The archaeal exosome localizes to the membrane

AbstractWe studied the cellular localization of the archaeal exosome, an RNA-processing protein complex containing orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. Fractionation of cell-free extracts of Sulfolobus solfataricus in sucrose density gradients revealed that DnaG and the active-site comprising subunit Rrp41 are enriched together with surface layer proteins in a yellow colored ring, implicating that the exosome is membrane-bound. In accordance with this assumption, DnaG and Rrp41 were detected at the periphery of the cell by immunofluorescence microscopy. Our finding suggests that RNA processing in Archaea is spatially organized.Structured summaryMINT-7891213: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by cosedimentation in solution (MI:0028)MINT-7891235: Rrp41 (uniprotkb:Q9UXC2), DnaG (uniprotkb:P95980) and SlaA (uniprotkb:Q2M1E7) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-7891278: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by fluorescence microscopy (MI:0416

Microbial functionality as affected by experimental warming of a temperate mountain forest soil—A metaproteomics survey

Soil microbes play an important role in terrestrial carbon (C) cycling, but their functional response to global warming remains yet unclear. Soil metaproteomics has the potential to contribute to a better understanding of warming effects on soil microbes as proteins specifically represent active microbes and their physiological functioning. To quantify warming effects on microbial proteins and their distribution among different functional and phylogenetic groups, we sampled forest soil that had been artificially warmed (+4 °C) during seven consecutive growing seasons and analyzed its metaproteomic fingerprint and linked to soil respiration as a fundamental ecosystem service. Bacterial protein abundances largely exceeded fungal abundances at the study site but protein abundances showed only subtle differences among control and warmed soil at the phylum and class level, i.e. a temperature-induced decrease in Firmicutes, an increase in Agaricomycetes and Actinobacteria, and a decrease in the Asco/Basidiomycota ratio. Community function in warmed soil showed a clear trend towards increased proteins involved in microbial energy production and conversion, related to the increased CO2 efflux from warmed soil as a result of stress environmental conditions. The differences in community function could be related to specific phyla using metaproteomics, indicating that microbial adaptation to long-term soil warming mainly changed microbial functions, which is related to enhanced soil respiration. The response of soil respiration to warming (+35% soil CO2 efflux during sampling) has not changed over time. Accordingly, potential long-term microbial adaptations to soil warming were too subtle to affect soil respiration rates or, were overlaid by other co-varying factors (e.g. substrate availability)

What’s a Biofilm?—How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation

Maternal hemoglobin depletion in a settled Northern Kenyan pastoral population

Objectives: This study examines maternal hemoglobin depletion in a cross-sectional sample of Ariaal women living in northern Kenya. Maternal hemoglobin depletion occurs when women do not have enough dietary iron to replace the high levels of iron allocated to the fetus during pregnancy. Methods: To study this phenomenon, reproductive histories, socioeconomic status, anthropometry, and hemoglobin levels were collected from a cross-section of 200 lactating Ariaal women in northern Kenya. Results: Ariaal women show increasing levels of hemoglobin with increasing time since birth and lower hemoglobin levels with increasing parity, indicating an incomplete repletion of dietary iron over women's reproductive lifetime. Women who lived in a more livestock-dependent village had higher hemoglobin levels and lower prevalence of clinical anemia than women who lived in villages more dependent on agriculture, indicating that differences in diet may alleviate the effects of iron depletion. Conclusions: These data demonstrate that Ariaal women are iron depleted due to pregnancy, incompletely replete hemoglobin during the course of lactation, and show depletion of hemoglobin with increasing parity. Women in this community may be able to improve their iron status through a greater reliance on food sources rich in dietary iron. Am. J. Hum. Biol., 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78218/1/21078_ftp.pd

(Meta)Proteomics analysis of bacterial adaptation mechanisms to infection-related and environmental stresses

