Biofilm formation in the thermoacidophilic crenarchaea Sulfolobus spp.

In dieser Studie wurden Biofilmanalysen an Crenarchaeota durchgeführt, welche die ersten tiefergehenden Untersuchungen an archaealen Biofilm überhaupt sind. Es wurden Methoden für die Analyse von Biofilm entwickelt, wie zum Beispiel der Mikrotiter Assay, CLSM und das Färben zur Detektion von Biofilm. Die verwandten Stämme S. solfataricus, S. acidocaldarius und S. tokodaii zeigten erhebliche Unterschiede in ihrer Biofilmarchitektur (von teppich- bis zu turmartigen Strukturen), im Protein- und Transkriptionsmuster, als auch im Bedarf von Zellanhängen für die Biofilmentwicklung. In der Biofilmmatrix konnten hohe Anteile an Zuckern (Mannose-, Glucose-, N-acetyl-D-glucosamin- und Galactosylreste) detektiert werden, wobei derzeit noch unklar ist, ob diese Zucker auf Exopolysaccharide, glykosylierte Proteine oder beides zurückzuführen sind. Zusätzlich wurden in der Biofilmmatrix geringe Mengen an eDNA nachgewiesen, die allerdings nicht für die Stabilität und Struktur des Biofilms benötigt werden. Auffällig war, dass alle Stämme unterschiedliche Reaktionen im Biofilm unter Stressbedingungen zeigten (Temperatur, pH und Eisen). Gene, die möglicherweise in Archaea generell eine Rolle in der Biofilmbildung spielen sind der Transkriptionsregulator Lrs14 und FabG, welches möglicherweise an einem neuartigen „quorum sensing system“ von Archaeen beteiligt ist. Weitere interessante Beobachtungen wurden bei der Analyse von Mutanten und dem Einfluss von Oberflächenstrukturen auf Biofilm Formation und Anheftung gemacht. Während S. solfataricus sowohl die Flagelle als auch den Ups-Pilus für die Anheftung an Oberflächen benötigt, sind diese für die weitere Biofilmformation weniger essentiell. Ein anderes Ergebnis wurde bei S. acidocaldarius erzielt, wo die Deletion von mindestens zwei Anhängen zu einer reduzierten Anheftung führte. Eine Ausnahme war hier das Anheften das bei Mutanten beobachtet wurde, die nur noch den Aap-Pilus besaßen (Steigerung um 150%). Die einzelnen Deletion von Oberflächenstrukturen hatte zudem auch Einfluss auf die Biofilmarchitekturen (drei verschiedene Phänotypen). Ein GFP wurde adaptiert und bietet nun die Möglichkeit für Biofilm Analysen von S. acidocaldarius. Abschließend hat eine in vivo Analyse der Ssα-man einen Einfluss auf die Zuckerzusammensetzung des EPS in S. solfataricus ergeben. Wobei aufgrund der erzielten Ergebnisse, nicht auszuschließen ist, dass dieses Protein in Sulfolobus spp. an einer möglichen Prozessierung des Glycan beteiligt ist

European microbiologically influenced corrosion network (EURO-MIC): new paths for science, sustainability and standards

ABSTRACT: Microbiologically influenced corrosion (MIC) is the corrosion of material caused or enhanced by microorganisms. It occurs directly or indirectly through their metabolic activities and can be accelerated 10 to 100 times, depending on the material. A wide range of materials can be affected by MIC, including metal, plastic, and concrete, impacting the entire infrastructure of society, including water and wastewater management systems, marine industrial facilities, and (on)offshore systems. One challenge of MIC common to all these sectors is the colonization of surfaces, where the presence of water is one of the basic requirements for biofilm to form. This phenomenon is a major global challenge caused by the growing world population and related industrial activities combined with climate change, and increasingly becoming a problem for our society [1] and [2]. The global cost of MIC is unambiguous and should almost certainly be underestimated. According to survey data, MIC is responsible for up to 20% of all corrosion found in aqueous systems, costing billions of dollars in rehabilitation costs alone [1]. In Europe, several research groups/ other industrial stakeholders are already dealing with MIC. Unfortunately, the discussions are fragmented, and the exchange of information is limited. A true transdisciplinary approach is hardly ever experienced, although this would be logical for this material/biology related challenge. Therefore, Europe needs to combine the efforts of experts in different fields and develop prevention measures according to the european rules, in close cooperation with industry, plant operators and owners of critical infrastructure to effectively contribute to this MIC challenge. In this context, our european MIC-network aims to provide the necessary interaction and communication, knowledge sharing, training of personnel and of researchers of different disciplines. Only in this Europe can get a leading role in this process, bringing ideas together on an equal level with other nations, and thereby considering the important values and attitudes for Europe (e.g., environmental protection) and resulting in a greater protection for people, property, and the environment. The working group structure of this Euro-MIC Cost Action, as well as specific objectives, ongoing activities, and expected impacts, will be presented.info:eu-repo/semantics/publishedVersio

