Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44

Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ～6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids.     

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media.

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm(3) H(2)O/cm(3) of sand, respectively), and at cell densities ranging between 1 x 10(6) and 8.5 x 10(8) cells/ml. Before O(2) availability became a limiting factor, the rate of light emission (L) increased with the square of time (t) and linearly with increasing cell density (c). A nonlinear model was developed that contains a "rate of increase in light emission" constant, B', which is determined directly from the slope of a plot of radical L/c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B' values were determined [49.0-57.6 x 10(-10) light units/(cell min(2))] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B'=61.6+/-3.2, log phase cells plus glucose, B'=63.2+/-7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.     

Siderophore cooperation of the bacterium Pseudomonas fluorescens in soil

While social interactions play an important role for the evolution of bacterial siderophore production in vitro, the extent to which siderophore production is a social trait in natural populations is less clear. Here, we demonstrate that siderophores act as public goods in a natural physical environment of Pseudomonas fluorescens: soil-based compost. We show that monocultures of siderophore producers grow better than non-producers in soil, but non-producers can exploit others'' siderophores, as shown by non-producers'' ability to invade populations of producers when rare. Despite this rare advantage, non-producers were unable to outcompete producers, suggesting that producers and non-producers may stably coexist in soil. Such coexistence is predicted to arise from the spatial structure associated with soil, and this is supported by increased fitness of non-producers when grown in a shaken soil–water mix. Our results suggest that both producers and non-producers should be observed in soil, as has been observed in marine environments and in clinical populations.     

一株荧光假单胞杆菌的分离鉴定与反硝化特性

【目的】从污水厂的活性污泥中获得一株高效反硝化细菌。【方法】采用低温驯化,进行初筛、复筛选取一株反硝化活性最高的菌株,命名为L2,通过形态学、生理生化特征及16S r RNA基因序列分析研究其分类地位,系统研究理化因素对该菌株反硝化性能的影响。【结果】菌株在低温条件下能够稳定高效地进行反硝化,鉴定该菌株为荧光假单胞杆菌(Pseudomonas fluorescens),其反硝化最适接种量为10%,温度为20°C,p H为7.0,盐浓度为0.5%,碳源为葡萄糖,C/N为5.0,能够耐受较高初始硝态氮浓度。【结论】菌株L2是一株耐低温、耐高浓度初始硝态氮、耐低C/N、兼性厌氧、高效反硝化的荧光假单胞杆菌。     

Automatic formation of hypotheses on the relationships between structure of naphthalene analogs and bioluminescence response of bioreporter <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HK44

Seven hypotheses on relationships between the structure of naphthalene analogs and bioluminescence response of bioreporter Pseudomonas fluorescens were formulated using GUHA (General Unary Hypotheses Automaton) on a training set of 37 compounds. Prediction of bioluminescence response of 12 new naphthalene analogs was successful in 69 % cases and resulted in rejection of single hypothesis. The results demonstrate applicability of GUHA in structure-activity research, especially for qualitative data.     

Cheating and resistance to cheating in natural populations of the bacterium Pseudomonas fluorescens

Bacteria perform cooperative behaviors that are exploitable by noncooperative cheats, and cheats frequently arise and coexist with cooperators in laboratory microcosms. However, evidence of competitive dynamics between cooperators and cheats in nature remains limited. Using the production of pyoverdine, an iron‐scavenging molecule, and natural soil populations of Pseudomonas fluorescens, we found that (1) nonproducers are present in the population; (2) they co‐occur (<1cm³) with pyoverdine producers; (3) they retain functional pyoverdine receptors; and (4) they can use the pyoverdine of on average 52% of producers. This suggests nonproducers can potentially act as social cheats in soil: utilizing the pyoverdine of others while producing little or none themselves. However, we found considerable variation in the extent to which nonproducers can exploit producers, as some isolates appear to produce exclusive forms of pyoverdine or kill nonproducers with toxins. We examined the consequences of this variation using theoretical modeling. We found variance in exploitability leads to some cheats gaining increased fitness benefits and others decreased benefits. However, the absolute gain in fitness from high exploitation is lower than the drop in fitness from low exploitation, decreasing the mean fitness of cheats and subsequently lowering the proportion of cheats maintained in the population. Our results suggest that although cooperator‐cheat dynamics can occur in soil, a range of mechanisms can prevent nonproducers from exploiting producers.     

Influence of different growth conditions on the survival and the efficacy of freeze-dried Pseudomonas fluorescens strain Pf153

High viability, storability and tolerance to variable environmental conditions are key factors in the development of microbial biological control agents (BCAs). The efficacy of microbial BCAs is influenced by the culture conditions and formulation process. Therefore, we investigated the influence of diverse growth conditions on the survival during freeze-drying and on the biocontrol efficacy of Pseudomonas fluorescens strain Pf153. Culture time, temperature and media, mild heat shock and pH change influenced the bacterium viability after freeze-drying. The best survival rate was reached by cultivation in King’s broth for 16 or 20 h. Growth temperatures of 25 and 30°C and a mild heat shock at 35°C for one hour influenced the survival rate positively. In all bioassays against Botrytis cinerea on Vicia faba leaves, Pf153 showed a significant increased efficacy compared to the untreated control. No differences of the efficacy between fresh and freeze-dried cells were observed. Furthermore, a growth temperature of 20°C increased the efficacy of Pf153 against B. cinerea. These results underline that the quality of the formulated product can be improved by manipulating the fermentation process.     

