Starting with the discovery of penicillin, other antibiotics, and insulin, the quest for understanding and use of biological systems, i. e. , microorganisms and ani mal tissue, for the production of value products has lead to a dramatic increase in microbiological and bioengineering research in the last decades. Chemical and pharmaceutical companies quickly realized the huge commercial potential of these bioproducts and have spent millions of US dollars on R &D as well as on a build up of production facilities. Although there was limited knowledge about the cell's molecular mechanisms, which are the basis for the formation of the desired products, products from fermentation and extraction of biological matrices were a success right from the start. R&D projects within industry and academia on the continuous improvement of production processes, especially microbial productivity and down stream processing, allowed a fast return of investment and secured competitiveness in the market. Whereas the focus of such research projects was mainly on the discovery of strains with higher pro ductivity for the product of interest, e. g. , antibiotics, a lot of expertise and knowledge was generated allowing the use of biotechnological products and processes outside the pharmaceutical arena. The tremendous increase in knowl edge and the technological developments in microbial genetics where driven by these research projects and, accompanied with the advancements in nucleotide chemistry leading to a much better understanding of intracellular processes, served as a basis for modern molecular biology and recombinant biotech nology.
Genomics and proteomics technologies have yielded volumes of data for more than 20 years, and they continues to produce data
at an astounding rate. Has all of this data helped us understand more about life, or it is just bogging us down in details
that cannot be assembled into meaningful ideas? This review of the proteomics efforts over the last couple of decades is meant
to emphasize that a new scientific discipline has emerged, Molecular Physiology, and that, indeed, this discipline is contributing
to our understanding of life. Molecular physiology offers the reductionisms details of individual cellular molecules and offers
the systems biology multivariant and high-dimensional datasets of cellular molecules.
The rapid growth of proteomics that has been built upon the available bacterial genome sequences has opened provided new approaches
to the analysis of bacterial functional genomics. In the study of pathogenic bacteria the combined technologies of genomics,
proteomics and bioinformatics has provided valuable tools for the study of complex phenomena determined by the action of multiple
gene sets. The review considers some of the recent developments in the establishment of proteomic databases as well as attempts
to define pathogenic determinants at the level of the proteome for some of the major human pathogens. Proteomics can also
provide practical applications through the identification of immunogenic proteins that may be potential vaccine targets as
well as in extending our understanding of antibiotic action. There is little doubt that proteomics has provided us with new
and valuable information on bacterial pathogens and will continue to be an important source of information in the coming years.
The rapid developments in the field of mass spectrometry have transformed it into a key technology in proteome research.
Increased sensitivity in mass spectrometry, as a result of more efficient ionisation techniques and better detectors, has
allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now quantitatively
sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification a closer
look at posttranslational modifications is now also possible. It is speculated that modifications like phosphorylation or
glycosylation exist on every second protein and that they are important for the protein function.
This review highlights the different mass spectrometric methods and gives a brief overview of strategies and methods used
to identify modifications.
... Porins are pore-forming proteins which are considered important for transport, both into and out of cells. General diffusion pores formed by porins allow the diffusion of hydrophilic molecules (,600 Da) without much substrate specificity [20]. Alkanes, despite their hydrophobic character also seem to have an effect on outer membrane porins which are usually known to form channels that enable the passage of hydrophilic molecules such as phenol [8,21], chlorophenol and antibiotics [22]. ...
Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log Pow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log Pow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94-8.67 and the molecular weights were in the range of 19.5-104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure.
... Porins are pore-forming proteins which are considered important for transport, both into and out of cells. General diffusion pores formed by porins allow the diffusion of hydrophilic molecules (,600 Da) without much substrate specificity [20]. Alkanes, despite their hydrophobic character also seem to have an effect on outer membrane porins which are usually known to form channels that enable the passage of hydrophilic molecules such as phenol [8,21], chlorophenol and antibiotics [22]. ...
Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log P ow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log P ow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94–8.67 and the molecular weights were in the range of 19.5–104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure.
... To provide such information, a new multi-parameter fluorescence-microscopy technique called MELK 1 was developed at Magdeburg University by WS and his coworkers; it is discussed in [Schubert W. et al., 2006] (see also [Dress A. et al., 2004;Schubert W., 2003Schubert W., , 2002Schubert W., , 2007aFriedenberger M. et al., 2007]). Details regarding this technology and the tasks associated with the data obtained by using it are discussed in [Cottingham K., 2008;Sage L., 2009]; for biological applications of MELK Technology, see [Schubert W. et al., 2009[Schubert W. et al., , 2008bBode M. et al., 2008;Schubert W., 2007b]. ...
In this note, we present a new method that allows us to determine threshold values for separating presence and absence of proteins in a stack of fluorescence images describing a spatial distribution of proteins across a biological object (like a slice of nervous tissue, a sample of blood cells, etc.). We apply this method to stacks of fluorescence images and find that the resulting threshold values are almost identical with threshold values found using completely independent methods based on technological and biological aspects of the images in question.
This dynamic proteome study describes the physiology of growth and survival of Deinococcus geothermalis, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. The industrial environment of this species differs from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. Quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins, from which 47 were assigned to a function. We propose that D. geothermalis grew and survived in aerobic conditions by channeling central carbon metabolism to pathways where mainly NADPH rather than NADH was retrieved from the carbon source. A major part of the carbon substrate was converted into succinate, which was not a fermentation product but likely served combating reactive oxygen species (ROS). Transition from growth to nongrowth resulted in downregulation of the oxidative phosphorylation observed as reduced expression of V-type ATPase responsible for ATP synthesis in D. geothermalis. The battle against oxidative stress was seen as upregulation of superoxide dismutase (Mn dependent) and catalase, as well as several protein repair enzymes, including FeS cluster assembly proteins of the iron-sulfur cluster assembly protein system, peptidylprolyl isomerase, and chaperones. Addition of soluble Mn reinitiated respiration and proliferation with concomitant acidification, indicating that aerobic metabolism was restricted by access to manganese. We conclude that D. geothermalis prefers to combat ROS using manganese-dependent enzymes, but when manganese is not available central carbon metabolism is used to produce ROS neutralizing metabolites at the expense of high utilization of carbon substrate.
With the genome sequences of several organisms now in public databases, the scientific community has realized that it is time to prepare for the next step: the understanding of biological systems or systems biology. Whereas genes contain the information for life, the encoded proteins and RNAs fulfill nearly all the functions, from replication to regulation. At present, there is a perceived demand for high-throughput and parallel analytical devices as research tools in systems biology, and, in addition, for new concepts to extract knowledge and value from these data. Protein biochips will play a decisive role in meeting this need in the future.
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