Browsing by Author "Albrecht, Dirk"

Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σ(B) regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction.

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species. Copyright (c) 2008 S. Karger AG, Basel

Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec-and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Staphylococcus aureus is both a prominent cause of nosocomial infections with significant morbidity and mortality and a commensal with nasal carriage in around 30% of the population. The rapid spread of multi-resistant strains necessitates novel therapeutic strategies, a challenging task because the species S. aureus and the host response against it are highly variable. In a prospective study among 2023 surgical and non-surgical patients, 12 patients developed S. aureus bacteremia. They were analysed in detail using a personalized approach. For each patient, the extracellular proteins of the infecting S. aureus strain were identified and the developing antibody response was assessed on 2-D immunoblots. S. aureus carriers showed clear evidence of strain-specific pre-immunization. In all immune-competent bacteremia patients, antibody binding increased strongly, in most cases already at diagnosis. In endogenous infections, the pattern of antibody binding was similar to the pre-infection pattern. In exogenous infections, in contrast, the pre-infection pattern was radically altered with the acquisition of new specificities. These were characteristic for individual patients. Nevertheless, a common signature of 11 conserved S. aureus proteins, recognized in at least half of the bacteremic patients, was identified. All patients mounted a dynamic antibody response to a subset of these proteins.

Background: Industrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided. Results: Whole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have been pointed out as potential optimization targets. Conclusions: The presented data give unprecedented insights into the complex adaptations of bacterial production strains to the changing physiological demands during an industrial-oriented fermentation. These are, to our knowledge, the first publicly available data that document quantifiable transcriptional responses of the commonly employed production strain B. licheniformis to changing conditions over the course of a typical fermentation process in such extensive depth.

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments. The ISME Journal (2012) 6, 766-776; doi: 10.1038/ismej.2011.137; published online 20 October 2011

The thermoacidophilic archaeon Picrophilus torridus belongs to the Thermoplasmatales order and is the most acidophilic organism known to date, growing under extremely acidic conditions around pH 0 (pH(opt) 1) and simultaneously at high temperatures up to 65 degrees C. Some genome features that may be responsible for survival under these harsh conditions have been concluded from the analysis of its 1.55 megabase genome sequence. A proteomic map was generated for P. torridus cells grown to the mid-exponential phase. The soluble fraction of the cells was separated by isoelectric focusing in the pH ranges 4-7 and 3-10, followed by a two dimension (2D) on SDS-PAGE gels. A total of 717 Coomassie collodial-stained protein spots from both pH ranges (pH 4-7 and 3-10) were excised and subjected to LC-MS/MS, leading to the identification of 665 soluble protein spots. Most of the enzymes of the central carbon metabolism were identified on the 2D gels, corroborating biochemically the metabolic pathways predicted from the P. torridus genome sequence. The 2D master gels elaborated in this study represent useful tools for physiological studies of this thermoacidophilic organism. Based on quantitative 2D gel electrophoresis, a proteome study was performed to find pH-or temperature-dependent differences in the proteome composition under changing growth conditions. The proteome expression patterns at two different temperatures (50 and 70 degrees C) and two different pH conditions (pH 0.5 and 1.8) were compared. Several proteins were up-regulated under most stress stimuli tested, pointing to general roles in coping with stress.

Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS.

The glucose and nitrogen starvation stimulons of Bacillus licheniformis were determined by transcriptome and proteome analyses. Under both starvation conditions, the main response of B. licheniformis was a switch to the usage of alternative nutrient sources. This was indicated by an induction of genes involved in the metabolism of C-2 substrates during glucose limitation. 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 observation is supported by the induction of a high number of genes coding for proteins involved in amino acid and lipid degradation. During nitrogen starvation, genes for several proteases and peptidases involved in nitrate and nitrite assimilation were induced, which enables this bacterium to recruit nitrogen from alternative sources. Both starvation conditions led to a down-regulation of transcription of most vegetative genes, which was subsequently reflected by a reduced synthesis of the corresponding proteins. A selected set of genes was induced by both starvation conditions. Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response.