Some factors affecting airborne survival of Pseudomonas fluorescens indoors

The effect of relative humidity (RH) on the airborne survival of a Pseudomonas fluorescens strain was studied at 20, 40, 60 and 80% RH indoors. The aero-stable spore of Bacillus subtilis subsp. niger , used as a tracer of physical losses was compared with a light scattering particle counter, as there were doubts about the reliability of the spore as tracer. The Rion counter was validated before use and found to give a good estimate of relative physical losses providing spray suspensions contained between 10⁷ and 10⁹ cfu ml⁻¹ cells and that the humidity was not more than 80% RH. Pseudomonas fluorescens , strain P⁺ S⁺, suspended in distilled water survived best at mid humidities and least at 80% RH. When suspended in 1% glycerol there was an apparent 'increase' in viability after an initial rapid reduction. This was thought to be due to delay in equilibration of glycerol in the cell membrane after concentration on dehydration. The cells were thought to be unstable and sensitive to the stress of rehydration before equilibration occurred. The findings are discussed in relation to Cox's theories of outer membrane damage on aerosolization.     

Influence of diverse nucleotide constituents of Pseudomonas fluorescens on the D-glucose-dehydrogenase activity of the bacterium

The D-glucose-dehydrogenase activity of P. fluorescens is enhanced or depressed by some nucleotides synthetised by this bacterium: stimulatory nucleotides have been separated from inhibitory. UTP exhibits a non-competitive inhibition upon the pure soluble enzyme.     

Bioluminescence of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HK44 in the course of encapsulation into silica gel. Effect of methanol

The bioluminescence (BLM) and colony-forming units (CFU) of Pseudomonas fluorescens HK44 were monitored during encapsulation into pre-polymerized Si(OMe)₄. The non-induced BLM of free cells was increased in the presence of 0.5–2.5 % MeOH. After mixing silica sol with the cell suspension, both BLM and CFU dropped to 1–3 and 8–18 %, respectively; both remained lowered as long as the silica biofilm contained residual MeOH. The kinetics of MeOH being released from silica biofilms (a thickness of 2–6 mm) were first-order. The decrease of bacterial activity due to encapsulation was proportional to the biofilm thickness. MeOH evolving during encapsulation is probably the principal stress factor but not the only one.     

Kinetics of Pseudomonas fluorescens growth under different conditions of culturing

The dynamics of Pseudomonas fluorescens VKM-1472 growth was studied under the conditions of batch and continuous cultivation, and the behaviour of the organism was investigated upon nutrition shifts and starvation. At D greater than 0.375 h-1, just as in the batch culture with a substrate excess, the strain realised excessive metabolism [15]: the yield in terms of the substrate fell down (38% for the batch culture and 40% for the continuous culture), the titre of viable cells decreased to a considerable extent, and maintenance expenditures increased (3 times for qgluc and 7.5 times for qO2). When the culture was incubated under the oligotrophous conditions, the biomass yield decreased fourfold within 8 days and the titre of viable cells dropped down twofold within the same period of time. However, the organism was still capable of oxidising a wide range of substrates (17 substrates were studied). As soon as a substrate was added, it was oxidised at a high rate and changes in the macromolecular composition were characteristic of an "up-jump" in the growth rate [11]. It is possible that the growth and behaviour of this organism are associated with its ecological niche occupied in natural systems.     

Integrated bioinformatic and phenotypic analysis of RpoN-dependent traits in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25

The alternative sigma factor RpoN is a key regulator in the acclimation of Pseudomonas to complex natural environments. In this study we show that RpoN is required for efficient colonization of sugar beet seedlings by the plant growth-promoting bacterium Pseudomonas fluorescens SBW25, and use phenotypic and bioinformatic approaches to profile the RpoN-dependent traits and genes of P. fluorescens SBW25. RpoN is required for flagellar biosynthesis and for assimilation of a wide variety of nutrient sources including inorganic nitrogen, amino acids, sugar alcohols and dicarboxylic acids. Chemosensitivity assays indicate that RpoN-regulated genes contribute to acid tolerance and resistance to some antibiotics, including tetracyclines and aminoglycosides. Gain of function changes associated with loss of RpoN included increased tolerance to hydroxyurea and Guanazole. Bioinformatic predictions of RpoN-regulated genes show a close correspondence with phenotypic analyses of RpoN-regulated traits and suggest novel functions for RpoN in P. fluorescens, including regulation of poly(A) polymerase. The reduced plant colonization ability observed for an rpoN mutant of P. fluorescens is therefore likely to be due to defects in multiple traits including nutrient assimilation, protein secretion and stress tolerance.     

Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens.

In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MF0 is maximal at a growth temperature of 17.5 degrees C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MF0, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5 degrees C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.     

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

Abstract

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm², and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.     

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.