Gene expression reprogramming of Pseudomonas alloputida in response to arginine through the transcriptional regulator ArgR.

Different bacteria change their life styles in response to specific amino acids. In Pseudomonas putida (now alloputida) KT2440, arginine acts both as an environmental and a metabolic indicator that modulates the turnover of the intracellular second messenger c-di-GMP, and expression of biofilm-related genes. The transcriptional regulator ArgR, belonging to the AraC/XylS family, is key for the physiological reprogramming in response to arginine, as it controls transport and metabolism of the amino acid. To further expand our knowledge on the roles of ArgR, a global transcriptomic analysis of KT2440 and a null argR mutant growing in the presence of arginine was carried out. Results indicate that this transcriptional regulator influences a variety of cellular functions beyond arginine metabolism and transport, thus widening its regulatory role. ArgR acts as positive or negative modulator of the expression of several metabolic routes and transport systems, respiratory chain and stress response elements, as well as biofilm-related functions. The partial overlap between the ArgR regulon and those corresponding to the global regulators RoxR and ANR is also discussed.

Exploring the Metabolic Response of Pseudomonas putida to L-arginine.

Beyond their role as protein-building units, amino acids are modulators of multiple behaviours in different microorganisms. In the root-colonizing beneficial bacterium Pseudomonas putida (recently proposed to be reclassified as alloputida) KT2440, current evidence suggests that arginine functions both as a metabolic indicator and as an environmental signal molecule, modulating processes such as chemotactic responses, siderophore-mediated iron uptake or the levels of the intracellular second messenger cyclic diguanylate (c-di-GMP). Using microcalorimetry and extracellular flux analysis, in this work we have studied the metabolic adaptation of P. putida KT2440 to the presence of L-arginine in the growth medium, and the influence of mutations related to arginine metabolism. Arginine causes rapid changes in the respiratory activity of P. putida, particularly magnified in a mutant lacking the transcriptional regulator ArgR. The metabolic activity of mutants affected in arginine transport and metabolism is also altered during biofilm formation in the presence of the amino acid. The results obtained here further support the role of arginine as a metabolic signal in P. putida and the relevance of ArgR in the adaptation to the amino acid. They also serve as proof of concept on the use of calorimetric and extracellular flux techniques to analyse metabolic responses in bacteria and the impact of different mutant backgrounds on such responses.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

The phosphodiesterase RmcA contributes to the adaptation of Pseudomonas putida to l-arginine.

Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3'-5'cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida, KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida, but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation.

Becoming settlers: Elements and mechanisms for surface colonization by Pseudomonas putida.

Pseudomonads are considered to be among the most widespread culturable bacteria in mesophilic environments. The evolutive success of Pseudomonas species can be attributed to their metabolic versatility, in combination with a set of additional functions that enhance their ability to colonize different niches. These include the production of secondary metabolites involved in iron acquisition or having a detrimental effect on potential competitors, different types of motility, and the capacity to establish and persist within biofilms. Although biofilm formation has been extensively studied using the opportunistic pathogen Pseudomonas aeruginosa as a model organism, a significant body of knowledge is also becoming available for non-pathogenic Pseudomonas. In this review, we focus on the mechanisms that allow Pseudomonas putida to colonize biotic and abiotic surfaces and adapt to sessile life, as a relevant persistence strategy in the environment. This species is of particular interest because it includes plant-beneficial strains, in which colonization of plant surfaces may be relevant, and strains used for environmental and biotechnological applications, where the design and functionality of biofilm-based bioreactors, for example, also have to take into account the efficiency of bacterial colonization of solid surfaces. This work reviews the current knowledge of mechanistic and regulatory aspects of biofilm formation by P. putida and pinpoints the prospects in this field.

Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas.

