Pseudomonas putida mediates bacterial killing, biofilm invasion and biocontrol with a type IVB secretion system

Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas putida strain IsoF is able to kill a wide range of soil and plant-associated Gram-negative bacteria with the aid of a type IVB secretion system (T4BSS) that delivers a toxic effector into bacterial competitors in a contact-dependent manner. This extends the range of targets of T4BSSs—so far thought to transfer effectors only into eukaryotic cells—to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by the kib gene cluster located within a rare genomic island, which was recently acquired by horizontal gene transfer. P. putida IsoF utilizes this secretion system not only as a defensive weapon to kill bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that strain IsoF can protect tomato plants against the phytopathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF can be exploited for pest control and sustainable agriculture

Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology

The Compound 2-Hexyl, 5-Propyl Resorcinol Has a Key Role in Biofilm Formation by the Biocontrol Rhizobacterium Pseudomonas chlororaphis PCL1606

The production of the compound 2-hexyl-5-propyl resorcinol (HPR) by the biocontrol rhizobacterium Pseudomonas chlororaphis PCL1606 (PcPCL1606) is crucial for fungal antagonism and biocontrol activity that protects plants against the phytopathogenic fungus Rosellinia necatrix. The production of HPR is also involved in avocado root colonization during the biocontrol process. This pleiotrophic response prompted us to study the potential role of HPR production in biofilm formation. The swimming motility of PcPLL1606 is enhanced by the disruption of HPR production. Mutants impaired in HPR production, revealed that adhesion, colony morphology, and typical air–liquid interphase pellicles were all dependent on HPR production. The role of HPR production in biofilm architecture was also analyzed in flow chamber experiments. These experiments revealed that the HPR mutant cells had less tight unions than those producing HPR, suggesting an involvement of HPR in the production of the biofilm matrix

Social behaviors of Pseudomonas putida IsoF

Members of the genus Pseudomonas are metabolically and physiologically extremely versatile. They inhabit a wide variety of habitats, including diverse terrestrial and aquatic niches. Some strains are of great interest because of their importance to cause plant and human diseases, as well as for their potential in biotechnological applications The diverse lifestyles of Pseudomonas sp. and the complexity of their interactions with multiple hosts have been the subject of numerous studies. In nature, most of bacteria live in close association with surfaces as complex communities referred to as biofilms. Bacteria growing in biofilms are usually more resistant to hostile conditions than those growing planktonically. Cell densities are very high in biofilm communities, and since the biofilm matrix also acts as a diffusion barrier for signal molecules, biofilms represent an ideal environment for the induction of cell-to-cell communication systems, generally named quorum sensing (QS) systems. The term QS is generally used to describe the phenomenon that bacteria are capable of perceiving and responding to self-generated signal molecules to coordinate their behavior at the group level. Apart from signaling molecules, pseudomonads are known to produce a variety of bioactive compounds, including biosurfactants, which contribute to their niche adaptation, e.g. bacterial competition or associations with certain plant and animal hosts. Bacteria face a constant battle for space and resources. On account of this rivalry they have evolved numerous strategies to deal with competitors. Phenotypic traits such as biofilm formation, cell-to-cell communication, secondary metabolites production, and bacterial competition are commonly associated with the lifestyle of these bacteria and their study may contribute to a better understanding of their success in the environment. The focus of this thesis was to investigate: i) the link between QS and biofilm formation, ii) biosurfactant production, and iii) bacterial competition, using the plant promoting bacterium, Pseudomonas putida IsoF as a model organism. My work showed that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced at early stages of P. putida biofilms, and act mainly as self-regulatory signals, triggering asocial motility of induced cells out of microcolonies. These findings broaden the perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behavior. In addition 1-7 found that two QS-regulated loci are responsible for biosynthesis of the cyclic lipopeptide (CLPs) biosurfactants, putisolvin I and II. One locus encodes the fatty acid CoA ligase PpuA which is required to attach the hexanoic acid chain to the peptide moiety synthesized by the psoABC operon. Finally I found that P. putida IsoF antagonizes several proteobacterial species by the aid of a type six secretion system (T6SS) and show that this antagonism is particularly effective in mixed-species biofilms. Mitglieder des Genus Pseudomonas sind metabolisch und physiologisch sehr vielseitig. Dadurch können sie eine Vielzahl an unterschiedlichen Habitaten besiedeln, wie etwa verschiedene terrestrische und aquatische Nischen. Einige Stämme sind aufgrund ihres Potenzials zur biotechnologischen Anwendung von großem Interesse, andere wiederum da sie Krankheitserreger von Pflanzen und Menschen sind. Die unterschiedlichen Lebensweisen von Pseudomonas sp. sowie die Komplexität ihrer Interaktionen mit verschiedenen Wirtsorganismen wurden schon in einer Vielzahl von Studien untersucht. In der Umwelt leben die meisten Bakterien in enger Assoziation mit Oberflächen als komplexe Gemeinschaften, so genannten Biofilmen. Bakterien in Biofilmen sind normalerweise resistenter gegenüber feindlichen Lebensbedingungen als planktonisch lebende Bakterien. Da die Zelldichte in Biofilmgemeinschaften enorm hoch ist und gleichzeitig die Biofilmmatrix als Diffusionsbarriere für Signalmoleküle wirkt, stellen Biofilme eine ideale Umgebung für die Induktion von Zell-Zell-Kommunikationssystemen dar, welche als Quorum-sensing (QS) Systeme bezeichnet werden. Die Bezeichnung QS beschreibt das Phänomen, dass Bakterien selbst generierte Signalmoleküle wahrnehmen und darauf reagieren können um ihr Gruppenverhalten zu koordinieren. Es ist bekannt, dass Pseudomonaden neben Signalmolekülen auch eine Vielzahl bioaktiver Verbindungen, wie zum Beispiel Biotenside, produzieren können. Diese tragen zur Nischenadaptation bei, wie etwa beim bakterielle Konkurrenzkampf oder der Assoziation mit bestimmten Pflanzen- und Tierwirten. Bakterien sind einem andauernden Kampf um Raum und Resourcen ausgesetzt weshalb sie unterschiedliche Strategien entwickelt haben um mit ihren Konkurrenten umzugehen. Phänotypische Merkmale wie Biofilmbildung, Zell-Zell- Kommunikation, Produktion von Sekundärmetaboliten und bakterielle Konkurrenz sind gewöhnlich mit der Lebensweise der Bakterien assoziiert. Das Studium dieser Phänotypen kann zum besseren Verständnis des Überlebenserfolgs in der Umwelt beitragen. Das Hauptaugenmerk dieser Arbeit war die Untersuchung i) der Verbindung zwischen QS und Biofilmbildung, ii) der Biotensidproduktion und iii) bakterieller Konkurrenz anhand des pflanzenwachstumsfördernden Modelorganismus Pseudomonas putida IsoF. Meine Arbeit zeigte, dass QS Signale (N-acyl-homoserine lactones, AHLs) stochastisch in den frühen Phasen von P. putida Biofilmen produziert werden und dabei hauptsächlich als selbst regulierende Signale wirken. Diese lösen asoziale Motilität in induzierten Zellen aus, welche daraufhin ihre Mikrokolonien verlassen. Diese Beobachtungen erweitern das Verständnis von QS indem gezeigt wird, dassAHLs die Expression von asozialen (selbst gerichtet) Merkmalen kontrolliert. Des Weiteren wird gezeigt, dass Heterogenität bei QS als Mechanismus zum Antrieb von phänotypischer Heterogenität in selbst gerichtetem Verhalten fungiert. Zusätzlich habe ich herausgefunden, dass zwei QS regulierte Loci für die Biosynthese der zyklischen lipopeptid-Biotenside, Putisolvin I und II verantwortlich sind. Einer der beiden Loci kodiert für die Fettsäure-CoA-Ligase PpuA, welche eine Hexansäurekette an die Peptid-Gruppe von Putisolvin anhängt, welche vom psoABC Operon synthetisiert wird. Schlussendlich habe ich noch herausgefunden, dass P. putida IsoF mehrere Proteobakterien mittels eines Typ 6 Sekretionssystems (T6SS) bekämpft. Dieser Antagonismus ist in Biofilmen mit gemischten Bakterienspezies besonders effektiv

Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms

The term ‘quorum sensing’ (QS) is generally used to describe the phenomenon that bacteria release and perceive signal molecules to coordinate cooperative behaviour in response to their population size. QS-based communication has therefore been considered a social trait. Here we show that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced in young biofilms of Pseudomonas putida and act mainly as self-regulatory signals rather than inducing neighbouring cells. We demonstrate that QS induces the expression of putisolvin biosurfactants that are not public goods, thereby triggering asocial motility of induced cells out of microcolonies. Phenotypic heterogeneity is most prominent in the early stages of biofilm development, whereas at later stages behaviour patterns across cells become more synchronized. Our findings broaden our perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behaviour

The Compound 2-Hexyl, 5-Propyl Resorcinol Has a Key Role in Biofilm Formation by the Biocontrol Rhizobacterium Pseudomonas chlororaphis PCL1606

The production of the compound 2-hexyl-5-propyl resorcinol (HPR) by the biocontrol rhizobacterium Pseudomonas chlororaphis PCL1606 (PcPCL1606) is crucial for fungal antagonism and biocontrol activity that protects plants against the phytopathogenic fungus Rosellinia necatrix. The production of HPR is also involved in avocado root colonization during the biocontrol process. This pleiotrophic response prompted us to study the potential role of HPR production in biofilm formation. The swimming motility of PcPLL1606 is enhanced by the disruption of HPR production. Mutants impaired in HPR production, revealed that adhesion, colony morphology, and typical air-liquid interphase pellicles were all dependent on HPR production. The role of HPR production in biofilm architecture was also analyzed in flow chamber experiments. These experiments revealed that the HPR mutant cells had less tight unions than those producing HPR, suggesting an involvement of HPR in the production of the biofilm matrix

σ54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ54 consensus sequence. The mapping of the σ54 regulon as well as the characterization of a σ54 mutant suggests an important role of σ54 not only in control of nitrogen metabolism but also in the virulence of this organism

Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis

It is unclear how Gram-positive bacteria, with a thick cell wall, can release membrane vesicles. Here, Toyofuku et al. show that a prophage-encoded endolysin can generate holes in the cell wall through which cytoplasmic membrane material protrudes and is released as vesicles

ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen P. aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP1(7)P2(7) tetradecamer that is required for proper biofilm development. PaClpP1(14) and PaClpP1(7)P2(7) complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development