Microbial infections can be either caused by a single species or complex multi-species consortia. One of the most prominent opportunistic human pathogens leading to mono- or mixed-species infections is the Gram-negative bacterium Pseudomonas aeruginosa. Understanding the molecular basis of its adaptation to infection-related stresses is an essential prerequisite for the prevention and treatment of P. aeruginosa infections. We therefore employed state-of-the-art proteomics approaches to elucidate the molecular adaptation mechanisms of P. aeruginosa to infection-related conditions. Moreover, structure, function and interaction of complex microbial consortia containing P. aeruginosa and causing catheter-associated urinary tract infections were investigated by metaproteomics analyses. Our investigations revealed that the adaptation of P. aeruginosa during infection is either based on gene expression changes caused by environmental signal integration or by gene mutations leading to a selective advantage in a particular host environment. In study I, investigating the proteome response of P. aeruginosa biofilms to the clinical relevant antibiotic ciprofloxacin, global changes in the protein profile were observed. Ciprofloxacin induced the expression of proteins involved in the Lex-induced SOS-response, drug efflux pumps and gene products of the ciprofloxacin-responsive prophage cluster and repressed the expression of porins and DNA-binding proteins. In study II the transcriptome and proteome of two clonal P. aeruginosa lineages during long-term colonization of cystic fibrosis (CF) patient’s lungs were analyzed. Point mutations in global regulator genes, i.e. retS, gacS, and gacA, were identified by genomic sequencing. Inactivation of RetS, found two years after the initial colonization, induced the expression of genes involved in chronic infections and coding for the type 6-secretion system (T6SS). Additional mutations in the GacS/GacA two-component regulatory system (TCS) were found to repress the expression of T6SS proteins and to induce the expression of proteins belonging to the type 3-secretion system (T3SS). In study III we elucidated the niche-specific adaptation of P. aeruginosa isolates from different infection sites by investigating their protein expression patterns and glucose metabolic fluxes. We could show that isolates from the urinary tract express a higher amount of proteins involved in the acquisition of micronutrients (i.e. iron) and carbohydrates compared to isolates from the CF lung. In study IV 16S rDNA sequencing and metaproteomics were employed to demonstrate that the investigated CAUTI-related biofilms consisted of two to five different species with one or two species dominating the mixed community. Following this line of research, we investigated in study V structure and function of a biofilm of a long-term catheterized patient, which was predominantly composed of P. aeruginosa and Morganella morganii, but also contained a minor proportion of the obligate anaerobe Bacteroides sp.. The comparison of in vivo and in vitro protein expression profiles of P. aeruginosa and M. morganii indicated that iron and carbohydrates are the major growth-limiting factors in the bladder. These results indicate different nutritional strategies of the two pathogens in the bladder environment. A comparison of urinary protein profiles of healthy persons and catheterized patients suggested that the human innate immune system is induced by CAUTIs. Moreover, numerous proteins involved in nutritional immunity, e.g. iron-, calcium- and magnesium-binding proteins, were found to be more abundant in the urine of catheterized patients. A follow-up (meta)proteomics study (study VI) aiming at the elucidation of interspecies interactions during multi-species infections indicated that the urease-positive uropathogen Proteus mirabilis induces the precipitation of metal ions by urine alkalization and thereby limits the availability of these important micronutrients for other co-infecting bacteria. This limitation seems to be sensed by the P. aeruginosa PhoP-PhoQ two-component system (TCS) leading to an increased resistance to antimicrobial peptides and biofilm-forming capacity of the pathogen. Also during co-cultivation of P. aeruginosa with Staphylococcus aureus a slight increase in the expression of the PhoP-PhoQ TCS and the alkaline protease could be observed (study VII). In study VIII a combined metagenomics and metaproteomics approach was employed to investigate structure and function of the lichen Lobaria pulmonaria, a complex consortium consisting of a fungus, an algal partner, cyanobacteria, and a highly diverse bacterial microbiome. The results presented in this work contribute to a better understanding of the manifold and complex bacterial adaptation mechanisms to infection-related and environmental stress and thereby foster the development of novel treatment and prevention