Negative regulation of bacterial quorum sensing tunes public goods cooperation

Bacterial quorum sensing (QS) often coordinates the expression of other, generally more costly public goods involved in virulence and nutrient acquisition. In many Proteobacteria, the basic QS circuitry consists of a synthase that produces a diffusible acyl-homoserine lactone and a cognate receptor that activates public goods expression. In some species, the circuitry also contains negative regulators that have the potential to modulate the timing and magnitude of activation. In this study, we experimentally investigated the contribution of this regulatory function to the evolutionary stability of public goods cooperation in the opportunistic pathogen Pseudomonas aeruginosa. We compared fitness and public goods expression rates of strains lacking either qteE or qscR, each encoding a distinct negative regulator, with those of the wild-type parent and a signal-blind receptor mutant under defined growth conditions. We found that (1) qteE and qscR mutations behave virtually identically and have a stronger effect on the magnitude than on the timing of expression, (2) high expression in qteE and qscR mutants imposes a metabolic burden under nutrient conditions that advance induction and (3) high expression in qteE and qscR mutants increases population growth when QS is required, but also permits invasion by both wild-type and receptor mutant strains. Our data indicate that negative regulation of QS balances the costs and benefits of public goods by attenuating expression after transition to the induced state. As the cells cannot accurately assess the amount of cooperation needed, such bet-hedging would be advantageous in changing parasitic and nonparasitic environments.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.     

Coevolution with phages does not influence the evolution of bacterial mutation rates in soil

Coevolution with phages drive the evolution of high bacterial mutation rates in vitro, but the relevance of this finding to natural populations is unclear. Here, we investigated how coevolution affects mutation rate evolution in soil, in the presence and absence of the rest of the natural microbial community. Although mutation rate on average increased threefold, neither coevolving phages nor the rest of natural community significantly affected mutation rates. Our results suggest that features of the soil over and above directly interacting organisms constrain the evolution of strong mutators, helping to explain their relatively low frequency compared with some laboratory and clinical settings.     

Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties

The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied. Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%). Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M. phaseolina exudate. In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM). The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M. phaseolina exudate. In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate. Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate. Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam. In general, filtration coefficient (lambda) of P. fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation. The horizontal movement of P. fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m. The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa). After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil. Motile isolates had significantly greater colonization of M. phaseolina sclerotia over the non-motile mutant.     

Proteomics of a plant growth-promoting rhizobacterium, Pseudomonas fluorescens MSP-393, subjected to salt shock

Summary Pseudomonas fluorescens MSP-393, a plant growth-promoting rhizobacterium is an efficient biocontrol agent in rice grown in saline soils of coastal ecosystems. To understand the mechanism of salt tolerance, proteome analysis of the bacterium was carried out employing two-dimensional gel electrophoresis and MALDI-TOF. This technique was used to investigate the regulation of gene product expression of P. fluorescens MSP-393 grown under high osmolarity and used peptide mass fingerprinting and in silico investigation to identify those proteins with altered expression. Among them 15 were assigned to proteins with known functions. Their roles in response to salt stress are discussed.     

Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

The pyoverdine from Pseudomonas chlororaphis D-TR133 showing mutual acceptance with the pyoverdine of Pseudomonas fluorescens CHA0

From Pseudomonas chlororaphis D-TR133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions. Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate. This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules. An unexpected feature is that the main pyoverdine of P. chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly. So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Revised structures of the pyoverdins from Pseudomonas putida CFBP 2461 and from Pseudomonas fluorescens CFBP 2392

Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, d- and l-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins.     

Styrene-catabolism regulation in Pseudomonas fluorescens ST: phosphorylation of StyR induces dimerization and cooperative DNA-binding

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation has been reported only for a very few response regulators. Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Furthermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.     

Fitness correlates with the extent of cheating in a bacterium

There is growing awareness of the importance of cooperative behaviours in microbial communities. Empirical support for this insight comes from experiments using mutant strains, termed ‘cheats’, which exploit the cooperative behaviour of wild‐type strains. However, little detailed work has gone into characterising the competitive dynamics of cooperative and cheating strains. We test three specific predictions about the fitness consequences of cheating to different extents by examining the production of the iron‐scavenging siderophore molecule, pyoverdin, in the bacterium Pseudomonas aeruginosa. We create a collection of mutants that differ in the amount of pyoverdin that they produce (from 1% to 96% of the production of paired wild types) and demonstrate that these production levels correlate with both gene activity and the ability to bind iron. Across these mutants, we found that (1) when grown in a mixed culture with a cooperative wild‐type strain, the relative fitness of a mutant is negatively correlated with the amount of pyoverdin that it produces; (2) the absolute and relative fitness of the wild‐type strain in the mixed culture is positively correlated with the amount of pyoverdin that the mutant produces; and (3) when grown in a monoculture, the absolute fitness of the mutant is positively correlated with the amount of pyoverdin that it produces. Overall, we demonstrate that cooperative pyoverdin production is exploitable and illustrate how variation in a social behaviour determines fitness differently, depending on the social environment.     

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.