Analysis of the BarA/UvrY Two-Component System in Shewanella oneidensis MR-1

The BarA/UvrY two-component system is well conserved in species of the γ-proteobacteria and regulates numerous processes predominantly by controlling the expression of a subset of noncoding small RNAs. In this study, we identified and characterized the BarA/UvrY two-component system in the gammaproteobacterium Shewanella oneidensis MR-1. Functional interaction of sensor kinase BarA and the cognate response regulator UvrY was indicated by in vitro phosphotransfer studies. The expression of two predicted small regulatory RNAs (sRNAs), CsrB1 and CsrB2, was dependent on UvrY. Transcriptomic analysis by microarrays revealed that UvrY is a global regulator and directly or indirectly affects transcript levels of more than 200 genes in S. oneidensis. Among these are genes encoding key enzymes of central carbon metabolism such as ackA, aceAB, and pflAB. As predicted of a signal transduction pathway that controls aspects of central metabolism, mutants lacking UvrY reach a significantly higher OD than the wild type during aerobic growth on N-acetylglucosamine (NAG) while under anaerobic conditions the mutant grew more slowly. A shorter lag phase occurred with lactate as carbon source. In contrast, significant growth phenotypes were absent in complex medium. Based on these studies we hypothesize that, in S. oneidensis MR-1, the global BarA/UvrY/Csr regulatory pathway is involved in central carbon metabolism processes

Characterization of ExeM, an Extracellular Nuclease of Shewanella oneidensis MR-1

Bacterial extracellular nucleases have multiple functions in processes as diverse as nutrient acquisition, natural transformation, biofilm formation, or defense against neutrophil extracellular traps (NETs). Here we explored the properties of ExeM in Shewanella oneidensis MR-1, an extracellular nuclease, which is widely conserved among species of Shewanella, Vibrio, Aeromonas, and others. In S. oneidensis, ExeM is crucial for normal biofilm formation. In vitro activity measurements on heterologously produced ExeM revealed that this enzyme is a sugar-unspecific endonuclease, which requires Ca2+ and Mg2+/Mn2+ as co-factors for full activity. ExeM was almost exclusively localized to the cytoplasmic membrane fraction, even when a putative C-terminal membrane anchor was deleted. In contrast, ExeM was not detected in medium supernatants. Based on the results we hypothesize that ExeM predominantly interacts with DNA in close proximity to the cell, e.g., to promote biofilm formation and defense against NETs, or to control uptake of DNA

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

Flagellar motor tuning - The hybrid motor in Shewanella oneidensis MR-1

Bacteria are exposed to constantly changing environments. An efficient way to navigate towards favourable conditions is flagella-mediated motility. Flagellar rotation is achieved by the bacterial flagellar motor, composed of the rotor and stator complexes which surround the rotor in a ring-like structure. As an exception among the Shewanella species, the fresh-water organism S. oneidensis MR-1 harbours two different stator complexes, the sodium-ion dependent PomAB and the proton-dependent MotAB, differentially supporting rotation of a single polar flagellum. Both PomAB and MotAB are simultaneously present and required for full speed under low sodium-ion conditions. Although tightly anchored to the cell wall, stators are constantly exchanged even during ongoing rotation. Moreover, sodium-ion and proton-dependent stators can function with the same rotor. This raises the question of how PomAB and MotAB contribute to rotation of a single flagellum and whether PomAB and MotAB coexist in the stator ring of S. oneidensis MR-1, forming a hybrid motor. Here, I report a novel model for the dynamic adaptation of the rotor-stator configuration in response to the environmental sodium ion level in S. oneidensis MR-1. Transcriptional fusions to lucB revealed that both pomAB and motAB are concurrently transcribed. By using fluorescence microscopy, functional fusions of mCherry to the B-subunits revealed that in sharp contrast to MotB, a fraction of PomB is polarly positioned independently of the sodium-ion concentration. At low sodium-ion concentration, PomB and MotB appear to coexist in the flagellar motor. However, in the absence of PomAB, MotB is recruited to the flagellated pole independently of the sodium-ion concentration. Interestingly, induced production of PomAB displaces polar MotB from the motor and confines it to the membrane. By quantifying single sfGfp molecules fused to PomB, I could show that the number of PomB in the stator ring is reduced from nine to five complexes when cells were shifted from a high to a low sodium-ion concentration. Thus, the incorporation efficiency of PomAB is directly modified in response to the sodium-ion concentration, whereas the association of MotAB into the stator ring rather depends on the presence of PomAB. Furthermore, two auxiliary proteins, MotX and MotY, were identified and shown to be essential for functionality of both PomAB and MotAB. Localisation studies revealed that, in contrast to Vibrio MotXY are not required for recruitment of the stator complexes to the flagellated pole. Taken together, my data support the model of dynamic stator swapping to tune the flagellar motor in response to environmental conditions, e.g. the availability of sodium ions. The concurrent presence of PomB and MotB at low sodium-ion concentration suggests the existence of a hybrid motor in S. oneidensis. Since it remains to be demonstrated whether MotAB stators are functionally incorporated in this hybrid motor, the second aim of this work was to biophysically analyse the contribution of MotAB and PomAB to motor rotation at the single cell level. To this end, a ‘bead assay’ and a ‘tethered cell assay’ were established. These set-ups required the delocalisation of the polar filament to a lateral position, the preparation of a highly specific antibody against the modified filament and, for the bead assay the attachment of polystyrene beads to the filament. While the bead assay was limited to short-term measurements, the tethered cell assay was optimised for long-term studies. The optimisation now permits a constant buffer exchange as well as the modulation of the stator complex level by an inducible promoter upstream of pomAB and motAB. Single cell analysis comparing the wild-type and the PomAB-driven motor revealed a significantly higher rotation speed for the wild-type motor at low sodium-ion concentration. Moreover, induced production of PomAB in a stator deletion background resurrected rotation speed in a stepwise manner, whereas production of MotAB in a PomAB-driven motor decreased rotation speed stepwise. These results strongly indicate that MotAB is incorporated into the force-generating PomAB-occupied stator ring, slowing down motor rotation. MotAB production in a stator deletion background did not restore rotation. However, swimming assays revealed that MotAB is sufficient to drive flagellar rotation in a subpopulation of cells, strongly suggesting that both stators are able to function together in a single motor. To clearly characterise the role of MotAB and PomAB in the hybrid motor of S. oneidensis MR-1 further biophysical studies are required. The genome wide bioinformatic analysis of all sequenced bacterial genomes revealed that dual or multiple stator complexes along with a single flagellar system are surprisingly widespread among bacterial species. Moreover, stator complex homology comparison in S. oneidensis MR-1 indicated that MotAB has recently been acquired by lateral gene transfer as a consequence of adaptation to a fresh-water environment. Thus, the flagellar motor might still be in a process of optimisation. Collectively, I hypothesize that S. oneidensis tunes its flagellar motor by exchanging stator complexes and that stator swapping represents a common mechanism applicable to other bacteria to adapt to changing environments