strategies.Mikrobielle Infektionskrankheiten können entweder durch eine pathogene Spezies oder durch komplexe Multi-Spezies-Gemeinschaften verursacht werden. Pseudomonas aeruginosa, einer der bedeutsamsten Gram-negativen opportunistisch humanpathogenen Bakterien, ist häufig an Multispezies-Infektionen beteiligt. Ein besseres Verständnis der molekularen Anpassung dieses Erregers an die Wirtsumgebung ist unerlässlich um neue Strategien für wirkungsvolle antiinfektiöse Therapien zu entwickeln. Eines der Hauptziele der vorliegenden Dissertation war es daher, die molekularen Anpassungsmechanismen von P. aeruginosa an infektionsrelevante Bedingungen mittels moderner Proteom-Analysen zu entschlüsseln. Des Weiteren sollten Struktur, Funktion und Interaktionen komplexer mikrobieller Gemeinschaften mittels neu etablierter Metaproteom-Analysen untersucht werden. Die Ergebnisse der vorliegenden Arbeit zeigen, dass sich P. aeruginosa durch Mutationen, die zu einem Selektionsvorteil führen, und/oder durch eine durch Umweltfaktoren bedingte Veränderung der Genexpressionsprofile an die Wirtsumgebung anpassen kann. Im Rahmen von Studie I wurde untersucht, wie sich das in der Klinik häufig eingesetzte Antibiotikum Ciprofloxacin auf die Proteinexpression von P. aeruginosa Biofilmen auswirkt. Die Ciprofloxacin-Behandlung induziert die Expression von Proteinen, die an der Lex-abhängigen SOS-Antwort und am Antibiotika-Export beteiligt sind, sowie von Prophagen-Proteinen und reprimiert die Expression von Porinen und DNA-bindenden Proteinen. In Studie II wurde die Langzeitanpassung von zwei klonalen P. aeruginosa Linien aus Mukoviszidose-Patienten mittels Proteom- und Transkriptomstudien untersucht. Eine zwei Jahre nach der initialen Besiedlung auftretende Mutation des RetS- Sensorkinase Gens führt zu einer verstärkten Expression von Genen deren Produkte an chronischen Infektionen beteiligt sind, darunter das Typ 6 Sekretionssystem (T6SS). Nach vier Jahren wurden weitere Mutationen in Genen des GacA/GacS Zweikomponentensystem (TCS) entdeckt, die zu einer Repression der Gene des T6SS führen, die Expression des Typ 3 Sekretionssystems (T3SS) jedoch induzieren. Die Expression des T3SS wird mit akuten Infektionen assoziiert. In Studie III wurde die Anpassung von P. aeruginosa an wirtsspezifische Nischen mittels Proteom- und Metabolom-Analysen verschiedener Isolate aus unterschiedlichen Infektionsgeschehen untersucht. Dabei konnte gezeigt werden, dass Isolate aus Harnwegsinfektionen im Vergleich zu Mukoviszidose-Isolaten mehr Proteine exprimieren die an der Aufnahme von Mikronährstoffen (z.B. Eisen) und Kohlenhydraten beteiligt sind. In Studie IV konnte mittels 16S rDNA Sequenzierung und Metaproteom-Analysen gezeigt werden, dass sich die untersuchten CAUTI-assoziierten Biofilme aus zwei bis fünf verschiedenen Spezies zusammensetzen, wobei ein oder zwei Spezies die Hauptmasse des Biofilms ausmachen. Im Rahmen einer Folgestudie (Studie V), in der der Biofilm eines langzeit-katheterisierten Patienten untersucht wurde, konnten P. aeruginosa, Morganella morganii, aber auch geringe Mengen des obligat anaeroben Bakteriums Bacteroides sp. identifiziert werden. Ein Vergleich der in vivo und in vitro Proteinexpressionsprofile der isolierten P. aeruginosa und M. morganii Stämme zeigte, dass Eisen und Kohlenhydrate limitierende Wachstumsfaktoren in der Blase darstellen und die beiden Pathogenen unterschiedliche Strategien zur Aufnahme von Nährstoffen aus dem Urin verfolgen. Vergleicht man die Proteinprofile des Urins gesunder Personen und katheterisierter Patienten, so kommen im Patientenurin verstärkt Proteine des angeborenen Immunsystems vor. Zudem finden sich im Patientenurin auch deutlich mehr Proteine die an der Bindung von Eisen, Kalzium und Magnesium beteiligt sind. In Studie VI wurden Interaktionen der Uropathogenen Proteus mirabilis und P. aeruginosa untersucht. P. mirabilis produziert große Mengen an Urease, was zu einer Alkalisierung des Mediums und damit zu einer Präzipitation von essentiellen Spurenelementen führt. Der Mangel an diesen Mikronährstoffen induziert das PhoP-PhoQ Zweikomponentensystem von P. aeruginosa, was letztendlich zu einer erhöhten Antibiotikaresistenz und Biofilmbildung führt. Studie VII, die die Interaktionen zwischen den CF-Pathogenen P. aeruginosa und Staphylococcus aureus untersuchte, zeigte, dass auch die Co-Kultivierung mit S. aureus die Expression von Genen des P. aeruginosa PhoP-PhoQ TCS induziert. In Studie VIII wurde die Struktur und Funktion der Flechte Lobaria pulmonaria mithilfe kombinierter Metagenom- und Metaproteom-Analysen untersucht. Die in der hier vorliegenden Arbeit erzielten Ergebnisse tragen wesentlich zu einem besseren Verständnis prokaryotischer Anpassungsmechanismen an infektionsrelevante Bedingungen oder Umweltstress bei und fördern damit die Entwicklung neuer Strategien zur Prävention und Bekämpfung mikrobieller Infektionskrankheiten

