Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole-cell screening approach

Due to their ability for direct electron transfer to electrodes, the utilization of rare earth metals as cofactor, and their periplasmic localization, pyrroloquinoline quinone‐dependent alcohol dehydrogenases (PQQ‐ADHs) represent an interesting class of biocatalysts for various biotechnological applications. For most biocatalysts protein stability is crucial, either to increase the performance of the protein under a given process condition or to maximize robustness of the protein towards mutational manipulations, which are often needed to enhance or introduce a functionality of interest. In this study, we describe a whole‐cell screening assay, suitable for probing PQQ‐ADH activities in Escherichia coli BL21(DE3) cells, and use this assay to screen smart mutant libraries for increased thermal stability of the PQQ‐ADH PedE (PP_2674) from Pseudomonas putida KT2440. Upon three consecutive rounds of screening, we identified three different amino acid positions, which significantly improve enzyme stability. The subsequent combination of the beneficial mutations finally results in the triple mutant R91D/E408P/N410K, which not only exhibits a 7°C increase in thermal stability but also a twofold increase in residual activity upon incubation with up to 50% dimethyl sulfoxide (DMSO), while showing no significant difference in enzymatic efficiency (kcat/KM)

SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions

© 2016 Colley, Dederer, Carnell, Kjelleberg, Rice and Klebensberger. Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions

Glucose starvation-induced dispersal of pseudomonas aeruginosa biofilms is camp and energy dependent

Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival. © 2012 Huynh et al

Matrix Polysaccharides and SiaD Diguanylate Cyclase Alter Community Structure and Competitiveness of Pseudomonas aeruginosa during Dual-Species Biofilm Development with Staphylococcus aureus

Copyright © 2018 Chew et al. Mixed-species biofilms display a number of emergent properties, including enhanced antimicrobial tolerance and communal metabolism. These properties may depend on interspecies relationships and the structure of the biofilm. However, the contribution of specific matrix components to emergent properties of mixed-species biofilms remains poorly understood. Using a dual-species biofilm community formed by the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, we found that whilst neither Pel nor Psl polysaccharides, produced by P. aeruginosa, affect relative species abundance in mature P. aeruginosa and S. aureus biofilms, Psl production is associated with increased P. aeruginosa abundance and reduced S. aureus aggregation in the early stages of biofilm formation. Our data suggest that the competitive effect of Psl is not associated with its structural role in cross-linking the matrix and adhering to P. aeruginosa cells but is instead mediated through the activation of the diguanylate cyclase SiaD. This regulatory control was also found to be independent of the siderophore pyoverdine and Pseudomonas quinolone signal, which have previously been proposed to reduce S. aureus viability by inducing lactic acid fermentation-based growth. In contrast to the effect mediated by Psl, Pel reduced the effective crosslinking of the biofilm matrix and facilitated superdiffusivity in microcolony regions. These changes in matrix cross-linking enhance biofilm surface spreading and expansion of microcolonies in the later stages of biofilm development, improving overall dual-species biofilm growth and increasing biovolume severalfold. Thus, the biofilm matrix and regulators associated with matrix production play essential roles in mixed-species biofilm interactions.IMPORTANCE Bacteria in natural and engineered environments form biofilms that include many different species. Microorganisms rely on a number of different strategies to manage social interactions with other species and to access resources, build biofilm consortia, and optimize growth. For example, Pseudomonasaeruginosa and Staphylococcus aureus are biofilm-forming bacteria that coinfect the lungs of cystic fibrosis patients and diabetic and chronic wounds. P. aeruginosa is known to antagonize S. aureus growth. However, many of the factors responsible for mixed-species interactions and outcomes such as infections are poorly understood. Biofilm bacteria are encased in a self-produced extracellular matrix that facilitates interspecies behavior and biofilm development. In this study, we examined the poorly understood roles of the major matrix biopolymers and their regulators in mixed-species biofilm interactions and development

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa

Magnesium Limitation Is an Environmental Trigger of the Pseudomonas aeruginosa Biofilm Lifestyle

Biofilm formation is a conserved strategy for long-term bacterial survival in nature and during infections. Biofilms are multicellular aggregates of cells enmeshed in an extracellular matrix. The RetS, GacS and LadS sensors control the switch from a planktonic to a biofilm mode of growth in Pseudomonas aeruginosa. Here we detail our approach to identify environmental triggers of biofilm formation by investigating environmental conditions that repress expression of the biofilm repressor RetS. Mg2+ limitation repressed the expression of retS leading to increased aggregation, exopolysaccharide (EPS) production and biofilm formation. Repression of retS expression under Mg2+ limitation corresponded with induced expression of the GacA-controlled small regulatory RNAs rsmZ and rsmY and the EPS biosynthesis operons pel and psl. We recently demonstrated that extracellular DNA sequesters Mg2+ cations and activates the cation-sensing PhoPQ two-component system, which leads to increased antimicrobial peptide resistance in biofilms. Here we show that exogenous DNA and EDTA, through their ability to chelate Mg2+, promoted biofilm formation. The repression of retS in low Mg2+ was directly controlled by PhoPQ. PhoP also directly controlled expression of rsmZ but not rsmY suggesting that PhoPQ controls the equilibrium of the small regulatory RNAs and thus fine-tunes the expression of genes in the RetS pathway. In summary, Mg2+ limitation is a biologically relevant environmental condition and the first bonafide environmental signal identified that results in transcriptional repression of retS and promotes P. aeruginosa biofilm formation

Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild

Detergenzien-induzierte Zellaggregation bei Pseudomonas aeruginosa

Pseudomonas aeruginosa Stamm PAO1 konnte mit dem toxischen anionischen Detergens Natriumdodecylsulfat (SDS) als einziger Kohlenstoff- und Energiequelle wachsen. Während des Wachstums auf oder in Gegenwart von SDS wurde die Bildung makroskopischer Zellaggregate beobachtet. Diese Aggregate bestanden aus beschädigten und unbeschädigten Zellen, welche in eine Matrix aus sauren Polysacchariden und DNA eingebettet waren. Diese Aggregate wurden gebildet, wenn frei suspendierte Zellen bei hoher Energieversorgung mit SDS versetzt wurden. Dabei aggregierte immer nur ein Teil der Gesamtpopulation. Bei stark limitierter Energieversorgung lysierten die Zellen in Gegenwart von SDS ohne die Bildung von Aggregaten. Diese physiologischen Untersuchungen zeigen, dass SDS toxisch für P. aeruginosa ist und die Bildung von Zellaggregaten einen aktiven und energieabhängigen Prozess darstellt.Aus Kulturen, die mit SDS wuchsen, wurde die nicht aggregierende Spontanmutante Stamm N isoliert. Dieser Stamm bildete glatte Kolonien bei Wachstum auf SDS-haltigen Agarplatten, wohingegen der Wildtyp raue und strukturierte Kolonien auf diesen Medien ausbildete. Aggregation in Flüssigkultur und Bildung rauer Kolonien bei Wachstum mit SDS konnten in Stamm N durch die Expression des Gens PA4929, welches eine putative Guanylatzyklase für die Synthese des Signalmoleküls cyclic di-guanosine monophosphate (c-di-GMP) codiert, wiederhergestellt werden. Die Expression der Phosphodiesterase CC3396 in Stamm PAO1, welche den Abbau von c-di-GMP katalysiert, führte zu einer stark verminderten Aggregation und einem teilweisen Verlust der rauen Koloniemorphologie während des Wachstums mit SDS. Die nicht aggregierenden Stämme N und PAO1[CC3396] wiesen unter starker Energielimitierung eine erheblich geringere Überlebensrate bei der Exposition gegenüber SDS auf. Die Überlebensrate dieser nicht aggregierenden Stämme konnte jedoch durch die Integration von Zellen in die Aggregate von Stamm PAO1 stark erhöht werden. Diese Untersuchungen zeigen, dass die Bildung von Aggregaten keine Voraussetzung für das Wachstum mit SDS ist, jedoch eine erhöhte Überlebensrate unter starker Energielimitierung gewährleistet.Um Gene zu finden, die an der SDS-induzierten Aggregation beteiligt sind, wurde eine Transposonmutagenese durchgeführt. Bei der Mehrzahl der nicht-aggregierenden Transposonmutanten waren zwei Gencluster, das psl- und das cupA Operon, betroffen, welche für die Anheftung von P. aeruginosa an Oberflächen benötigt werden. Das psl Operon kodiert für Proteine, die für die Biosynthese eines extrazellulären Polysaccharids benötigt werden, dessen Monomere vorwiegend aus Mannose und Glucose bestehen. Durch Confocal Laser Scanning Microscopy von Aggregaten, die mit einem spezifischen Lektin gefärbt worden waren, konnten zahlreiche Regionen lokalisiert werden, die Mannose und Glukose enthielten.Das cupA Operon kodiert für Proteine, die für die Bildung adhäsiver Fimbrien benötigt werden. Northern Blot Analysen zeigten eine starke Zunahme des cupA1 Transkriptes in SDS-gewachsenen Zellen von Stamm PAO1 im Vergleich zu Succinat-gewachsenen Zellen. Eine nicht-aggregierende Transposonmutante mit einem Defekt innerhalb des Gens PA0172 zeigte diese Erhöhung des cupA1 Transkriptes nicht. Das Gen PA0172 ist Teil eines Clusters (PA0172-PA0169), dessen Funktion bisher unbekannt war. Durch gezielte Mutation der Gene PA0172 und PA0169 konnte gezeigt werden, dass diese Gene Teil eines putativen c-di-GMP-abhängigen Signaltransduktionswegs sind, der an der transkriptionellen Regulation des cupA Operons beteiligt ist. Sequenzanalysen legen dabei nahe, dass innerhalb eines solchen Signaltransduktionsweges das Genprodukt von PA0172 ein Rezeptor für einen noch unbekannten Reiz darstellt. Die konservierte GGDEF-Domäne des Genprodukts von PA0169 lässt vermuten, dass es sich bei diesem Protein potentiell um eine Guanylatzyklase handelt, die diesen Reiz über das intrazelluläre Signal c-di-GMP weiterleitet. Durch Transkriptionsanalysen wurde die Beteiligung dieser Gene an der transkriptionellen Aktivierung des cupA Operons nachgewiesen. Auch wurden Hinweise gefunden, dass diese Gene zudem an der posttranskriptionellen Stabilisierung der mRNA des cupA Operons beteiligt sind.Die vorliegende Arbeit beschreibt eine neuartige Stressantwort gegenüber toxischen Detergentien, die sowohl physiologisch als auch molekularbiologisch charakterisiert wurde. Dabei konnten durch Identifizierung eines bisher unbekannten Signaltransduktionswegs und dessen Beteiligung an der transkriptionellen Regulation von adhäsiven Oberflächenstukturen neue Erkenntnisse über die komplexen Vorgänge der Aggregation bzw. Biofilmbildung gewonnen werden. Anhand dieser Ergebnisse kann die SDS-induzierte Aggregation einer Teilpopulation als eine adaptive Strategie von Pseudomonas aeruginosa aufgefasst werden, die das Überleben der Gesamtpopulation unter variierenden Umweltbedingungen gewährleistet