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The present work consists of four parts, containing experimental data obtained from analysis of 'Bacillus subtilis' specific and general defense strategies against reactive oxygen species. In the first part, the peroxide and superoxide stress stimulons ob 'B. subtilis' were analyzed by means of transcriptomics and proteomics. Oxidative stress responsive genes were classified into two groups: the gene expression pattern was either similar after both stresses or the genes primarily responded to one stimulus. The high induction observed for members of the PerR-regulon after both stimuli supported the assumption that activation of the peroxide specific PerR-regulon represented the primary stress response after superoxide and peroxide stress. The second part focuses on protein carbonylation in 'B. subtilis' wild-type and 'sigB' mutant cells. The introduction of carbonyl groups into amino acid side chains of proteins represents one possible form of protein modification after attack by reactive oxygen species. Carbonyl groups are readily detectable and the observed amounts can thus serve as an indicator for the severity of protein damage. The resultsdemonstrate clearly that 'B. subtilis' proteins are susceptible to hydrogen peroxide (H2O2) mediated carbonylation damage. The application of low concentrations of H2O2 prior to the exposure to otherwise lethal levels of peroxide reduced markedly the degree of protein carbonylation, which also held true for glucose starved cells. Artificial preloading with general stress proteins resulted in a lower level of protein carbonylation when cells were subjected to oxidative stress, but no differences were detected between wild-type and 'sigB' mutant cells. In the third part, strains with mutations in genes encoding general stress proteins were screenedfor decreased resistance after H2O2 challenge. It was demonstrated that resistance to H2O2 challenge. It was demonstrated that resistance to H2O2 after transient heat treatment, likewise to conditions of glucose starvation, was at least partly mediated by the sB-dependent general stress response. The screening of mutants in sB-controlled genes revealed an important role for the deoxyribonucleic acid (DNA)-binding protein Dps in the context of sB-mediated resistance to oxidative stress underlining previous reports. Therefore, the experimental strategy opens a global view on the importance of DNA integrity in 'B. subtilis' under conditions of oxidative stress. The fourth part includes analysis of a 'B. subtilis' thioredoxin conditional mutant. The thiol-disulfide oxidoreductase TrxA is an essential protein in 'B. subtilis' that is suggested to be involved in maintaining the cytoplasmic thiol-disulfide state even under conditions of oxidative stress. To investigate the physiological role of TrxA, growth experiments and two-dimensional gel electrophoresis were carried out with exponentially growing cells that were depleted of TrxA. The observations indicate that TrxA essentially involved in the re-reduction of phosphoadenosyl phosphosulfate reductase CysH within the sulfate assimilation pathway of 'B. subtilis'.
The toluene-degrading and solvent-tolerant strain Pseudomonas putida DOT-T1E was investigated with respect to its suitability and economic efficiency as biocatalyst in aqueous-organic two-phase systems with aliphatic solvents as organic phase (Rojas et al. 2004, chapter 4 and 5) and to its adaptive responses to the solvent decanol. The adaptive changes on the level of cell morphology (chapter 2), membrane fatty acids and permeability (chapter 3), as well as energetics and surface properties (chapter 5) of P. putida DOT-T1E have been investigated in order to ascertain information about the strain's suitability for two-phase biotransformation systems (chapter 4). The morphological adaptation to the presence of solvents was observable in changes of the cell size of P. putida DOT-T1E. Those changes were dependent on the cellular activity and occurred only after addition of non-lethal solvent concentrations. The cells reacted to the presence of organic solvents by decreasing the ratio between surface and volume of the cells and therefore reducing their relative surfaces (chapter 2). The cell surface and especially the cytoplasmic membrane are the major targets for toxic effects of membrane-active compounds like solvents. The mechanism of the cis-trans isomerisation of unsaturated fatty acids counteracts the fluidizing effect of solvents by increase the ordering of the membrane and therefore its rigidity. By comparing the responses of the cells to a series of stress factors (like solvents), a direct correlation between the activation of this mechanism and the well investigated K+-uptake pumps was observed (chapter 3). Huertas et al. (1998) reported that this strain tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10 % (vol/vol). 1-decanol is, in comparison to toluene, less hazardous and volatile, and it possesses good extraction properties for the desired fine chemical products. In further investigations of possible biotechnological processes, it was discovered that decanol is also a more suitable solvent as organic phase (chapter 4). Although the cells of P. putida DOT-T1E needed additional energy for their adaptation to the presence of the solvent decanol, they were able to maintain or activate their electron transport phosphorylation allowing homeostasis of ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities converging with more negative cell surface charges were observed in cells grown in the presence of 1-decanol (chapter 5). It is however important to note that all the cell’s properties observed are closely linked to each other since they are all part of the adaptive response of the cells. It can be concluded that the easy adaptability and good growth properties of Pseudomonas putida DOT-T1E in the presence of the organic solvent 1-decanol make this system an excellent candidate for two-phase fermentation processes. Moreover, the absence of differences in the energetics of the bacteria during exposure to 1-decanol as compared to bacteria that grew in the absence of 1-decanol, support that this organism can be used for the industrial production of fine chemicals in an economically sound manner.
