Search and discovery strategies for biotechnology: the paradigm shift.
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Profound changes are occurring in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. The advances that have been made in the past decade in areas such as combinatorial chemistry, combinatorial biosynthesis, metabolic pathway engineering, gene shuffling, and directed evolution of proteins have caused some companies to consider withdrawing from natural product screening. In this review we examine the paradigm shift from traditional biology to bioinformatics that is revolutionizing exploitable biology. We conclude that the reinvigorated means of detecting novel organisms, novel chemical structures, and novel biocatalytic activities will ensure that natural products will continue to be a primary resource for biotechnology. The paradigm shift has been driven by a convergence of complementary technologies, exemplified by DNA sequencing and amplification, genome sequencing and annotation, proteome analysis, and phenotypic inventorying, resulting in the establishment of huge databases that can be mined in order to generate useful knowledge such as the identity and characterization of organisms and the identity of biotechnology targets. Concurrently there have been major advances in understanding the extent of microbial diversity, how uncultured organisms might be grown, and how expression of the metabolic potential of microorganisms can be maximized. The integration of information from complementary databases presents a significant challenge. Such integration should facilitate answers to complex questions involving sequence, biochemical, physiological, taxonomic, and ecological information of the sort posed in exploitable biology. The paradigm shift which we discuss is not absolute in the sense that it will replace established microbiology; rather, it reinforces our view that innovative microbiology is essential for releasing the potential of microbial diversity for biotechnology penetration throughout industry. Various of these issues are considered with reference to deep-sea microbiology and biotechnology. (+info)
Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.
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Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics. (+info)
The Fas-induced apoptosis analyzed by high throughput proteome analysis.
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The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo. (+info)
Proteomics of Synechocystis sp. strain PCC 6803. Identification of periplasmic proteins in cells grown at low and high salt concentrations.
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Periplasmic proteins isolated by cold osmotic shock of Synechocystis sp. PCC 6803 cells were identified using 2D PAGE, MS and genome analysis. Most of the periplasmic proteins represent 'hypothetical proteins' with unknown function. A number of proteases of different specificity, and several enzymes involved in cell wall biosynthesis were also found. In salt-adapted cells, six proteins were greatly enhanced and three proteins were newly induced. Most of the salt-enhanced proteins are involved in the alteration of cell wall structure of salt-adapted cells. The precursors of all 57 periplasmic proteins identified have a signal peptide; 47 of them contain a typical Sec-dependent signal peptide, whereas 10 contain a putative twin-arginine signal peptide. (+info)
The transcriptional activator Cat8p provides a major contribution to the reprogramming of carbon metabolism during the diauxic shift in Saccharomyces cerevisiae.
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In yeast, the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression and protein synthesis. The zinc cluster protein Cat8p is required for the derepression of nine genes under nonfermentative growth conditions (ACS1, FBP1, ICL1, IDP2, JEN1, MLS1, PCK1, SFC1, and SIP4). To investigate whether the transcriptional control mediated by Cat8p can be extended to other genes and whether this control is the main control for the changes in the synthesis of the respective proteins during the adaptation to growth on ethanol, we analyzed the transcriptome and the proteome of a cat8 Delta strain during the diauxic shift. In this report, we demonstrate that, in addition to the nine genes known as Cat8p-dependent, there are 25 other genes or open reading frames whose expression at the diauxic shift is altered in the absence of Cat8p. For all of the genes characterized here, the Cat8p-dependent control results in a parallel alteration in mRNA and protein synthesis. It appears that the biochemical functions of the proteins encoded by Cat8p-dependent genes are essentially related to the first steps of ethanol utilization, the glyoxylate cycle, and gluconeogenesis. Interestingly, no function involved in the tricarboxylic cycle and the oxidative phosphorylation seems to be controlled by Cat8p. (+info)
The dual origin of the yeast mitochondrial proteome.
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We propose a scheme for the origin of mitochondria based on phylogenetic reconstructions with more than 400 yeast nuclear genes that encode mitochondrial proteins. Half of the yeast mitochondrial proteins have no discernable bacterial homologues, while one-tenth are unequivocally of alpha-proteobacterial origin. These data suggest that the majority of genes encoding yeast mitochondrial proteins are descendants of two different genomic lineages that have evolved in different modes. First, the ancestral free-living alpha-proteobacterium evolved into an endosymbiont of an anaerobic host. Most of the ancestral bacterial genes were lost, but a small fraction of genes supporting bioenergetic and translational processes were retained and eventually transferred to what became the host nuclear genome. In a second, parallel mode, a larger number of novel mitochondrial genes were recruited from the nuclear genome to complement the remaining genes from the bacterial ancestor. These eukaryotic genes, which are primarily involved in transport and regulatory functions, transformed the endosymbiont into an ATP-exporting organelle. (+info)
Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes.
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Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies. (+info)
A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen.
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A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified. (+info)