Transcriptome analysis of early surface-associated growth of Shewanella oneidensis MR-1.

Bacterial biofilm formation starts with single cells attaching to a surface, however, little is known about the initial attachment steps and the adaptation to the surface-associated life style. Here, we describe a hydrodynamic system that allows easy harvest of cells at very early biofilm stages. Using the metal ion-reducing gammaproteobacterium Shewanella oneidensis MR-1 as a model organism, we analyzed the transcriptional changes occurring during surface-associated growth between 15 and 60 minutes after attachment. 230 genes were significantly upregulated and 333 were downregulated by a factor of ≥ 2. Main functional categories of the corresponding gene products comprise metabolism, uptake and transport, regulation, and hypothetical proteins. Among the genes highly upregulated those implicated in iron uptake are highly overrepresented, strongly indicating that S. oneidensis MR-1 has a high demand for iron during surface attachment and initial biofilm stages. Subsequent microscopic analysis of biofilm formation under hydrodynamic conditions revealed that addition of Fe(II) significantly stimulated biofilm formation of S. oneidensis MR-1 while planktonic growth was not affected. Our approach to harvest cells for transcriptional analysis of early biofilm stages is expected to be easily adapted to other bacterial species

Flagellen-vermittelte Motilität in Shewanella : Mechanismen zur effektiven Fortbewegung in S. putrefaciens CN-32 und S. oneidensis MR-1

