Proteomic analysis of carbohydrate catabolism and regulation in the marine bacterium Rhodopirellula baltica

The marine bacterium Rhodopirellula baltica is a model organism for aerobic carbohydrate degradation in marine systems, where polysaccharides represent the dominant components of biomass. On the basis of the genome sequence and a 2-D map of soluble proteins, the central catabolic routes of R. baltica were reconstructed. Almost all enzymes of glycolysis and TCA cycle were identified. In addition, almost all enzymes of the oxidative branch of the pentose phosphate cycle were detected. This proteomic reconstruction was corroborated by determination of selected enzymatic activities. To study substrate-dependent regulation in R. baltica, cells were adapted to growth with eight different carbohydrates and profiled with 2-DE for changes in protein patterns. Relative abundances of regulated proteins were determined using the 2-D DIGE technology and protein identification was achieved by PMF. Most of the up-regulated proteins were either dehydrogenases/oxidoreductases or proteins of unknown function which are unique for R. baltica. For only some of the regulated proteins, the coding genes are located in a physiologically meaningful genomic context. e.g., a ribose-induced alcohol dehydrogenase is encoded within an operon-like structure together with genes coding for a ribose-specific ABC-transporter. However, most of the regulated genes are randomly distributed across the genome.     

Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein,the initiator of chromosomal DNA replication

DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s).     

PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells

PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Dynamic proteome changes of Shigella flexneri 2a during transition from exponential growth to stationary phase

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.     

Towards the proteome of the marine bacterium Rhodopirellula baltica: mapping the soluble proteins

The marine bacterium Rhodopirellula baltica, a member of the phylum Planctomycetes, has distinct morphological properties and contributes to remineralization of biomass in the natural environment. On the basis of its recently determined complete genome we investigated its proteome by 2-DE and established a reference 2-DE gel for the soluble protein fraction. Approximately 1000 protein spots were excised from a colloidal Coomassie-stained gel (pH 4-7), analyzed by MALDI-MS and identified by PMF. The non-redundant data set contained 626 distinct protein spots, corresponding to 558 different genes. The identified proteins were classified into role categories according to their predicted functions. The experimentally determined and the theoretically predicted proteomes were compared. Proteins, which were most abundant in 2-DE gels and the coding genes of which were also predicted to be highly expressed, could be linked mainly to housekeeping functions in glycolysis, tricarboxic acid cycle, amino acid biosynthesis, protein quality control and translation. Absence of predictable signal peptides indicated a localization of these proteins in the intracellular compartment, the pirellulosome. Among the identified proteins, 146 contained a predicted signal peptide suggesting their translocation. Some proteins were detected in more than one spot on the gel, indicating post-translational modification. In addition to identifying proteins present in the published sequence database for R. baltica, an alternative approach was used, in which the mass spectrometric data was searched against a maximal ORF set, allowing the identification of four previously unpredicted ORFs. The 2-DE reference map presented here will serve as framework for further experiments to study differential gene expression of R. baltica in response to external stimuli or cellular development and compartmentalization.     

Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells

Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/10⁶ cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.     

A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.     

Ralstonia eutropha H16 flagellation changes according to nutrient supply and state of poly(3-hydroxybutyrate) accumulation

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella.     

Proteomic analysis of lactose-starved Lactobacillus casei during stationary growth phase

Aims:  Starvation stress is a condition that nonstarter lactic acid bacteria (NSLAB) normally encounter. This study was aimed to investigate starvation-induced proteins in Lactobacillus casei during stationary growth phase.
Methods and Results:  The impact of carbohydrate starvation on L. casei GCRL163 was investigated using two different media (a modified de Man, Rogosa and Sharpe broth and a semi-defined medium). Cells were grown in the presence of excess lactose (1%) or starvation (0%) and differences in the patterns of one-dimensional sodum dodecyl sulfate–polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cytosolic protein fractions were investigated. Differentially regulated proteins were identified by MALDI-TOF/TOF mass spectrometry. Many differentially regulated proteins were enzymes of various metabolic pathways involved in carbohydrate metabolism to yield energy. Differences in protein expression were also observed in the two culture conditions tested in this experiment.
Conclusion:  Numerous glycolytic enzymes were differentially regulated under lactose starvation. The differential expression of these glycolytic enzymes suggests a potential survival strategy under harsh growth conditions (i.e. lactose starvation).
Significance and Impact of the Study:  This paper reports improved understanding of stress responses and survival mechanism of NSLAB under lactose-depleted cheese-ripening condition. This knowledge of how NSLAB bacteria adapt to lactose starvation could be applied to predict the performances of bacteria in other industrial applications.     

Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts

The activities of 6 folate enzymes were measured in extracts of human diploid skin fibroblasts during the lag, log and stationary phases of the culture cycle. The levels of 4 folate enzymes involved in nucleic acid biosynthesis, $\text{viz.}$, folate reductase, serine hydroxymethyltransferase, thymidylate synthetase and 10-formyl-THF synthetase, increased from 2–20 fold during the log phase of growth. In contrast, the levels of 2 enzymes, $\text{viz.}$, methylene-THF reductase and 5-methyl-THF: homocysteine methyltransferase, involved in regulating the levels of 5-methyl-THF, the major tissue and serum folate compound, decreased 3–4 fold during log growth, returning to high levels again only after the cells had been in the stationary phase for 5 and 20 days respectively. This reciprocal pattern of change is consistent with the known or postulated functions of these folate enzymes.     

Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare

Background

Clostridium thermocellum produces H₂ and ethanol, as well as CO₂, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.

Results

Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.

Conclusions

Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H₂ synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.     

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two‐dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5‐fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule‐bound starch synthase 1, Os03g0842900 (putative steroleosin‐B), N‐carbamoylputrescine amidase, spermidine synthase 1, tubulin α‐1 chain and glutelin type‐A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.