Deciphering functional diversification within the lichen microbiota by meta-omics

Abstract Background Recent evidence of specific bacterial communities extended the traditional concept of fungal-algal lichen symbioses by a further organismal kingdom. Although functional roles were already assigned to dominant members of the highly diversified microbiota, a substantial fraction of the ubiquitous colonizers remained unexplored. We employed a multi-omics approach to further characterize functional guilds in an unconventional model system. Results The general community structure of the lichen-associated microbiota was shown to be highly similar irrespective of the employed omics approach. Five highly abundant bacterial orders—Sphingomonadales, Rhodospirillales, Myxococcales, Chthoniobacterales, and Sphingobacteriales—harbor functions that are of substantial importance for the holobiome. Identified functions range from the provision of vitamins and cofactors to the degradation of phenolic compounds like phenylpropanoid, xylenols, and cresols. Conclusions Functions that facilitate the persistence of Lobaria pulmonaria under unfavorable conditions were present in previously overlooked fractions of the microbiota. So far, unrecognized groups like Chthoniobacterales (Verrucomicrobia) emerged as functional protectors in the lichen microbiome. By combining multi-omics and imaging techniques, we highlight previously overlooked participants in the complex microenvironment of the lichens

Response of Microbial Communities and Their Metabolic Functions to Drying–Rewetting Stress in a Temperate Forest Soil

Global climate change is predicted to alter drought–precipitation patterns, which will likely affect soil microbial communities and their functions, ultimately shifting microbially-mediated biogeochemical cycles. The present study aims to investigate the simultaneous variation of microbial community compositions and functions in response to drought and following rewetting events, using a soil metaproteomics approach. For this, an established field experiment located in an Austrian forest with two levels (moderate and severe stress) of precipitation manipulation was evaluated. The results showed that fungi were more strongly influenced by drying and rewetting (DRW) than bacteria, and that there was a drastic shift in the fungal community towards a more Ascomycota-dominated community. In terms of functional responses, a larger number of proteins and a higher functional diversity were observed in both moderate and severe DRW treatments compared to the control. Furthermore, in both DRW treatments a rise in proteins assigned to “translation, ribosomal structure, and biogenesis„ and “protein synthesis„ suggests a boost in microbial cell growth after rewetting. We also found that the changes within intracellular functions were associated to specific phyla, indicating that responses of microbial communities to DRW primarily shifted microbial functions. Microbial communities seem to respond to different levels of DRW stress by changing their functional potential, which may feed back to biogeochemical cycles

Effects of adult temperature on gene expression in a butterfly: identifying pathways associated with thermal acclimation

Abstract Background Phenotypic plasticity is a pervasive property of all organisms and considered to be of key importance for dealing with environmental variation. Plastic responses to temperature, which is one of the most important ecological factors, have received much attention over recent decades. A recurrent pattern of temperature-induced adaptive plasticity includes increased heat tolerance after exposure to warmer temperatures and increased cold tolerance after exposure to cooler temperatures. However, the mechanisms underlying these plastic responses are hitherto not well understood. Therefore, we here investigate effects of adult acclimation on gene expression in the tropical butterfly Bicyclus anynana, using an RNAseq approach. Results We show that several antioxidant markers (e.g. peroxidase, cytochrome P450) were up-regulated at a higher temperature compared with a lower adult temperature, which might play an important role in the acclamatory responses subsequently providing increased heat tolerance. Furthermore, several metabolic pathways were up-regulated at the higher temperature, likely reflecting increased metabolic rates. In contrast, we found no evidence for a decisive role of the heat shock response. Conclusions Although the important role of antioxidant defence mechanisms in alleviating detrimental effects of oxidative stress is firmly established, we speculate that its potentially important role in mediating heat tolerance and survival under stress has been underestimated thus far and thus deserves more attention

Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes

The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e., chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes