Characterizing the flavodoxin landscape in Clostridioides difficile.

Clostridioides difficile infections have become a major challenge in medical facilities. The bacterium is capable of spore formation allowing the survival of antibiotic treatment. Therefore, research on the physiology of C. difficile is important for the development of alternative treatment strategies. In this study, we investigated eight putative flavodoxins of C. difficile 630. Flavodoxins are small electron transfer proteins of specifically low potential. The unusually high number of flavodoxins in C. difficile suggests that they are expressed under different conditions. We determined high transcription levels for several flavodoxins during the exponential growth phase, especially for floX. Since flavodoxins are capable of replacing ferredoxins under iron deficiency conditions in other bacteria, we also examined their expression in C. difficile under low iron and no iron levels. In particular, the amount of fldX increased with decreasing iron concentration and thus could possibly replace ferredoxins. Moreover, we demonstrated that fldX is increasingly expressed under different oxidative stress conditions and thus may play an important role in the oxidative stress response. While increased fldX expression was detectable at both RNA and protein level, CD2825 showed increased expression only at mRNA level under H2O2 stress with sufficient iron availability and may indicate hydroxyl radical-dependent transcription. Although the exact function of the individual flavodoxins in C. difficile needs to be further investigated, the present study shows that flavodoxins could play an important role in several physiological processes and under infection-relevant conditions. IMPORTANCE: The gram-positive, anaerobic, and spore-forming bacterium Clostridioides difficile has become a vast problem in human health care facilities. The antibiotic-associated infection with this intestinal pathogen causes serious and recurrent inflammation of the intestinal epithelium, in many cases with a severe course. To come up with novel targeted therapies against C. difficile infections, a more detailed knowledge on the pathogen's physiology is mandatory. Eight putative flavodoxins, an extraordinarily high copy number of this type of small electron transfer proteins, are annotated for C. difficile. Flavodoxins are known to be essential electron carriers in other bacteria, for instance, during infection-relevant conditions such as iron limitation and oxidative stress. This work is a first and comprehensive overview on characteristics and expression profiles of the putative flavodoxins in the pathogen C. difficile.

From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins.

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis. IMPORTANCE: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

Revealing the small proteome of Haloferax volcanii by combining ribosome profiling and small-protein optimized mass spectrometry.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.

The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection.

Clostridioides difficile infections (CDIs) remain a healthcare problem due to high rates of relapsing/recurrent CDIs (rCDIs). Breakdown of colonization resistance promoted by broad-spectrum antibiotics and the persistence of spores contribute to rCDI. Here, we demonstrate antimicrobial activity of the natural product class of chlorotonils against C. difficile. In contrast to vancomycin, chlorotonil A (ChA) efficiently inhibits disease and prevents rCDI in mice. Notably, ChA affects the murine and porcine microbiota to a lesser extent than vancomycin, largely preserving microbiota composition and minimally impacting the intestinal metabolome. Correspondingly, ChA treatment does not break colonization resistance against C. difficile and is linked to faster recovery of the microbiota after CDI. Additionally, ChA accumulates in the spore and inhibits outgrowth of C. difficile spores, thus potentially contributing to lower rates of rCDI. We conclude that chlorotonils have unique antimicrobial properties targeting critical steps in the infection cycle of C. difficile.

CdrS Is a Global Transcriptional Regulator Influencing Cell Division in Haloferax volcanii.

Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPR interference (CRISPRi)-mediated repression of the cdrS gene caused slow growth and cell division defects and changed the expression of multiple genes and their products associated with cell division, protein degradation, and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) identified 18 DNA-binding sites of the CdrS protein, including one upstream of the promoter for a cell division gene, ftsZ1, and another upstream of the essential gene dacZ, encoding diadenylate cyclase involved in c-di-AMP signaling, which is implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. IMPORTANCE Cell division is a central mechanism of life and is essential for growth and development. Members of the Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in members of the Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in members of the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here, we identify the small protein CdrS as essential for survival and a central regulator of cell division in the euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways, including synthesis of signaling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.

Bottom-up and top-down proteomic approaches for the identification, characterization, and quantification of the low molecular weight proteome with focus on short open reading frame-encoded peptides.

The recent discovery of alternative open reading frames creates a need for suitable analytical approaches to verify their translation and to characterize the corresponding gene products at the molecular level. As the analysis of small proteins within a background proteome by means of classical bottom-up proteomics is challenging, method development for the analysis of small open reading frame encoded peptides (SEPs) have become a focal point for research. Here, we highlight bottom-up and top-down proteomics approaches established for the analysis of SEPs in both pro- and eukaryotes. Major steps of analysis, including sample preparation and (small) proteome isolation, separation and mass spectrometry, data interpretation and quality control, quantification, the analysis of post-translational modifications, and exploration of functional aspects of the SEPs by means of proteomics technologies are described. These methods do not exclusively cover the analytics of SEPs but simultaneously include the low molecular weight proteome, and moreover, can also be used for the proteome-wide analysis of proteolytic processing events.

A Small RNA Is Linking CRISPR-Cas and Zinc Transport.

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon Haloferax volcanii, a plethora of sRNAs have been identified; however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from Haloferax volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, and the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild-type Haloferax cells and the deletion mutant revealed upregulation of six genes in the deletion strain, showing that this sRNA has a clearly defined function. Three of the six upregulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, and ZnuC1) suggesting the involvement of s479 in the regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild-type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes, but the mature s479 contains a crRNA-like 5' handle, and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together, this might suggest that the CRISPR-Cas system is involved in s479 function.

Metabolic Labeling of Clostridioides difficile Proteins.

The introduction of stable isotopes in vivo via metabolic labeling approaches (SILAC or 15N-labeling) allows, after combination of differentially treated labeled and unlabeled cells or protein extracts, for correction of protein quantification errors implemented during elaborated sample preparation workflows. The SILAC-based approach uses heavy arginine and lysine to incorporate the label into bacterial strains and cell lines, whereas 15N-metabolic labeling is achieved by cultivation in 15N-salt containing media. In case of Clostridioides difficile, the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism makes metabolic labeling challenging. Here, a step-by-step guideline for the metabolic labeling of C. difficile is described, which combines cultivation in liquid 15N-substituted medium followed by cultivation steps on solid 15N-substituted medium. The described procedure results in a label incorporation rate higher than 97%. Cells prepared by the following method can be used as standard for relative quantification approaches of, e.g., the membrane or surface proteome of C. difficile.

Proteomic Charting of Imipenem Adaptive Responses in a Highly Carbapenem Resistant Clinical Enterobacter roggenkampii Isolate.

Gram-negative bacteria belonging to the Enterobacter cloacae complex are increasingly implicated in difficult-to-treat nosocomial infections, as exemplified by a recently characterized highly carbapenem-resistant clinical Enterobacter roggenkampii isolate with sequence type (ST) 232. While mechanisms of carbapenem resistance are well-understood, little is known about the responses of highly drug-resistant bacteria to these antibiotics. Our present study was therefore aimed at charting the responses of the E. roggenkampii ST232 isolate to the carbapenem imipenem, using a 'stable isotope labeling of amino acids in cell culture' approach for quantitative mass spectrometry. This unveiled diverse responses of E. roggenkampii ST232 to imipenem, especially altered levels of proteins for cell wall biogenesis, central carbon metabolism, respiration, iron-sulfur cluster synthesis, and metal homeostasis. These observations suggest a scenario where imipenem-challenged bacteria reduce metabolic activity to save resources otherwise used for cell wall biogenesis, and to limit formation of detrimental reactive oxygen species at the cytoplasmic membrane due to respiration and Fenton chemistry. We consider these observations important, because knowing the adaptive responses of a highly resistant bacterium of the E. cloacae complex to last-resort antibiotics, such as imipenem, provides a 'sneak preview' into the future development of antibiotic resistance in this emerging group of pathogens.

Multi-protease Approach for the Improved Identification and Molecular Characterization of Small Proteins and Short Open Reading Frame-Encoded Peptides.

The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEPs), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins, with a focus on SEPs, in the archaeon Methanosarcina mazei. Combining the data of all proteases, we identified 63 small proteins and additional 28 SEPs with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEPs, a complete sequence coverage was achieved. Moreover, for five SEPs, incorrectly predicted translation start points or potential in vivo proteolytic processing were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach allows to improve the identification and molecular characterization of small proteins and SEPs. LC-MS data: ProteomeXchange PXD023921.

Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea but can also result in more serious, life-threatening conditions. The incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing. Against this background antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Information on the effects of AMPs on C. difficile will not only enhance the knowledge for possible biomedical application but may also provide insights into mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelope but also resulted in pronounced changes in central cellular processes such as carbohydrate metabolism. Further, the number of flagella per cell was increased during the adaptation process. The potential involvement of flagella in nisin adaptation was supported by a more resistant phenotype exhibited by a non-motile but hyper-flagellated mutant.

Double trouble: Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment.

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

Optimized Proteomics Workflow for the Detection of Small Proteins.

Small open reading frame encoded proteins (SEPs) gained increasing interest during the last few years because of their broad range of important functions in both prokaryotes and eukaryotes. In bacteria, signaling, virulence, and regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased toward the identification of larger proteins. Here, we present a workflow that allows enhanced identification of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest, and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small-pore-sized solid-phase material increased the number of identified SEPs by a factor of 2, and utilization of alternative proteases to trypsin reduced the spectral counts for larger proteins. The application of the optimized protocol allowed the detection of 210 already annotated proteins up to 100 amino acids (aa) length, including 16 proteins below 51 aa in the Gram-positive model organism Bacillus subtilis. Moreover, 12% of all identified proteins were up to 100 aa, which is a significantly larger fraction than that reported in studies involving traditional proteomics workflows. Finally, the application of an integrated proteogenomics search database and extensive subsequent validation resulted in the confident identification of three novel, not yet annotated, SEPs, which are 21, 26, and 42 aa long.

Membrane Modulation of Super-Secreting "midiBacillus" Expressing the Major Staphylococcus aureus Antigen - A Mass-Spectrometry-Based Absolute Quantification Approach.

Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain ("midiBacillus") expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell's array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell.

Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP.

Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).

Proteomic response of Streptococcus pneumoniae to iron limitation.

Iron is an essential trace element and involved in various key metabolic pathways in bacterial lifestyle. Within the human host, iron is extremely limited. Hence, the ability of bacteria to acquire iron from the environment is critical for a successful infection. Streptococcus pneumoniae (the pneumococcus) is a human pathobiont colonizing symptomless the human respiratory tract, but can also cause various local and invasive infections. To survive and proliferate pneumococci have therefore to adapt their metabolism and virulence factor repertoire to different host compartments. In this study, the response of S. pneumoniae to iron limitation as infection-relevant condition was investigated on the proteome level. The iron limitation was induced by application of the iron chelator 2,2'-bipyridine (BIP) in two different media mimicking different physiological traits. Under these conditions, the influence of the initial iron concentration on pneumococcal protein expression in response to limited iron availability was analyzed. Interestingly, one major difference between these two iron limitation experiments is the regulation of proteins involved in pneumococcal pathogenesis. In iron-poor medium several proteins of this group were downregulated whereas these proteins are upregulated in iron-rich medium. However, iron limitation in both environments led to a strong upregulation of the iron uptake protein PiuA and the significant downregulation of the non-heme iron-containing ferritin Dpr. Based on the results, it is shown that the pneumococcal proteome response to iron limitation is strongly dependent on the initial iron concentration in the medium or the environment.

Picking vanished proteins from the void: how to collect and ship/share extremely dilute proteins in a reproducible and highly efficient manner.

Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches.

Metalloproteome of the prostate: carcinoma cell line DU-145 in comparison to healthy rat tissue.

AIM: To study the metalloproteome of DU-145 prostate carcinoma cells in comparison to prostate from control and selenium-deficient rats. MATERIALS AND METHODS: Total proteome of the samples was compared by two-dimensional electrophoresis (2-DE) and metalloproteome was analysed by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP/MS). Immunotests were used to quantify protein expression of superoxide dismutase, thioredoxin reductase and metallothionein. RESULTS: There was no general relation between protein expression and metal load. SEC-ICP/MS spectra for many metals varied significantly in terms of peak number and intensity between individuals of the same sample group. However, nickel and zinc peaks were consistently suppressed in DU-145 cells under selenium deficiency. Concurrent redistribution of cobalt binding to a low molecular weight fraction (presumably cobalamin) was observed. CONCLUSION: Metal load of proteins in comparison to their expression might point to yet unknown mechanisms of oncogenesis and may lead to new biomarkers of cancer.