Crenarchaeal Biofilm Formation under Extreme Conditions

Background: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. Methodology: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. Conclusion: The study gives first insights into formation and development of crenarchaeal biofilms in extrem

Macromolecular Fingerprinting of Sulfolobus Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis

Microorganisms in nature often live in surfaceassociated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development

The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T) is important for swarming and biofilm formation.

Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB).Paenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA.This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T)

MotX and MotY Are Required for Flagellar Rotation in Shewanella oneidensis MR-1▿ †

The single polar flagellum of Shewanella oneidensis MR-1 is powered by two different stator complexes, the sodium-dependent PomAB and the proton-driven MotAB. In addition, Shewanella harbors two genes with homology to motX and motY of Vibrio species. In Vibrio, the products of these genes are crucial for sodium-dependent flagellar rotation. Resequencing of S. oneidensis MR-1 motY revealed that the gene does not harbor an authentic frameshift as was originally reported. Mutational analysis demonstrated that both MotX and MotY are critical for flagellar rotation of S. oneidensis MR-1 for both sodium- and proton-dependent stator systems but do not affect assembly of the flagellar filament. Fluorescence tagging of MotX and MotY to mCherry revealed that both proteins localize to the flagellated cell pole depending on the presence of the basal flagellar structure. Functional localization of MotX requires MotY, whereas MotY localizes independently of MotX. In contrast to the case in Vibrio, neither protein is crucial for the recruitment of the PomAB or MotAB stator complexes to the flagellated cell pole, nor do they play a major role in the stator selection process. Thus, MotX and MotY are not exclusive features of sodium-dependent flagellar systems. Furthermore, MotX and MotY in Shewanella, and possibly also in other genera, must have functions beyond the recruitment of the stator complexes

<i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A cells lose the ability to swarm on LB-agar plates.

<p>The upper panel shows swarming cells of wild-type (first column), <i>P. alvei</i> Δ<i>slh</i>A (second column), <i>P. alvei</i> Δhag (third column), wild-type (pEXALV) (fourth column), <i>P. alvei</i> Δ<i>slh</i>A (pEXALV) (fifth column) and the complemented strain <i>P. alvei</i> Δ<i>slh</i>A_comp (sixth column) on 0.4% (upper panel), 1% (middle panel) and 1.5% (lower panel) LB-agar plates. The pictures represent one of three independent experiments.</p

Knockout of <i>slhA</i> and <i>hag</i> decreases biofilm formation of <i>P. alvei</i> CCM 2051^T cells.

<p>(A) Evaluation of the ability of cells of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i>, wild-type (pEXALV), Δ<i>slh</i>A (pEXALV) carrying the pEXALV vector, and the complemented strain <i>P. alvei</i> Δ<i>slh</i>A_comp for biofilm formation using Crystal violet (CV) staining. Data represent mean values <u>+</u> SD of at least four independent experiments with each four replicates and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) SEM analysis of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i> and the complemented strain Δ<i>slh</i>A_comp showing an overview and enlarged view of the biofilm. Size bars are 20 µm for the upper panel and 5 µm for the lower panel. (C) CSLM analysis of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i> and the complemented strain Δ<i>slh</i>A_comp stained with Hoechst 33258 showing a diagonally above view (upper panel) and a side view (lower panel) of a three day biofilm. Size bars are 20 µm.</p