Proteomic signatures select the physiology state of the cell. By using 2-D technique, proteome signature of Bacillus subtilis under different stresses and starvations are analyzed. Consequently, a proteomic map of Bacillus subtilis in non-growing phase was created. The ammonium and tryptophan as well as phenol and catechol stress are analyzed using both of proteomics and transcriptomics. And the proteomic map represents a good application in the prediction of the mode of action of phenol and catechol stress.
The Gram-positive bacterium Bacillus licheniformis is an important industrial host for the production of enzymes. Genomic DNA arrays and proteomics are being used to investigate the physiology of this bacterium. A genome-wide transcriptional profiling analysis of the adaptation of B. licheniformis to phosphate starvation shows more than 100 induced genes. Most of strongly induced genes belong to the putative Pho regulon. The data of the transcriptome analysis have been verified by the analysis of the extracellular and cytoplasmic proteome. The main response of B. licheniformis to glucose starvation was a switch to the usage of alternative carbon sources. In addition, B. licheniformis seems to be using other organic substances like amino acids and lipids as carbon sources when subjected to glucose starvation. This was indicated by the induction of a high number of genes the proteins of which are involved in amino acid and lipid degradation. During nitrogen starvation genes necessary for the recruitment of nitrogen from alternative sources were induced, e.g. genes for nitrate and nitrite assimilation, several proteases and peptidases. Both starvation conditions led to a down-regulation of the transcription of most vegetative genes and subsequently to a reduced synthesis of the corresponding proteins. Only a few genes were induced by both starvation conditions like yvyD, citA and the methylcitrate shunt genes mmgD, mmgE and yqiQ. Data of this study use to better understand the physiology of this bacterium during fermentation processes and thus to identify and circumvent bottlenecks of B. licheniformis based bioprocesses. In addition, the phytase promoter was tested for the construction of an alternative phosphate regulated expression system for B. licheniformis.
Degradation of branched chain aliphatic and aromatic petroleum hydrocarbons by microorganisms
(2008)
The overall aim of the work was to investigate the ability of several Gram-positive bacteria including Mycocbacterium neoaurum SBUG 109, Nocardia cyriacigeorgica SBUG 1472 and Rhodococcus ruber SBUG 82 and the yeast Trichosporon mucoides SBUG-Y 801 to degrade and transform branched chain hydrocarbons which occur in petroleum and its fraction products such as gasoline or gas oil and which are known as important and recalcitrant environmental pollutants. Pristane, iso-pentylbenzene and sec-octylbenzene were used in this work as model compounds. These compounds represent significant groups of petroleum constituents (branched chain alkanes and aromatic hydrocarbons). Three bacteria and the yeast T. mucoides SBUG-Y 801 were selected in a screen of 16 hydrocarbon-utilizing strains in the SBUG collection and from 21 isolated hydrocarbon-utilizing strains from oil-contaminated habitats of Saudi Arabian Desert and of Vietnam. The bacteria were identified in cooperation with DSZM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) as M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472, R. ruber SBUG 82. These bacterial and yeast strains were shown to possess high potential for degrading and transforming pristane, iso-pentylbenzene and sec-octylbenzene. The intermediates produced by these bacteria during incubation with pristane were analyzed by GC and GC/MS. The products 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid and 2,6,10,14–tetramethyl-pentadecan–3–one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10–trimethylundecanoic acid; 3,7-dimethyl decanedioic acid and 2,6,10,14–tetramethyl–pentadecan–3-one were identified. In N. cyriacigeorgica, 2-methylpentanedioic acid; 4,8-dimethylnonanedioic acid; 2,6-dimethylheptanedioic acid and pristanic acid were found. The detection of 11 intermediates during pristane degradation by the three Gram-positive bacteria provided sufficient information to elucidate in detail three degradative pathways of pristane involving mono-, di- and sub-terminal oxidations. The sub-terminal oxidation by M. neoaurum and R. ruber was demonstrated for the first time. This occurence of a sub-terminal oxidation in these strains was strengthened by further results of aromatic compounds transformation (see below). During this pathway, ketone mono-oxygenation reactions seem to be involved. Because of this it will be of interest to look more closely at the catalytic processes involved and their possible extension to the bio-degradation of other branched chain hydrocarbons. Since in the present study 59 %, 51 % and 84 % of pristane were degraded in 3 weeks by M. neoaurum, R. ruber and N. cyriacigeorgica, this illustrated that the degradation rates of this isoprenoid alkane were high. The bacteria we studied were not only effective degraders of multiple branched chain alkane but also useful transformers of aromatic hydrocarbons. The intermediates produced were analyzed by comparing the retention times and UV/Vis spectra of the HPLC elution profile as well as the retention times and mass spectra of the GC/MS with those of available standards. Using iso-pentylbenzene as a substrate, 8 metabolites were generated by M. neoaurum transformation including product A (phenylacetic acid), B (acetophenone), D (iso-valerophenone), E (succinic acid), F (benzoic acid), G [(2-hydroxy-phenyl)-acetic acid] and H (2-methyl-4-phenyl-butyric acid). We additionally identified an alkyl hydroxylated iso-pentylbenzene derivative as 2-methyl-4-phenyl-butan-2-ol or 2-methyl-4-phenyl-butan-1-ol. Two metabolites (C and D) were detected by N. cyriacigeorgica transformation and three metabolites (A, D and F) were identified by R. ruber transformation which led to the complete biotransformation of this substance. iso-Pentylbenzene transformation by M. neoaurum was initiated by attack on the alkyl side chain followed by ring cleavage. The appearance of iso-valeorophenone confirmed the occurrence of a sub-terminal oxidation mechanism in M. neoaurum and R. ruber. In addition to products A, C, D and G, the identification X-(3–methyl–butyl)-phenol (X means that position of the hydroxy group on the aromatic ring system, such as 2, 3 or 4 remained unclear) in T. mucoides cultivation demonstrated for the first time the capacity of alkyl side chain attack by this organism which was hitherto known only for its ability of ring cleavage. The detection of 15 degradation products of sec-octylbenzene (including 2-phenylpropionic acid, 3-phenylbutyric acid, ß-methylcinnamic acid, 5-phenylhexanoic acid, acetophenone, 2-hydroxy-acetophenone, 2,3-dihydroxy-benzoic acid, succinic acid, 7-phenyloctan-2-one, benzoic acid, phenylacetic acid, 7-phenyl-octan-2-ol, hydroxy-phenylacetic acid and 2-hydroxybenzoic acid), in the studied bacteria pointed to an effective sec-octylbenzene degradation pathway in which dehydrogenation of 3-phenylbutyric acid to form ß-methylcinnamic acid is a newly described option. The identification of 2-phenylpropionic acid and 3-phenylbutyric acid in sec-octylbenzene transformation experiments by T. mucoides confirmed the possibility of alkyl side chain attack by this yeast. Summarizing the results, we describe for the first time in detail the biotransformation of sec-octylbenzene by M. neoaurum, N. cyriacigeorgica, R. ruber and T. mucoides. Our results suggest that these microorganisms may be useful as potential strains for hydrocarbon degradation and it may be of interest to investigate their suitability to solve specific environmental pollutant problems associated with branched chain aliphatic and alkyl-branched compounds which contribute to the persistence of hydrocarbon fractions in the environment.
Understanding of the regulatory mechanisms controlling stress gene expression of S.aureus in response to environmental stress is very essential in studying its fitness and virulence. In this work, the changes in protein expression profiles as well as the gene transcription of S.aureus after heat exposure, osmotic stress and in response to the antibiotic puromycin were studied in order to provide detailed insights into the response of S.aureus to various kinds of environmental stress under in vitro conditions, namely: (1) to investigate the global response of S.aureus to heat stress conditions using transcriptomic and proteomic analyses. (2) to study the transcriptome and proteome of S.aureus in response to antibiotic substance puromycin. (3) to define the proteome signatures of S.aureus under NaCl stress condition. (4) to complete the proteome map of cytoplasmic proteins of S.aureus by identifying proteins exclusively synthesized during the exposure to stress. Firstly, the high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF-MS and a DNA array approach were used to investigate the cellular response of S.aureus to heat stress. A switch from normal growth temperature to high temperature condition revealed complex changes in the protein expression pattern as well as the genes expression profile. The effect of puromycin stress on S.aureus cells was analyzed, using a gel-based proteomic approach and transcriptomic analyses with DNA microarrays. We compared the protein synthesis pattern as well as the transcription data of S.aureus in response to puromycin stress with that in response to heat shock. The results demonstrated that both stress conditions induced specific, overlapping and general responses. Finally, the protein expression profile of S.aureus in response to NaCl stress was analyzed with 2D gel based proteomic approach. Our proteome analyses revealed the repression of the synthesis of many enzymes belong to different metabolism pathways . In summary, the signatures for stress or starvation stimuli can be used as diagnostic tools for the prediction of the mode of action of new antibiotics or for studying the physiological state of cells grown. Expression of the respective genes under in vivo conditions could provide some ideas on the environmental signals that specifically influence the survival of S.aureus within and outside the host.
The introduction of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) enabled the separation and visualization of a substantial fraction of an organism’s entire proteome, and when mass spectrometry entered protein science, these proteins became even amenable to identification on a grand scale. Nevertheless, important classes of proteins elude a separation on classical 2 D gels, as the ones showing extremes in isoelectric point or molecular weight, and foremost very hydrophobic proteins naturally embedded in lipid membranes. This thesis aimed at the establishment and adaptation of alternatives to 2-D PAGE. New techniques allowing for an identification and quantification of critical protein classes were designed and adopted to physiological questions in the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. In a comprehensive study on cytoplasmic proteins of S. aureus COL the number of proteins identified by a 2-D gel based approach could be extended by 650 proteins employing gel free technologies. Application of these complementary methods resulted in the establishment of a comprehensive reference map of the cytosolic proteome in growing and non-growing S. aureus cells which can serve as basis for further physiological investigations. Gel free separation of complex protein digests was likewise used in a quantitative study on heat stress in B. subtilis. By implementation of the iTRAQ® technology four different physiological states could be relatively quantified in one experiment. A parallel generation of 2-D gel based data enabled the depiction of strengths and weaknesses of protein quantitation by both, spot intensities on 2-D gels and iTRAQ® signal intensities in MS/MS spectra. Furthermore, new insights into heat sensitivity of pivotal enzymes involved in amino acid biosynthesis could be delivered. The institution of gel free approaches and advancements in 2-D PAGE provide the tools to penetrate into yet unamenable scopes of proteomes. A review on proteome coverage in B. subtilis gives an overview on the strategies which have been explored for most comprehensive protein identification in various sub-proteomes. Although more than one third of B. subtilis’ open reading frames could be demonstrated on protein level, one has to be aware of the fact that it still is a long way to achieve complete coverage of its proteome. Integral membrane proteins make up about one quarter of the entirety of proteins in a cell. Despite their large portion they are clearly understudied due to the intricacy of identification. Their low abundance and non-accessibility of membrane-spanning domains represent major experimental difficulties. The establishment of a protocol efficiently depleting cytosolic proteins by membrane shaving and targeting trans-membrane peptides by novel digestion strategies essentially facilitated identification of highly hydrophobic integral membrane proteins. This protocol was not only successfully applied to the membrane proteome of growing S. aureus cells, but was shown to be applicable in B. subtilis as well. Both studies displayed the novel membrane shaving approach to be highly complementary to a previously established separation of membrane proteins via 1 D PAGE. A combination of the two techniques resulted in identification of about half of the theoretical membrane proteome in both bacteria, and hence layed the foundation for advanced and quantitative analyses. In this regard, 14N/15N metabolically labeled membrane samples of growing and non-growing cells of S. aureus COL were relatively quantified revealing a significant difference in amount for more than one third of the proteins. A corresponding experimental setup was used to compare the membrane proteomes of S. aureus SA113 and its mutant deficient in the lysylphosphatidylglycerol synthetase MprF. Interesting quantitative differences were obtained for proteins most likely involved in the regulation of cellular surface net charge as well as for virulence-associated proteins.
Deciphering the entire protein complement of a living cell together with the elucidation of dynamic processes on protein level are the main goals of proteomics as it is used today. To achieve this goal, namely the elucidation of dynamic processes of the entire bacterial cell, we have developed strategies and distinct workflows to cover the most proteins in different subcellular localizations in bacteria together with a stable isotopes labeling approach to follow temporal and spatial changes in different proteomic subfractions. In this work, it has been shown that the use of mass spectrometry based in vivo quantitation techniques and the application of subcellular and chromatographic fractionation has lead to a new level of qualitative and quantitative proteomics data. Emphasizing on the studies revealing the dynamics of the bacterial physiology on a time resolved base, both spatial and temporal processes can be monitored to obtain knowledge on physiological processes in a depth that has not been reached before in comparable global studies.