Bacterial biofilm represents a multicellular community embedded within an extracellular matrix attached to a surface. This lifestyle confers to bacterial cells protection against hostile environments, such as antibiotic treatment and host immune response in case of infections. The Pseudomonas genus is characterised by species producing strong biofilms difficult to be eradicated and by an extraordinary metabolic versatility which may support energy and carbon/nitrogen assimilation under multiple environmental conditions. Nutrient availability can be perceived by a Pseudomonas biofilm which, in turn, readapts its metabolism to finally tune its own formation and dispersion. A growing number of papers is now focusing on the mechanism of nutrient perception as a possible strategy to weaken the biofilm barrier by environmental cues. One of the most important nutrients is amino acid L-arginine, a crucial metabolite sustaining bacterial growth both as a carbon and a nitrogen source. Under low-oxygen conditions, L-arginine may also serve for ATP production, thus allowing bacteria to survive in anaerobic environments. L-arginine has been associated with biofilms, virulence, and antibiotic resistance. L-arginine is also a key precursor of regulatory molecules such as polyamines, whose involvement in biofilm homeostasis is reported. Given the biomedical and biotechnological relevance of biofilm control, the state of the art on the effects mediated by the L-arginine nutrient on biofilm modulation is presented, with a special focus on the Pseudomonas biofilm. Possible biotechnological and biomedical applications are also discussed.

Pseudomonas putida and its close relatives: mixing and mastering the perfect tune for plants.

Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms of utmost interest in agricultural biotechnology for their stimulatory and protective effects on plants. Among the various PGPR species, some Pseudomonas putida strains combine outstanding traits such as phytohormone synthesis, nutrient solubilization, adaptation to different stress conditions, and excellent root colonization ability. In this review, we summarize the state of the art and the most relevant findings related to P. putida and its close relatives as PGPR, and we have compiled a detailed list of P. putida sensu stricto, sensu lato, and close relative strains that have been studied for their plant growth-promoting characteristics. However, the mere in vitro analysis of these characteristics does not guarantee correct plant performance under in vivo or field conditions. Therefore, the importance of studying adhesion and survival in the rhizosphere, as well as responses to environmental factors, is emphasized. Although numerous strains of this species have shown good performance in field trials, their use in commercial products is still very limited. Thus, we also analyze the opportunities and challenges related to the formulation and application of bioproducts based on these bacteria. KEY POINTS: •The mini-review updates the knowledge on Pseudomonas putida as a PGPR. • Some rhizosphere strains are able to improve plant growth under stress conditions. • The metabolic versatility of this species encourages the development of a bioproduct.

Role of the Transcriptional Regulator ArgR in the Connection between Arginine Metabolism and c-di-GMP Signaling in Pseudomonas putida.

The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an argR-null mutant grows poorly with arginine as the sole carbon or nitrogen source and also displays increased biofilm formation and reduced surface motility. Modulation of c-di-GMP levels by exogenous arginine requires ArgR. The expression of certain biofilm matrix components, namely, the adhesin LapF and the exopolysaccharide Pea, as well as the diguanylate cyclase CfcR is influenced by ArgR, likely through the alternative sigma factor RpoS. Our data indicate the existence of a regulatory feedback loop between ArgR and c-di-GMP mediated by FleQ. IMPORTANCE Identifying the molecular mechanisms by which metabolic and environmental signals influence the turnover of the second messenger c-di-GMP is key to understanding the regulation of bacterial lifestyles. The results presented here point at the transcriptional regulator ArgR as a central node linking arginine metabolism and c-di-GMP signaling and indicate the existence of a complex balancing mechanism that connects cellular arginine contents and second messenger levels, ultimately controlling the lifestyles of Pseudomonas putida.

C-di-GMP and biofilm are regulated in Pseudomonas putida by the CfcA/CfcR two-component system in response to salts.

In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system.

Genome-Wide Analysis of Targets for Post-Transcriptional Regulation by Rsm Proteins in Pseudomonas putida.

Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins.

The architecture of a mixed fungal-bacterial biofilm is modulated by quorum-sensing signals.

Interkingdom communication is of particular relevance in polymicrobial biofilms. In this work, the ability of the fungus Ophiostoma piceae to form biofilms individually and in consortium with the bacterium Pseudomonas putida, as well as the effect of fungal and bacterial signal molecules on the architecture of the biofilms was evaluated. Pseudomonas putida KT2440 is able to form biofilms through the secretion of exopolysaccharides and two large extracellular adhesion proteins, LapA and LapF. It has two intercellular signalling systems, one mediated by dodecanoic acid and an orphan LuxR receptor that could participate in the response to AHL-type quorum sensing molecules (QSMs). Furthermore, the dimorphic fungus O. piceae uses farnesol as QSM to control its yeast to hyphae morphological transition. Results show for the first time the ability of this fungus to form biofilms alone and in mixed cultures with the bacterium. Biofilms were induced by bacterial and fungal QSMs. The essential role of LapA-LapF proteins in the architecture of biofilms was corroborated, LapA was induced by farnesol and dodecanol, while LapF by 3-oxo-C6-HSL and 3-oxo-C12-HSL. Our results indicate that fungal signals can induce a transient rise in the levels of the secondary messenger c-di-GMP, which control biofilm formation and architecture.

