation (¹8O labeling). Also, these techniques can be cost or time prohibitive or not amenable to the biological system of interest (i.e., metabolic labeling of clinical samples, most animals, or fungi). This is the case with the quantification of fungal proteomes, which often require auxotroph mutants to fully metabolically label. Alternatively, label-free strategies for protein quantification such as using integrated ion abundance and spectral counting have been demonstrated for quantification affording over 2 orders of magnitude of dynamic range which is comparable to metabolic labeling strategies. Direct comparisons of these quantitative techniques are largely lacking in the literature but are highly warranted in order to evaluate the capabilities, limitations, and analytical variability of available quantitative strategies. Here, we present the direct comparison of SILAC to label-free quantification by spectral counting of an identical set of data from the bottom-up proteomic analysis of human embryonic stem cells, which are readily able to be quantified using both strategies, finding that both strategies result in a similar number of protein identifications. We also discuss necessary constraints for accurate quantification using spectral counting and assess the potential of this label-free strategy as a viable alternative for quantitative proteomics.

Whole-genome transcriptional profiling of Bradyrhizobium japonicum during chemoautotrophic growth.

Bradyrhizobium japonicum is a facultative chemoautotroph capable of utilizing hydrogen gas as an electron donor in a respiratory chain terminated by oxygen to provide energy for cellular processes and carbon dioxide assimilation via a reductive pentose phosphate pathway. A transcriptomic analysis of B. japonicum cultured chemoautotrophically identified 1,485 transcripts, representing 17.5% of the genome, as differentially expressed when compared to heterotrophic cultures. Genetic determinants required for hydrogen utilization and carbon fixation, including the uptake hydrogenase system and components of the Calvin-Benson-Bassham cycle, were strongly induced in chemoautotrophically cultured cells. A putative isocitrate lyase (aceA; blr2455) was among the most strongly upregulated genes, suggesting a role for the glyoxylate cycle during chemoautotrophic growth. Addition of arabinose to chemoautotrophic cultures of B. japonicum did not significantly alter transcript profiles. Furthermore, a subset of nitrogen fixation genes was moderately induced during chemoautotrophic growth. In order to specifically address the role of isocitrate lyase and nitrogenase in chemoautotrophic growth, we cultured aceA, nifD, and nifH mutants under chemoautotrophic conditions. Growth of each mutant was similar to that of the wild type, indicating that the glyoxylate bypass and nitrogenase activity are not essential components of chemoautotrophy in B. japonicum.

An oligonucleotide microarray resource for transcriptional profiling of Bradyrhizobium japonicum.

A DNA microarray, comprising 70-mer oligonucleotides, representing 8,453 open reading frames (ORFs), was constructed based on the Bradyrhizobium japonicum strain USDA110 genomic sequence. New annotation predicted 199 additional genes, which were added to the microarray and were shown to be transcribed. These arrays were used to profile transcription in cells under a variety of conditions, including growth in minimal versus rich medium, osmotic stress, and free-living cells versus bacteroids. Increased expression was seen for genes involved in translation, motility, and cell envelope synthesis in rich medium whereas expression increased in minimal medium for genes involved in vitamin biosynthesis and stress responses. Treatment with 50 mM NaCl activated stress-inducible genes but repressed genes involved in chemotaxis and motility. Strikingly, no known transport systems for accumulation of compatible solutes or osmoprotectants were induced in response to osmotic stress. A number of nif, fix, and hup genes, but not all, were upregulated in bacteroids. The B. japonicum type III secretion system, known to be important in early nodulation, was downregulated in bacteroids. The availability of a reliable, low-cost B. japonicum microarray provides a useful tool for functional genomic studies of one of the most agriculturally important bacteria.

Transcriptional and physiological responses of Bradyrhizobium japonicum to desiccation-induced stress.

The growth and persistence of rhizobia and bradyrhizobia in soils are negatively impacted by drought conditions. In this study, we used genome-wide transcriptional analyses to obtain a comprehensive understanding of the response of Bradyrhizobium japonicum to drought. Desiccation of cells resulted in the differential expression of 15 to 20% of the 8,453 [corrected] B. japonicum open reading frames, with considerable differentiation between early (after 4 h) and late (after 24 and 72 h) expressed genes. While 225 genes were universally up-regulated at all three incubation times in response to desiccation, an additional 43 and 403 up-regulated genes were common to the 4/24- and 24/72-h incubation times, respectively. Desiccating conditions resulted in the significant induction (>2.0-fold) of the trehalose-6-phosphate synthetase (otsA), trehalose-6-phosphate phosphatase (otsB), and trehalose synthase (treS) genes, which encode two of the three trehalose synthesis pathways found in B. japonicum. Gene induction was correlated with an elevated intracellular concentration of trehalose and increased activity of trehalose-6-phosphate synthetase, collectively supporting the hypothesis that this disaccharide plays a prominent and important role in promoting desiccation tolerance in B. japonicum. Microarray data also indicated that sigma(54)- and sigma(24)-associated transcriptional regulators and genes encoding isocitrate lyase, oxidative stress responses, the synthesis and transport of exopolysaccharides, heat shock response proteins, enzymes for the modification and repair of nucleic acids, and the synthesis of pili and flagella are also involved in the response of B. japonicum to desiccation. Polyethylene glycol-generated osmotic stress induced significantly fewer genes than those transcriptionally activated by desiccation. However, 67 genes were commonly induced under both conditions. Taken together, these results suggest that B. japonicum directly responds to desiccation by adapting to changes imparted by reduced water activity, such as the synthesis of trehalose and polysaccharides and, secondarily, by the induction of a wide variety of proteins involved in protection of the cell membrane, repair of DNA damage, stability and integrity of proteins, and oxidative stress responses.

Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

Complex regulation of arsenite oxidation in Agrobacterium tumefaciens.

Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc2and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.

Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov.

Two moderately thermophilic, Gram-positive, spore-forming bacteria were isolated from different geographical locations and sources; strain GS5-97(T) from a beet sugar factory in Leopoldsdorf, Lower Austria, and strain YNP10 from a geothermally heated soil, Yellowstone National Park, USA. The sequences of their 16S rRNA genes were found to be 99.8% identical, and DNA-DNA hybridization experiments revealed that strains GS5-97(T) and YNP10 share 89.9 mol% similarity to each other, but only 34.3 and 39.2 mol% similarity, respectively, to Geobacillus caldoxylosilyticus DSM 12041(T), which is their closest related type strain. A polyphasic analysis showed that these two isolates were more similar to each other than to other characterized geobacilli. Their DNA G+C content was 43.2 and 42.4 mol%, respectively, and they were identical with respect to many phenotypic features (e.g. T(opt) 55 degrees C; pH(opt) 7.0). Both strains clearly displayed best growth when cultured aerobically. They differed slightly in their cellular fatty acid profiles and polar lipid pattern, and genotypically they could also be distinguished based on randomly amplified polymorphic DNA fingerprints and internal transcribed spacer analysis. Freeze-etching experiments revealed oblique surface layer (S-layer) lattices in both strains, and biochemical analyses of the purified S-layer proteins indicated the occurrence of glycosylation. Based on the properties of these organisms relative to those currently documented for the genus Geobacillus and for the various sister genera in the Bacillus radiation, a novel species is proposed, Geobacillus tepidamans sp. nov., with GS5-97(T) (=ATCC BAA-942(T)=DSM 16325(T)) as the type strain. Strain YNP10 has been deposited in the American Type Culture Collection as ATCC BAA-943.