Molecular Control of Extracellular DNA Release and Degradation in Shewanella oneidensis MR-1 Biofilms: The Role of Phages and Nucleases

Bakterien bilden unter natürlichen Bedingungen häufig oberflächen-assoziierte multizelluläre Gemeinschaften, welche allgemein als Biofilme bezeichnet werden. Die Bildung von Biofilmen ist ein komplexer und präzise regulierter Prozess, der es Bakterien ermöglicht, beinahe jede Art von Oberfläche zu besiedeln und dadurch physikalischen Stressfaktoren, Nährstoffmangel und Antibiotika standzuhalten. Des Weiteren kann oberflächenassoziiertes Wachstum die Virulenz von pathogenen Bakterien erhöhen und Umweltkeimen die Erschließung von Oberflächen als Nährstoff- und Energiequelle ermöglichen. Aus diesem Grund hat sich gezeigt, dass bakterielle Biofilmbildung von großer medizinischer, ökologischer und ökonomischer Relevanz ist. Ein wichtiger Bestandteil von Biofilmen ist die extrazelluläre polymere Matrix welche sich typischerweise aus Exopolysacchariden, Proteinen und extrazellulärer DNA (eDNA) zusammensetzt. Die Bedeutung der eDNA für Biofilme war lange unklar, jedoch konnte durch eine Reihe von Studien gezeigt werden, dass eDNA für die meisten Bakterienspezies, darunter Shewanella oneidensis MR-1, von essentieller Bedeutung für die strukturelle Entwicklung der Biofilme ist. Vielfach unbekannt sind jedoch Mechanismen, welche die Freisetzung von eDNA regulieren bzw. ausführen und solche, die an der Modulation und am Abbau (z.B. zur endogen induzierten Auflösung von Biofilmen oder zur Erschließung von eDNA als Nährstoffquelle) beteiligt sind. In der vorliegenden Arbeit wurde diese Mechanismen molekularbiologisch, mikroskopisch und biochemisch untersucht

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

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

Molecular Control of Extracellular DNA Release and Degradation in Shewanella oneidensis MR-1 Biofilms: The Role of Phages and Nucleases

Bakterien bilden unter natürlichen Bedingungen häufig oberflächen-assoziierte multizelluläre Gemeinschaften, welche allgemein als Biofilme bezeichnet werden. Die Bildung von Biofilmen ist ein komplexer und präzise regulierter Prozess, der es Bakterien ermöglicht, beinahe jede Art von Oberfläche zu besiedeln und dadurch physikalischen Stressfaktoren, Nährstoffmangel und Antibiotika standzuhalten. Des Weiteren kann oberflächenassoziiertes Wachstum die Virulenz von pathogenen Bakterien erhöhen und Umweltkeimen die Erschließung von Oberflächen als Nährstoff- und Energiequelle ermöglichen. Aus diesem Grund hat sich gezeigt, dass bakterielle Biofilmbildung von großer medizinischer, ökologischer und ökonomischer Relevanz ist. Ein wichtiger Bestandteil von Biofilmen ist die extrazelluläre polymere Matrix welche sich typischerweise aus Exopolysacchariden, Proteinen und extrazellulärer DNA (eDNA) zusammensetzt. Die Bedeutung der eDNA für Biofilme war lange unklar, jedoch konnte durch eine Reihe von Studien gezeigt werden, dass eDNA für die meisten Bakterienspezies, darunter Shewanella oneidensis MR-1, von essentieller Bedeutung für die strukturelle Entwicklung der Biofilme ist. Vielfach unbekannt sind jedoch Mechanismen, welche die Freisetzung von eDNA regulieren bzw. ausführen und solche, die an der Modulation und am Abbau (z.B. zur endogen induzierten Auflösung von Biofilmen oder zur Erschließung von eDNA als Nährstoffquelle) beteiligt sind. In der vorliegenden Arbeit wurde diese Mechanismen molekularbiologisch, mikroskopisch und biochemisch untersucht

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

Differentially regulated genes related to regulation.

<p>Differentially regulated genes related to regulation.</p

Influence of iron addition to <i>S. oneidensis</i> MR-1 biofilm formation under hydrodynamic conditions.

<p>Gfp-tagged <i>S. oneidensis</i> MR-1 wild-type cells were incubated in flow chambers in LM medium (upper panel) that was supplemented with iron at the indicated concentrations (lower panels). Biofilm formation was analyzed by CLSM at the indicated time points, displayed are three-dimensional shadow projections. The numbers below the images recorded after 0.5 and 5 hours represent the average surface coverage. The lateral edge of each image is 250 µm in length.</p

Coverage of a glass surface after inoculation and medium flow for 15 (upper image) and 60 minutes (lower image).

<p>Displayed are images taken by confocal laser scanning microscopy in flow chambers at the indicated time points. The lateral edge of each image equals 250 µm. The values given represent the average and standard deviation of surface coverage in percent of 6 randomly selected spots in three different channels of a flow chamber.</p

Differentially regulated genes related to “metabolic adaptation” – amino acid transport and metabolism.

<p>Differentially regulated genes related to “metabolic adaptation” – amino acid transport and metabolism.</p