Arginine as an environmental and metabolic cue for cyclic diguanylate signalling and biofilm formation in Pseudomonas putida.

Cyclic diguanylate (c-di-GMP) is a broadly conserved intracellular second messenger that influences different bacterial processes, including virulence, stress tolerance or social behaviours and biofilm development. Although in most cases the environmental cue that initiates the signal transduction cascade leading to changes in cellular c-di-GMP levels remains unknown, certain L- and D-amino acids have been described to modulate c-di-GMP turnover in some bacteria. In this work, we have analysed the influence of L-amino acids on c-di-GMP levels in the plant-beneficial bacterium Pseudomonas putida KT2440, identifying L-arginine as the main one causing a significant increase in c-di-GMP. Both exogenous (environmental) and endogenous (biosynthetic) L-arginine influence biofilm formation by P. putida through changes in c-di-GMP content and altered expression of structural elements of the biofilm extracellular matrix. The contribution of periplasmic binding proteins forming part of amino acid transport systems to the response to environmental L-arginine was also studied. Contrary to what has been described in other bacteria, in P. putida these proteins seem not to be directly responsible for signal transduction. Rather, their contribution to global L-arginine pools appears to determine changes in c-di-GMP turnover. We propose that arginine plays a connecting role between cellular metabolism and c-di-GMP signalling in P. putida.

Plant growth promotion by Pseudomonas putida KT2440 under saline stress: role of eptA.

New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria in agricultural practices. The non-pathogenic bacterium Pseudomonas putida KT2440 is an excellent root colonizer of crops of agronomical importance and has been shown to activate the induced systemic resistance of plants in response to certain foliar pathogens. In this work, we have analyzed additional plant growth promotion features of this strain. We show it can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization. Inoculation with P. putida KT2440 significantly improved seed germination and root and stem length of soybean and corn plants under saline conditions compared to uninoculated plants, whereas the effects were minor under non-saline conditions. Also, random transposon mutagenesis was used for preliminary identification of KT2440 genes involved in bacterial tolerance to saline stress. One of the obtained mutants was analyzed in detail. The disrupted gene encodes a predicted phosphoethanolamine-lipid A transferase (EptA), an enzyme described to be involved in the modification of lipid A during lipopolysaccharide (LPS) biosynthesis. This mutant showed changes in exopolysaccharide (EPS) production, low salinity tolerance, and reduced competitive fitness in the rhizosphere.

Characterization of the bacteriocins and the PrtR regulator in a plant-associated Pseudomonas strain.

The emergence of antibiotic resistant bacterial strains demands the development of new antimicrobial agents. In the last decades, bacteriocins have gained significant interest due to their potential application as biopreservatives in the food industry and as therapeutic agents in medicine. Recent studies project the use of these antimicrobials in agriculture as biocontrol agents. The characterization of bacteriocins and their genetic regulation, however, have been scarcely studied in plant-associated bacteria. In this report, an in-silico and proteomic analysis was performed to identify the bacteriocins produced by Pseudomonas fluorescens SF4c. More than one functional bacteriocin was detected in this strain (S-type bacteriocins and phage-tail-like bacteriocins [tailocins]). It is known that the regulator PrtR represses bacteriocin production in P. aeruginosa under normal condition. However, the mechanism for tailocin regulation remains unknown in plant-associated pseudomonads. In this work, an orthologue of the prtR of P. aeruginosa was identified in the SF4c-tailocin cluster and a prtR null mutant constructed. The expression and production of tailocins was abolished in this mutant; thus evidencing that, unlike P. aeruginosa, PrtR is a positive regulator of tailocins expression in P. fluorescens.

Arginine Biosynthesis Modulates Pyoverdine Production and Release in Pseudomonas putida as Part of the Mechanism of Adaptation to Oxidative Stress.

Iron is essential for most life forms. Under iron-limiting conditions, many bacteria produce and release siderophores-molecules with high affinity for iron-which are then transported into the cell in their iron-bound form, allowing incorporation of the metal into a wide range of cellular processes. However, free iron can also be a source of reactive oxygen species that cause DNA, protein, and lipid damage. Not surprisingly, iron capture is finely regulated and linked to oxidative-stress responses. Here, we provide evidence indicating that in the plant-beneficial bacterium Pseudomonas putida KT2440, the amino acid l-arginine is a metabolic connector between iron capture and oxidative stress. Mutants defective in arginine biosynthesis show reduced production and release of the siderophore pyoverdine and altered expression of certain pyoverdine-related genes, resulting in higher sensitivity to iron limitation. Although the amino acid is not part of the siderophore side chain, addition of exogenous l-arginine restores pyoverdine release in the mutants, and increased pyoverdine production is observed in the presence of polyamines (agmatine and spermidine), of which arginine is a precursor. Spermidine also has a protective role against hydrogen peroxide in P. putida, whereas defects in arginine and pyoverdine synthesis result in increased production of reactive oxygen species.IMPORTANCE The results of this study show a previously unidentified connection between arginine metabolism, siderophore turnover, and oxidative stress in Pseudomonas putida Although the precise molecular mechanisms involved have yet to be characterized in full detail, our data are consistent with a model in which arginine biosynthesis and the derived pathway leading to polyamine production function as a homeostasis mechanism that helps maintain the balance between iron uptake and oxidative-stress response systems.

The Pseudomonas putida CsrA/RsmA homologues negatively affect c-di-GMP pools and biofilm formation through the GGDEF/EAL response regulator CfcR.

Expression of cfcR, encoding the only GGDEF/EAL response regulator in Pseudomonas putida, is transcriptionally regulated by RpoS, ANR and FleQ, and the functionality of CfcR as a diguanylate cyclase requires the multisensor CHASE3/GAF hybrid histidine kinase named CfcA. Here an additional level of cfcR control, operating post-transcriptionally via the RNA-binding proteins RsmA, RsmE and RsmI, is unraveled. Specific binding of the three proteins to an Rsm-binding motif (5'CANGGANG3') encompassing the translational start codon of cfcR was confirmed. Although RsmA exhibited the highest binding affinity to the cfcR transcript, single deletions of rsmA, rsmE or rsmI caused minor derepression in CfcR translation compared to a ΔrsmIEA triple mutant. RsmA also showed a negative impact on c-di-GMP levels in a double mutant ΔrsmIE through the control of cfcR, which is responsible for most of the free c-di-GMP during stationary phase in static conditions. In addition, a CfcR-dependent c-di-GMP boost was observed during this stage in ΔrsmIEA confirming the negative effect of Rsm proteins on CfcR translation and explaining the increased biofilm formation in this mutant compared to the wild type. Overall, these results suggest that CfcR is a key player in biofilm formation regulation by the Rsm proteins in P. putida.

Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures.

Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 µM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.

FleQ of Pseudomonas putida KT2440 is a multimeric cyclic diguanylate binding protein that differentially regulates expression of biofilm matrix components.

The intracellular signal molecule cyclic di-GMP (c-di-GMP) is an important element in regulation of biofilm formation by bacteria. In Pseudomonas aeruginosa, FleQ functions as a c-di-GMP-dependent transcriptional regulator of expression of flagellar genes and the exopolysaccharide (EPS) Pel, a component of the biofilm extracellular matrix. In the plant-beneficial bacterium Pseudomonas putida KT2440, a mutation in fleQ reduces biofilm formation and colonization of plant surfaces. Using isothermal titration calorimetry and electrophoretic mobility shift assays, we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes. Data obtained by analytical gel filtration and ultracentrifugation indicate that FleQ is in multiple oligomeric states in solution (dimers, tetramers and hexamers), which do not show altered equilibrium in the presence of c-di-GMP. DNA binding is independent of c-diGMP, although it is favored by the second messenger in the case of the promoter of the operon responsible for synthesis of the species-specific EPS Pea. Analysis of expression using transcriptional fusions showed an influence of FleQ upon two of the four EPS operons under regular growth conditions. Finally, a consensus sequence for promoter recognition by FleQ in P. putida is also proposed.