Protein quality control systems are essential for the viability and growth of all living organisms. They protect the cell from irreversible protein aggregation. Because the frequency of protein misfolding, which ultimately results in protein aggregation, varies with the environmental conditions, the amount and activity of protein quality systems have to be accurately adapted to the rate of protein misfolding. The main goal of this thesis was to gain detailed molecular insights into the transcriptional and post-translational regulation of these protein quality control networks in the ecologically, medically and industrially important phylum of low GC, Gram-positive bacteria. In these bacteria the core protein quality control systems are under the transcriptional control of the global repressor CtsR. In a first study it was demonstrated that the arginine kinase McsB is not responsible for the regulation of CtsR activity during heat stress, as was concluded by others on the basis of previous in vitro data. Rather, it was demonstrated that CtsR acts as an intrinsic thermosensor that adapts its activity to the surrounding temperature. CtsR displays a decreased DNA binding at higher temperatures, which leads to induction of transcription of the protein quality control systems under these conditions. This CtsR feature is conserved in all low GC, Gram-positive bacteria. However, the CtsR proteins of various low GC, Gram-positive species do not have the same temperature optima. CtsR responds to heat in a species-specific manner according to their corresponding growth temperature. Detailed analysis revealed that a highly conserved tetra-glycine loop within the winged helix-turn-helix domain of CtsR is responsible for thermosensing. Dual control of CtsR activity during different stresses was demonstrated for the first time in this work. In addition to heat-dependent de-repression, CtsR is inactivated by thiol-specific stress conditions. This latter de-repression depends on a molecular redox-switch that is independent of CtsR auto-regulation. In Bacillus subtilis and its closest relatives the McsA/McsB stress-sensing complex is responsible for CtsR de-repression during redox stress conditions. McsA is able to sense the redox state of the cell via its highly conserved cysteine residues. When these cysteines are reduced, McsA is able to bind and inhibit McsB. But when these cysteine residues are oxidized, McsB is released from McsA. Thereby, McsB is activated and removes CtsR from the DNA. However, the McsA/McsB complex is not present in all low GC, Gram-positive bacteria. In the species lacking this complex, ClpE is able to act as a redox-sensor probably via its highly conserved N-terminal zinc finger domain. When these cysteine residues are oxidized, ClpE is activated which results in CtsR de-repression. In addition to the transcriptional regulation of CtsR low GC, Gram-positive protein quality control systems are regulated post-transcriptionally. The expression of the McsA/McsB adaptor pair is regulated by CtsR. However, McsB activity is also tightly regulated by three different regulatory proteins (McsA/ClpC/YwlE). McsB is needed to target specific substrates to ClpC, either for refolding or degradation by the ClpCP protease. It was demonstrated that only the auto- phosphorylated form of McsB is able to bind to its substrates. This McsB function is inhibited in non-stressed cells by a direct interaction with ClpC. Consequently, McsB is activated by a release from ClpC during protein stress. In addition, McsB activation depends on the presence of its activator McsA. Accordingly, McsB cannot be activated as an adaptor protein during thiol-specific stress because McsA is no longer able to bind to McsB under these conditions. However, also active McsB is subject to post-translational control. Activated McsB is either de-phosphorylated by McaP or degraded by ClpCP ensuring an appropriate shut-down of the McsB adaptor. Both McaP and ClpC inhibit McsB activity with different intensities. ClpC possesses a stronger impact on McsB activity than McaP but both proteins are needed for an adequate silencing of McsB activity. In addition, it was shown for the first time that B. subtilis McsB is a global adaptor that influences the stability of multiple proteins. The B. subtilis ClpC protein is unlike most members of the Hsp100 family because it not only requires several adaptor proteins for substrate recognition but also for its general ATP- dependent activity. Biochemical analysis revealed how ClpC is activated by distinct adaptor proteins. McsB modulates ClpC activity by regulatory phosphorylation of arginine residues. Moreover, McaP (formerly YwlE) was identified as an arginine phosphatase that modulates the McsB mediated ClpC activity. MecA, another known adaptor protein for ClpC, activates ClpC independently of these arginine phosphorylations, which demonstrates the existence of multiple pathways for ClpC activation.