Bakterien können sich mittels der Rotation helikaler Proteinfilamente – den Flagellen – höchst effizient durch ihre Umwelt bewegen. In einer sich ständig verändernden Umgebung ermöglicht diese Art der Fortbewegung eine gerichtete Bewegung hin zu optimalen Bedingungen. Über ein breites chemosensorisches Potential nehmen Bakterien spezifische Reize wahr und reagieren durch eine Modulierung der Bewegungsmaschinerie, die Stimulus-abhängige Chemotaxis entlang von Gradienten ermöglicht. Verschiedene Habitate führen in Mikroorganismen zu einer spezifischen Anpassung der Flagellensysteme, was zu einer hohen Variabilität der Charakteristika von Flagellensystemen unterschiedlicher Organismen führt. Manche Bakterien besitzen neben dem primären, meist polaren, Flagellensystem auch sekundäre laterale Flagellen, die eine Fortbewegung unter Bedingungen ermöglichen, welche die Funktion der polaren Flagelle einschränken können. So gewährleisten diese zum Beispiel die Fortbewegung in Umgebungen mit hoher Viskosität oder das Schwärmen von Zellen über Oberflächen. Die Produktion des kostspieligen zweiten Flagellen-systems unterliegt dabei einer strikten regulatorischen Kontrolle. Shewanella putrefaciens CN-32 besitzt, neben einer Na+-getriebenen polaren Flagelle, ebenfalls ein sekundäres H+-getriebenes Flagellensystem, das Ähnlichkeit zu lateralen Flagellensystemen anderer Organismen aufweist. Erstaunlicherweise produziert eine Subpopulation in S. putrefaciens CN-32 ein bis zwei laterale, zufällig lokalisierende Flagellen bereits während des exponentiellen Wachstums in planktonischer Kultur. Phänotypische und fluoreszenzmikroskopische Analysen de-monstrierten, dass beide Flagellensysteme in S. putrefaciens CN-32 auf struktureller Ebene hoch-spezifisch sind. Ein Chemotaxissystem induziert spezifisch den Richtungswechsel der bidirektional rotierenden polaren Flagelle, aber nicht den der unidirektional drehenden lateralen Flagelle. Die Rotation der polaren Flagelle ist ausreichend, um maximale Schwimmgeschwindigkeit in planktonischer Kultur zu erreichen. Dennoch zeigen Zellen, die ein bis zwei zusätzliche laterale Flagellen besitzen, eine effektivere, gerichtete Schwimmbewegung, die vermutlich mit der effizienten Neuorientierung der Zellkörper einhergeht. Eine solche Eigenschaft für ein laterales Flagellensystem wurde bisher noch nicht beschrieben und könnte daher ein neuartiges System zur erfolgreichen Exploration neuer Habitate darstellen. Die Präsenz von dualen Flagellensystemen in einer Vielzahl von aquatisch vorkommenden Bakterien ist ein Indiz für ähnliche Funktionsweisen der Flagellensysteme in anderen Organismen. Im Gegensatz zu S. putrefaciens CN-32 nutzt Shewanella oneidensis MR-1 nur eine einzelne polare Flagelle mit einem dualen Statorsystem, um sich bei unterschiedlichen Natriumkonzen-trationen fortbewegen zu können. Das Flagellenfilament besteht aus den homologen Flagellinen FlaA und FlaB, deren posttranslationale Modifizierung essentiell für die Assemblierung der Flagelle ist. MS-Untersuchungen, sowie im Rahmen dieser Arbeit durchgeführte Genort-spezifische Muta-tionen zeigten, dass FlaA und FlaB an mindestens vier Serinen über O-glykosidische Bindungen modifiziert werden. Detaillierte Analysen der Modifizierung wiesen darauf hin, dass diese sich aus einem Pseudaminsäure-Derivat (Pse) und einer strukturell unbekannten, 264 Da schweren Einheit zusammensetzt. Partiell konservierte Gene innerhalb der Modifizierungsregion neben variablen Bereichen, wie z.B. dem sfmABCDE-Operon, lassen einen allgemeinen Mechanismus der Flagellin-Glykosylierung mit Spezies-spezifischen Zucker-Resten in anderen Shewanella-Vertretern vermuten. Meine Ergebnisse zeigen, dass ein synchron funktionierendes duales Flagellensystem in S. putrefaciens CN-32 effektives gerichtetes Schwimmverhalten vermitteln kann und demonstrieren die essentielle Bedeutung der Glykosylierung von Flagellin mit Pse für die Motilität von S. oneidensis MR-1. Die Erkenntnisse dieser Arbeit erweitern das Wissen über Flagellen-vermittelte Fortbewe-gung in Bakterien und verdeutlichen die Komplexität und Variabilität der Flagellensysteme in Bezug auf die evolutionäre Anpassung homologer Proteinkomplexe und regulatorischer Netzwerke an die Umwelt

Wrapped Up: The Motility of Polarly Flagellated Bacteria

A huge number of bacterial species are motile by flagella, which allow them to actively move toward favorable environments and away from hazardous areas and to conquer new habitats. The general perception of flagellum-mediated movement and chemotaxis is dominated by the Escherichia coli paradigm, with its peritrichous flagellation and its famous run-and-tumble navigation pattern, which has shaped the view on how bacteria swim and navigate in chemical gradients. However, a significant amount-more likely the majority-of bacterial species exhibit a (bi)polar flagellar localization pattern instead of lateral flagella. Accordingly, these species have evolved very different mechanisms for navigation and chemotaxis. Here, we review the earlier and recent findings on the various modes of motility mediated by polar flagella

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA

ArcS, the Cognate Sensor Kinase in an Atypical Arc System of Shewanella oneidensis MR-1▿ †

The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment

Antibiotic Drug screening and Image Characterization Toolbox (A.D.I.C.T.): test data for workflow deployment and reproduction

Data in this repository is part of the A.D.I.C.T. toolbox published in F100