Search

The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. Although these are mostly focussed on the genetic components alone, the context in which the circuit performs is crucial for its outcome. We characterise 20 genetic NOT logic gates in up to 7 bacterial-based contexts each, to generate 135 different functions. The contexts we focus on are combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each gate shows seven different dynamic behaviours, depending on the context. That is, gates can be fine-tuned by changing only contextual parameters, thus improving the compatibility between gates. Finally, we analyse portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expands functionality, and advocate for considering context as a fundamental design parameter.

Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli , was performed in a sandwich reaction with E. coli -specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 10 4 CFU mL -1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis , Enterobacter agglomerans , Pseudomonas putida , Staphylococcus aureus , and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ± 0.03 μM/μl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ± 0.02 μM/μl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes. We also explore the role of the transmembrane (TM) and PAS domains of CbrA in the signal recognition. A ΔcbrXA mutant lacking its TM domains is uncoupled in its growth in histidine and citrate as carbon sources, but its overexpression restores the ability to grow in such carbon sources. In these conditions ΔTM-CbrA is able to respond to carbon availability, thus suggesting an intracellular nature for the signal sensed.

Identifying the zinc (Zn) ligation and coordination environment in complex biological and environmental systems is crucial to understand the role of Zn as a biologically essential but sometimes toxic metal. Most studies on Zn coordination in biological or environmental samples rely on the extended X-ray absorption fine structure (EXAFS) region of a Zn K-edge X-ray absorption spectroscopy (XAS) spectrum. However, EXAFS analysis cannot identify unique nearest neighbors with similar atomic number (i.e., O versus N) and provides little information on Zn ligation. Herein, we demonstrate that high energy resolution-X-ray absorption near edge structure (HR-XANES) spectroscopy enables the direct determination of Zn ligation in whole cell bacteria, providing additional insights lost from EXAFS analysis at a fraction of the scan time and Zn concentration. HR-XANES is a relatively new technique that has improved our understanding of trace metals (e.g., Hg, Cu, and Ce) in dilute systems. This study is the first to show that HR-XANES can unambiguously detect Zn coordination to carboxyl, phosphoryl, imidazole, and/or thiol moieties in model microorganisms.

Eradication of P. putida F1 was investigated as a function of current density in pulsed electric fields of 6.7, 4, 2.8, 2 and 1 kV/cm. The pulse numbers were 200, 2000, 5000 and 10,000 and were performed by a series of trains of 500 pulses (except for the 200 pulses). The frequency was 100 Hz and pulse durations were 10 μs or 20 μs as indicated for each experiment. The current density range was 0.02 ± 0.01 to 5.2 ± 0.5 Acm -2 . A clear tendency for increasing bacterial death was found as a result of increasing the current density in each of the tested electric field strengths. The total bacterial eradication when the electric field was reduced from 4 to 1 kV/cm was obtained at a higher current density, 2 ± 0.2 and 5.2 ± 0.5 Acm -2 , respectively. Increasing the current density led to higher cell permeability and larger bacteria size. The percentage of propidium iodide permeability in an electric field of 1 kV/cm at a current density of 5.2 ± 0.5 Acm -2 was 65 ± 0.3% compared to the control that was only 10 ± 0.9%. The cell size at 1 kV/cm in a current density of 5.2 ± 0.5 Acm -2 was about 3-fold higher compared to untreated cells. To the best of our knowledge, this is the first study that evaluated the influence of increasing current density on bacterial eradication in moderate electric fields., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sepsis is a common and often deadly systemic response to an infection, usually caused by bacteria. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality. To replace the time-consuming blood culture we are developing a method to directly separate bacteria from red and white blood cells to enable faster bacteria identification. The blood cells are moved from the sample flow into a parallel stream using acoustophoresis. Due to their smaller size, the bacteria are not affected by the acoustic field and therefore remain in the blood plasma flow and can be directed to a separate outlet. When optimizing for sample throughput, 1 ml of undiluted whole blood equivalent can be processed within 12.5 min, while maintaining the bacteria recovery at 90% and the blood cell removal above 99%. That makes this the fastest label-free microfluidic continuous flow method per channel to separate bacteria from blood with high bacteria recovery (>80%). The high throughput was achieved by matching the acoustic impedance of the parallel stream to that of the blood sample, to avoid that acoustic forces relocate the fluid streams.

Microbe-associated aluminum (Al) hydroxides occur naturally in aquatic and geologic environments and they might play a crucial role in the sequestration of trace metals because these composite solids comprise both reactive mineral and organic surface, but how they do it still remains unknown. Here we replicate Al hydroxide organo-mineral composite formation in soil and sediments by synthesising composites using Pseudomonas putida cells, during coprecipitation with Al hydroxide. Morphological and ATR-FTIR analysis show closely attached nano-sized Al hydroxides on the bacterial surface. For composites dominated by either bacteria or Al hydroxide, an enhanced metal adsorption is observed on the composites than on pure Al hydroxide at pH < 6. Cd uptake by the mainly Al mineral composite is approximately additive, i.e., the sum of the end-member metal adsorptivities, whereas that on the mainly bacteria composite is non-additive. This non-additive sorption is not only due to the blockage of surface reactive sorption sites, but more importantly the changes of surface charge when bacteria and Al mineral are interacted. EXAFS results show that Cd is predominately sorbed as a bidentate corner-sharing complex on the amorphous Al hydroxide surface and a carboxyl-binding on the bacterial surface. This study has important implications for understanding both Al and trace metal cycling in microbe-rich geologic environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacteria swim in sequences of straight runs that are interrupted by turning events. They drive their swimming locomotion with the help of rotating helical flagella. Depending on the number of flagella and their arrangement across the cell body, different run-and-turn patterns can be observed. Here, we present fluorescence microscopy recordings showing that cells of the soil bacterium Pseudomonas putida that are decorated with a polar tuft of helical flagella, can alternate between two distinct swimming patterns. On the one hand, they can undergo a classical push-pull-push cycle that is well known from monopolarly flagellated bacteria but has not been reported for species with a polar bundle of multiple flagella. Alternatively, upon leaving the pulling mode, they can enter a third slow swimming phase, where they propel themselves with their helical bundle wrapped around the cell body. A theoretical estimate based on a random-walk model shows that the spreading of a population of swimmers is strongly enhanced when cycling through a sequence of pushing, pulling, and wrapped flagellar configurations as compared to the simple push-pull-push pattern.

This paper evaluated the external gelation technique for preparing microcapsules. The microcapsules were consisted of Pseudomonas putida Rs-198 (Rs-198) core and sodium alginate (NaAlg)-bentonite (Bent) shell. Different emulsification rotation speeds and core/shell ratios were used to prepare the microcapsules of each formulation. The near-spherical microcapsules were monodisperse with a mean diameter of 25-100 μm and wrinkled surfaces. Fourier transform infrared spectrophotometry (FTIR) and thermogravimetric analysis (TGA) revealed the physical mixture of the wall material and the superior thermal stability of the microcapsules. Percentage yield, water content, and encapsulation efficiency were evaluated and correlated with the changes in emulsification rotation speed and core/shell ratio. In vitro release experiments demonstrated that 60% of the bacteria were released from the NaAlg-Bent microcapsules within three days. Considerably better survival was observed for encapsulated cells compared to free cells, especially in pH 4.0 and 10.0. In summary, the desired properties of microcapsules can be obtained by external gelation technique and the microcapsules on the bacteria had a good protective effect.

Terpenes are a class of natural compounds that have recently moved into the focus as a bio-based resource for chemical production, owing to their abundance, their mostly cyclic structures, and the presence of olefin or single hydroxy groups. To apply this raw material in new industrial fields, a second hydroxy group is inserted into borneol by cytochrome P450cam (CYP101) enzymes in a whole-cell catalytic biotransformation with Pseudomonas putida KT2440. Next, a semi-continuous batch system was developed to produce 5-exo-hydroxyborneol with a final concentration of 0.54 g L -1 . The bifunctional terpene was then used for the synthesis of a bio-based polyester by a solvent-free polycondensation reaction. The resulting polymer showed a glass transition temperature of around 70 °C and a molecular weight in the range of 2000-4000 g mol -1 (M w ). These results show that whole-cell catalytic biotransformation of terpenes could lead to bio-based, higher-functionalized monomers, which might be basic raw materials for different fields of application, such as biopolymers., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many bacteria perform a run-and-tumble random walk to explore their surrounding and to perform chemotaxis. In this article we present a novel method to infer the relevant parameters of bacterial motion from experimental trajectories including the tumbling events. We introduce a stochastic model for the orientation angle, where a shot-noise process initiates tumbles, and analytically calculate conditional moments, reminiscent of Kramers-Moyal coefficients. Matching them with the moments calculated from experimental trajectories of the bacteria E. coli and Pseudomonas putida, we are able to infer their respective tumble rates, the rotational diffusion constants, and the distributions of tumble angles in good agreement with results from conventional tumble recognizers. We also define a novel tumble recognizer, which explicitly quantifies the error in recognizing tumbles. In the presence of a chemical gradient we condition the moments on the bacterial direction of motion and thereby explore the chemotaxis strategy. For both bacteria we recover and quantify the classical chemotactic strategy, where the tumble rate is smallest along the chemical gradient. In addition, for E. coli we detect some cells, which bias their mean tumble angle towards smaller values. Our findings are supported by a scaling analysis of appropriate ratios of conditional moments, which are directly calculated from experimental data., Competing Interests: The authors have declared that no competing interests exist.

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida., Competing Interests: The authors have declared that no competing interests exist.

Efflux pumps are critically important membrane components that play a crucial role in strain tolerance in Pseudomonas putida to antibiotics and aromatic hydrocarbons that result in these toxicants being expelled from the bacteria. Here, the effect of propranolol on P. putida was examined by sudden addition of 0.2, 0.4 and 0.6 mg mL-1 of this β-blocker to several strains of P. putida, including the wild type DOT-T1E and the efflux pump knockout mutants DOT-T1E-PS28 and DOT-T1E-18. Bacterial viability measurements reveal that the efflux pump TtgABC plays a more important role than the TtgGHI pump in strain tolerance to propranolol. Mid-infrared (MIR) spectroscopy was then used as a rapid, high-throughput screening tool to investigate any phenotypic changes resulting from exposure to varying levels of propranolol. Multivariate statistical analysis of these MIR data revealed gradient trends in resultant ordination scores plots, which were related to the concentration of propranolol. MIR illustrated phenotypic changes associated with the presence of this drug within the cell that could be assigned to significant changes that occurred within the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during the growth of bacteria following toxic perturbation with the same concentration levels of propranolol. Metabolic profiling revealed that ornithine, which was only produced by P. putida cells in the presence of propranolol, presents itself as a major metabolic feature that has important functions in propranolol stress tolerance mechanisms within this highly significant and environmentally relevant species of bacteria.

Bacteria typically form biofilms under natural conditions. To elucidate the effect of the carriage of carbazole-degradative plasmid pCAR1 on biofilm formation by host bacteria, we compared the biofilm morphology, using confocal laser scanning microscopy, of three pCAR1-free and pCAR1-carrying Pseudomonas hosts: P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Although pCAR1 did not significantly affect biofilm formation by PAO1 or Pf0-1, pCAR1-carrying KT2440 became filamentous and formed flat biofilms, whereas pCAR1-free KT2440 formed mushroom-like biofilms. pCAR1 contains three genes encoding nucleoid-associated proteins (NAPs), namely, Pmr, Pnd and Phu. The enhanced filamentous morphology was observed in two double mutants [KT2440(pCAR1ΔpmrΔpnd) and KT2440(pCAR1ΔpmrΔphu)], suggesting that these NAPs are involved in modulating the filamentous phenotype. Transcriptome analyses of the double mutants identified 32 candidate genes that may be involved in filamentation of KT2440. Overexpression of PP&#95;2193 in KT2440 induced filamentation and overexpression of PP&#95;0308 or PP&#95;0309 in KT2440(pCAR1) enhanced filamentation of cells over time. This suggests that pCAR1 induces development of an abnormal filamentous morphology by KT2440 via a process involving overexpression of several genes, such as PP&#95;2193. In addition, pCAR1-encoded NAPs partly suppress too much filamentation of KT2440(pCAR1) by repressing transcription of some genes, such as PP&#95;0308 and PP&#95;0309., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.

Chemotaxis has been shown to be beneficial for the migration of soil-inhabiting bacteria towards industrial chemical pollutants, which they degrade. Many studies have demonstrated the importance of this microbial property under various circumstances; however, few quantitative analyses have been undertaken to measure the two essential parameters that characterize the chemotaxis of bioremediation bacteria: the chemotactic sensitivity coefficient χ(0) and the chemotactic receptor constant K(c). The main challenge to determine these parameters is that χ(0) and K(c) are coupled together in non-linear mathematical models used to evaluate them. In this study we developed a method to accurately measure these parameters for Pseudomonas putida in the presence of toluene, an important pollutant in groundwater contamination. Our approach uses a multilayer microfluidic device to expose bacteria to a convection-free linear chemical gradient of toluene that is stable over time. The bacterial distribution within the gradient is measured in terms of fluorescence intensity, and is then used to fit the parameters Kc and χ(0) with mathematical models. Critically, bacterial distributions under chemical gradients at two different concentrations were used to solve for both parameters independently. To validate the approach, the chemotaxis parameters of Escherichia coli strains towards α-methylaspartate were experimentally derived and were found to be consistent with published results from related work., (© 2014 Wiley Periodicals, Inc.)

Optical long-term observation of individual cells, combined with modern data analysis tools, allows for a detailed study of cell-to-cell variability, heredity, and differentiation. We developed a microfluidic device featuring facile cell loading, simple and robust operation, and which is amenable to high-resolution life-cell imaging. Different cell strains can be grown in parallel in the device under constant or changing media perfusion without cross-talk between the cell ensembles. The culturing chamber has been optimized for use with nonadherent cells, such as Saccharomyces cerevisiae, and enables controlled colony growth over multiple generations under aerobic or anaerobic conditions. Small changes in the layout will make the device also useable with bacteria or mammalian cells. The platform can be readily set up in every laboratory with minimal additional requirements and can be operated without technology training.

Pseudomonas putida N, a poly-3-hydroxyalkonate (PHA)-producing bacterium, showing ampicillin resistance, is an unusual strain. In the presence of this antibiotic, it grows as giant cells (25-50μm) forming complex networks inter-connected by micro-tubular structures. The transformation of this bacterium with a plasmid containing the gene phaF, which encodes a phasin involved in the molecular architecture of the PHA-granules, (i) restores the wild-type phenotype by reducing both bacterial size and length (coco-bacilli ranging between 0.5 and 3μm), and (ii) increases ampicillin resistance by more than 100-fold., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A novel fluorescence probe capable of assessing the cytoplasmic entry of siderophore-based conjugates was synthesized and evaluated by photochemical characterization and cell-based assays. The specific responsiveness to the cytoplasmic entry of the probe was implemented by adopting a disulfide linker, whose cleavage under the reducing conditions of the cytoplasm induced the display of a distinctive fluorescence signal.

In microcystin-LR (MC-LR) degradation system, the change in surface characteristics and cell viability of Pseudomonas putida was studied. The purpose of this study was to reveal the influence of MC-LR on P. putida and elucidate the toxicity of MC-LR on microorganisms. The result demonstrated that MC-LR enhanced the cytoplasmic membrane permeability, as well as affected the ion metabolism and protein release of P. putida. The soluble sugar and Na+, Cl-release increased with the rising concentration of MC-LR ranging from 0 mg x L(-1) to 2.0 mg x L(-1). Flow Cytometry Method(FCM) analysis revealed that MC-LR accelerated the death of P. putida, and the death rate increased with the ascending concentration of MC-LR. Compared with the control, the death rate on day 5 increased by nearly 30% when 2.5 mg x L(-1) MC-LR was added. Scanning electron microscopy (SEM) analysis showed that the cells were deformed under the toxicity of MC-LR. After 5-day exposure to 2.5 mg x L(-1) MC-LR, the majority of the cells were ruptured and the intracellular materials flew out. The cellular structure was severely damaged under this condition.

Intensive microbial growth typically observed in laboratory rarely occurs in nature. Because of severe nutrient deficiency, natural populations exhibit near-zero growth (NZG). There is a long-standing controversy about sustained NZG, specifically whether there is a minimum growth rate below which cells die or whether cells enter a non-growing maintenance state. Using chemostat with cell retention (CCR) of Pseudomonas putida, we resolve this controversy and show that under NZG conditions, bacteria differentiate into growing and VBNC (viable but not non-culturable) forms, the latter preserving measurable catabolic activity. The proliferating cells attained a steady state, their slow growth balanced by VBNC production. Proteomic analysis revealed upregulated (transporters, stress response, self-degrading enzymes and extracellular polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic enzymes) proteins in the CCR versus batch culture. Based on these profiles, we identified intracellular processes associated with NZG and generated a mathematical model that simulated the observations. We conclude that NZG requires controlled partial self-digestion and deep reconfiguration of the metabolic machinery that results in the biosynthesis of new products and development of broad stress resistance. CCR allows efficient on-line control of NZG including VBNC production. A well-nuanced understanding of NZG is important to understand microbial processes in situ and for optimal design of environmental technologies., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4 was studied. Through using PEG as porogen, the environmental tolerance as well as the biocatalytic reaction efficiency of immobilized cells was greatly improved, i.e., Ca-alginate-PEG immobilized cells got better temperature and substrate concentration tolerance than Ca-alginate immobilized cells and showed similar efficiency with free cells, suggesting that the intrinsic mass transfer resistance of immobilization obviously decreased. It was also observed that the pore diameter and porosity of immobilization beads were related with the molecular weight of PEG. PEG400 was found to be a relatively suitable porogen for Ca-alginate-PEG immobilized cells catalyzed hydrolysis of glycinonitrile. It was noteworthy that the Ca-alginate-PEG immobilized cells could be reused more than 18 times with little loss of enzyme activity which had shown good operation ability and great application potential.

Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd) resistant and sensitive bacteria, respectively to study the effect of Cd on physicochemical surface properties which include the study of surface charge and hydrophobicity which are subjected to vary under stress conditions. In this research work, effective concentration 50 (EC50 ) was calculated to exclude the doubt that dead cells were also responding and used as reference point to study the changes in cell surface properties in the presence of Cd. EC50 of C. metallidurans CH34 was found to be 2.5 and 0.25 mM for P. putida mt2. The zeta potential analysis showed that CH34 cells were slightly less unstable than mt2 cells as CH34 cells exhibited -8.5 mV more negative potential than mt2 cells in the presence of Cd in growth medium. Cd made P. putida mt2 surface to behave as intermediate hydrophilic (θw  = 25.32°) while C. metallidurans CH34 as hydrophobic (θw  = 57.26°) at their respective EC50 . Although belonging to the same gram-negative group, both bacteria behaved differently in terms of changes in membrane fluidity. Expression of trans fatty acids was observed in mt2 strain (0.45%) but not in CH34 strain (0%). Similarly, cyclopropane fatty acids were observed more in mt2 strain (0.06-0.14%) but less in CH34 strain (0.01-0.02%). Degree of saturation of fatty acids decreased in P. putida mt2 (36.8-33.75%) while increased in C. metallidurans CH34 (35.6-39.3%). Homeoviscous adaptation is a survival strategy in harsh environments which includes expression of trans fatty acids and cyclo fatty acids in addition to altered degree of saturation. Different bacteria show different approaches to homeoviscous adaptation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reporter genes are widely used to quantify promoter activity, which controls production of mRNA through the interplay with RNA polymerases and transcription factors. Some of such reporters have either diffuse (lux) or focused (GFP) optical outputs that allow description of transcriptional activity in populations and in single cells. This chapter discusses the use of a dual reporter system GFP-luxCDABE that is placed in broad-host-range plasmids having origins of replication from RK2 and pBBR1. The value of this system is shown in Pseudomonas putida by characterizing the activity of the Pb promoter, which drives an operon for benzoate biodegradation in this bacterium. To this end we compare in the same cells bioluminescence as the output signal of the whole population and single cell-bound fluorescence caused by GFP expression and revealed by flow cytometry assays.

Atomic force microscopy (AFM) in conjunction with a bioprobe developed using a polydopamine wet adhesive was used to directly measure the adhesive force between bacteria and different polymeric membrane surfaces. Bacterial cells of Pseudomonas putida and Bacillus subtilis were immobilized onto the tip of a standard AFM cantilever, and force measurements made using the modified cantilever on various membranes. Interaction forces measured with the bacterial probe were compared, qualitatively, to predictions by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory with steric interactions included. The XDLVO theory predicted attractive interactions between low energy hydrophobic membranes with high energy hydrophilic bacterium (P. putida). It also predicted a shallow primary maximum with the most hydrophilic bacterium, B. subtilis . Discrepancies between predictions using the XDLVO theory and theory require involvement of factors such as bridging effects. Differences in interaction between P. putida and B. subtilis are attributed to acid-base interactions and steric interactions. P. putida is Gram negative with lipopolysaccharides present in the outer cell membrane. A variation in forces of adhesion for bacteria on polymeric membranes studied was interpreted in terms of hydrophilicity and interfacial surface potential calculated from physicochemical properties.

Initial bacterial attachment, likely affected by local shear, could influence biofilm formation. However, there are contradictory reports for the shear effects on attachment of different bacteria onto different surfaces. In this study, four bacteria, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, were examined for their attachment to glass and octadecyltrichlorosilane (OTS) modified glass under different shears. Polystyrene particles were used to verify that their shear dependent attachment on glass and OTS could be interpreted using an analysis based on the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. In particular, the critical shear force (F(c-shear)) could correlate with the maximum attractive force (F(MAX)(XDLVO)) toward the secondary energy minimum as F(c-shear)=cF(MAX)(XDLVO). For these particles, c of ~1 was obtained, the value was within the coefficient range (0.1-1) of substances sliding over glass. For S. epidermidis, E. coli and P. aeruginosa on glass, c was 0.3, <0.6 and 0.2, respectively. When considering potential protein adsorption on OTS during bacterial attachment, c of these species on OTS was slightly above 1. A greatly enhanced attachment of P. aeruginosa on OTS was also observed, probably due to the presence of flagella. For P. putida, the attachment first decreased slightly or maintained with shear and then increased. Such behaviors were probably caused by the increased secretion of extracellular polymeric substances (EPS) at higher shears by P. putida. The results from this study suggested that, without complications from surface features/EPS, the analysis based on the XDLVO theory could provide a basis for understanding shear effect on initial bacterial attachment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled.

We have developed a program that can accurately analyze the dynamic properties of tethered bacterial cells. The program works especially well with cells that tend to give rise to unstable rotations, such as polar-flagellated bacteria. The program has two novel components. The first dynamically adjusts the center of the cell's rotational trajectories. The second applies piecewise linear approximation to the accumulated rotation curve to reduce noise and separate the motion of bacteria into phases. Thus, it can separate counterclockwise (CCW) and clockwise (CW) rotations distinctly and measure rotational speed accurately. Using this program, we analyzed the properties of tethered Pseudomonas aeruginosa and Pseudomonas putida cells for the first time. We found that the Pseudomonas flagellar motor spends equal time in both CCW and CW phases and that it rotates with the same speed in both phases. In addition, we discovered that the cell body can remain stationary for short periods of time, leading to the existence of a third phase of the flagellar motor which we call "pause." In addition, P. aeruginosa cells adopt longer run lengths, fewer pause frequencies, and shorter pause durations as part of their chemotactic response. We propose that one purpose of the pause phase is to allow the cells to turn at a large angle, where we show that pause durations in free-swimming cells positively correlate with turn angle sizes. Taken together, our results suggest a new "run-reverse-turn" paradigm for polar-flagellated Pseudomonas motility that is different from the "run-and-tumble" paradigm established for peritrichous Escherichia coli.

We report a salvage pathway in Gram-negative bacteria that bypasses de novo biosynthesis of UDP N-acetylmuramic acid (UDP-MurNAc), the first committed peptidoglycan precursor, and thus provides a rationale for intrinsic fosfomycin resistance. The anomeric sugar kinase AmgK and the MurNAc α-1-phosphate uridylyl transferase MurU, defining this new cell wall sugar-recycling route in Pseudomonas putida, were characterized and engineered into Escherichia coli, channeling external MurNAc directly to peptidoglycan biosynthesis.

Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram-negative bacteria, including mcl-polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl-PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non-producing bacteria., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Bacterial attachment on solid surfaces has various implications in environmental, industrial and medical applications. In this study, the effects of rhamnolipid biosurfactants on initial attachment of bacteria on hydrophilic glass and hydrophobic octadecyltrichlorosilane (OTS) modified glass were evaluated under continuous-flow conditions. The bacteria investigated were three Gram-negative species Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, and two Gram-positive species Staphylcoccus epidermidis and Bacillus subtilis. Rhamnolipids, at 10 and 200 mg/l, significantly reduced the attachment of all but S. epidermidis on both glass and OTS-modified glass. For S. epidermidis rhamnolipids reduced the attachment on OTS-modified glass but not on glass. Studies were further done to identify the mechanism(s) by which rhamnolipids reduced the cell attachment. The following potential properties of rhamnolipids were investigated: inhibition of microbial growth, change of cell surface hydrophobicity, easier detachment of cells already attached to substratum, and modification of substratum surface properties. Results showed that rhamnolipids were ineffective for the latter two effects. Rhamnolipids, up to 200mg/l, inhibited the growth of B. subtilis, S. epidermidis and P. aeruginosa PAO1 but not the growth of E. coli, P. putida and P. aeruginosa E0340. Also, rhamnolipids tended to increase the hydrophobicity of P. aeruginosa PAO1 and E. coli, decrease the hydrophobicity of P. putida and S. epidermidis, and have no clear effect on the hydrophobicity of B. subtillis. These trends however did not correlate with the observed trend of cell attachment reduction. The responsible mechanism(s) remained unknown., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. This study assessed whether P. putida filamentation could confer survival advantages. Filamentation of P. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed., Results: P. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. Proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein IbpA and recombinase RecA in filament-inducing conditions. Our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of RecA., Conclusions: This study provides insights into the altered metabolism of P. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by P. putida as a survival strategy in its hostile, recurrently changing habitat.

A biofilter packed with walnut shells was used to eliminate ethyl acetate from an air stream. The shells treated with NaOH were used as medium for immobilization of Pseudomonas putida PTCC 1694. At an empty bed residence time (EBRT) of 60s, a removal efficiency of 99% was achieved at inlet concentrations lower than 430ppm of ethyl acetate. The removal efficiency decreased below 80% with an increase in inlet concentration of ethyl acetate. When the EBRT was increased to 75 s, the removal efficiency remained above 80% even though the inlet loading rate was increased to 421g/m(3)h. Michaelis-Menten type and zero-order diffusion limited models were employed and the predicted data perfectly matched the experimental data. Thus P. putida immobilized on walnut shell has potential for the removal of ethyl acetate from air streams., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chemotaxis toward chemical pollutants provides a mechanism for bacteria to migrate to locations of high contamination, which may improve the effectiveness of bioremediation. A microfluidic device was designed to mimic the dissolution of an organic-phase contaminant from a single pore into a larger macropore representing a preferred pathway for microorganisms that are carried along by groundwater flow. The glass windows of the microfluidic device allowed direct image analysis of bacterial distributions within the vicinity of the organic contaminant. Concentrations of chemotactic bacteria P. putida F1 near the organic/aqueous interface were 25% greater than those of a nonchemotactic mutant in the vicinity of toluene for a fluid velocity of 0.5 m/d. For E. coli responding to phenol, the bacterial concentrations were 60% greater than the controls, also at a velocity of 0.5 m/d. Velocities in the macropore were varied over a range from 0.5 to 10 m/d, the lower end of which is typical of groundwater velocities. The accumulation of chemotactic bacteria near the NAPL chemoattractant source decreased as the fluid velocity increased. Good agreement between computer-based simulations, generated using reasonable values of the model parameters, and the experimental data for P. putida strains confirmed the contribution due to chemotaxis. The experimental data for E. coli required a larger chemotactic sensitivity coefficient than that for P. putida, which was consistent with parameter values reported in the literature., (Copyright © 2012 Wiley Periodicals, Inc.)

The biocatalytic activity of periplasmic dehydrogenase of Pseudomonas putida G7 strain to catalyse limonin was enhanced when whole cells were permeabilized with EDTA (1 μM) lysozyme (100 μg/ml). The treated cells were entrapped in dialysis membranes to increase the stability. Permeabilized cells (1 g dry weight) entrapped in dialysis membrane could biotransform 73.67% of limonin in unpasteurized mandarin juices in a single-batch cycle of 3 h. Furthermore, permeabilized cells stored for 45 days in phosphate buffered saline (at 4 or 30°C) retained enzyme activity and were reusable for up to eight batch cycles of limonin reduction. The results of this study suggest a potential application of permeabilized P. putida G7 cells for reducing limonin levels in mandarin juices.

A good part of the contemporary synthetic biology agenda aims at reprogramming microorganisms to enhance existing functions and/or perform new tasks. Moreover, the functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavors rely on the availability of genetic tools to successfully modify á la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for the production of directed mutants and allelic replacements, random mutant libraries to discover new functions, and means to stably implant larger genetic networks into the genome of specific hosts. The list of gram-negative species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available molecular techniques to do so is very limited beyond Escherichia coli. In this chapter, utilization of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of gram-negative microorganisms.

Background: Disaccharides are, in general, the first choice as formulation compounds when freeze-drying microorganisms. Although polysaccharides and other biopolymers are considered too large to stabilise and interact with cell components in the same beneficial way as disaccharides, polymers have been reported to support cell survival. In the present study we compare the efficiency of sucrose and the polymers Ficoll, hydroxyethylcellulose, hydroxypropylmethylcellulose and polyvinylalcohol to support the survival of three bacterial strains during freeze drying. The initial osmotic conditions were adjusted to be similar for all formulations. Formulation characterisation was used to interpret the impact that different compound properties had on cell survival., Results: Despite differences in molecular size, both sucrose and the sucrose-based polymer Ficoll supported cell survival after freeze drying equally well. All formulations became amorphous upon dehydration. Scanning electron microscopy and X-ray diffraction data showed that the discerned differences in structure of the dry formulations had little impact on the survival rates. The capability of the polymers to support cell survival correlated with the surface activity of the polymers in a similar way for all investigated bacterial strains., Conclusion: Polymer-based formulations can support cell survival as effectively as disaccharides if formulation properties of importance for maintaining cell viability are identified and controlled., (Copyright © 2011 Society of Chemical Industry.)

The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 µM (for Mg(2+)) to 3 µM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed., (Copyright © 2011 John Wiley & Sons, Ltd.)

The effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption was investigated using a combination of batch adsorption, potentiometric titrations, Fourier transform infrared spectroscopy. Both the potentiometric titrations and the Cu(II) adsorption experiments indicated that the presence of EPS in a biomass sample significantly enhance Cu(II) adsorption capacity. Surface complexation modeling showed that the pKa values for the three functional groups (carboxyl, phosphate and hydroxyl) were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition. However, site concentrations on the untreated cell surface were found to be significantly higher than those on the EPS-free cell surface. Infrared analysis provided supporting evidence and demonstrated that carboxyl and phosphate groups are responsible for Cu(II) adsorption on the native and EPS-free cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bacteriological studies of cadaveric blood for the presence of Pseudomonas putida and Pseudomonas fluorescence have demonstrated that identification of these microorganisms may be considered as an evidence of death by drowning. These bacteria are typical "aqueous" organisms non-pathogenic for man. They do not grow at a temperature of 41 degrees C and therefore are usually absent in normal human microflora. It is concluded that the establishment of the fact and location of death by drowning based on the analysis of plankton composition should be supplemented by identification of various forms of Pseudomonas in cadaveric blood.

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T(m)) and unfolding enthalpy (DeltaH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

Antibiotics can induce cell death via a variety of action modes, including the inhibition of transcription, ribosomal function, and cell wall biosynthesis. In this study, we demonstrated directly that iron availability is important to the action of antibiotics, and the ferric reductases of Pseudomonas putida and Pseudomonas aeruginosa could accelerate antibiotic-mediated cell death by promoting the Fenton reaction. The modulation of reduced nicotinamide-adenine dinucleotide (NADH) levels and iron chelation affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death. The results of transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron homeostasis is crucial for bacterial cell survival under antibiotics and should constitute a significant target for boosting the action of antibiotics.

Bacterial pesticide degraders are generally heterogeneously distributed in soils, leaving soil volumes devoid of degradation potential. This is expected to have an impact on degradation rates because the degradation of pollutant molecules in such zones will be contingent either on degraders colonizing these zones or on pollutant mass transfer to neighboring zones containing degraders. In a model system, we quantified the role exerted by water on mineralization rate in the context of a heterogeneously distributed degradation potential. Alginate beads colonized by Pseudomonas putida KT2440 were inserted at prescribed locations in sand microcosms so that the initial spatial distribution of the mineralization potential was controlled. The mineralization rate was strongly affected by the matric potential (decreasing rate with decreasing matric potential) and by the initial distribution of the degraders (more aggregated distributions being associated with lower rates). The mineralization was diffusion-limited, as confirmed with a mathematical model. In wet conditions, extensive cell dispersal was observed for the flagellated wild type and, albeit to a lesser extent, for a nonflagellated mutant, partially relieving the diffusion limitation. Dry conditions, however, sustained low mineralization rates through the combined effects of low pollutant diffusivity and limited degrader dispersal.

Adhesion of Pseudomonas putida F1 onto agave-fiber/recycled-polyethylene foamed composites was studied under different controlled conditions. The adhesion process was analyzed in batch experiments controlling factors such as pH, contact time, temperature, initial biomass concentration and ionic strength; and was verified by scanning electron microscopy (SEM). The number of adhered bacteria after the experimental time was determined by difference between concentration of suspended cells in NaCl solution contained in two different Erlenmeyer flasks, one of the flasks with composite pellets and the other one without them. The concentration of cells in each flask was obtained using the serial dilution technique. Experimental data analysis showed that adsorption follows first-order kinetics. And it was further corroborated to be an irreversible process. For the first time, an equation is proposed here to predict the correlation between adhered bacteria and aqueous pH. In addition to the obvious reuse of waste material, these results suggested that agave-fiber/polymer foamed composites could be used as support for bacterial immobilization to be applied, among others in environmental processes such as bioremediation and biofiltration of gases with almost limitless possibilities.

Aquifer recharge for the wastewater reuse has been considered and studied as a promising process to cope with the worldwide water scarcity. Soil clogging by an excessive growth of bacteria is often accompanied with the aquifer recharge. In this study, biofilm morphology and hydraulic conductivity were concurrently characterized at two flow rates and two levels of substrate concentrations. The experiments were conducted using a biofilm flow cell that was filled with glass beads. The biofilm images taken by confocal laser scanning microscopy (CLSM) were quantified by textural, areal, and fractal parameters. Hydraulic conductivity was monitored during the experiments. The flow velocity influenced the superficial morphology of biofilms and initial clogging time, while the substrate concentration affected biofilm density and clogging rate. Three different clogging mechanisms were suggested depending on the flow rate and substrate concentration: (1) clogging at a high flow rate can be accelerated by entrapped and accumulated biofilms, and can be easily eliminated by high shear force, (2) clogging at a low flow rate can be delayed for the time of local biofilm growths in the narrow pore necks, but the biofilm is rigid enough not to be sloughed, and (3) clogging in a solution with high substrate concentrations cannot be easily eliminated because of the growth of dense biofilms. The depicted biological clogging mechanisms will play a role in supporting studies about aquifer recharge., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water-replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water-limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water-limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water-replete and solute stress conditions. There was greater spatial stratification of dead cells under water-limiting than water-replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water-limiting conditions, ROS accumulation is greater in an DeltaalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration-mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water-replete and water-limiting conditions.

Adsorption and desorption of salmon sperm DNA on bacteria (Bacillus thuringiensis, Pseudomonas putida), two different colloidal fractions (organic and inorganic clay) from an Alfisol, minerals (montmorillonite, kaolinite and goethite) and colloid-bacteria composites were studied. Similar adsorption capacity and affinity of DNA were observed on two bacterial cells. However, the two bacterial strains played different roles in affecting the adsorption of DNA on the composites of soil colloidal particles with bacteria. The introduction of B. thuringiensis in soil colloids and minerals systems dramatically promoted DNA adsorption on colloidal particles especially organic clay, while P. putida decreased the adsorption of DNA on kaolinite and goethite. Electrostatic force and ligand exchange are regarded to be the major driving forces involved in the adsorption of DNA on bacterial cells, montmorillonite, soil colloids and goethite. Presence of bacteria enhanced the proportion of DNA adsorption on soil colloidal particles by electrostatic force and depressed that by ligand exchange process. Information obtained in this study is of fundamental significance for the understanding of the ultimate fate of extracellular DNA in soil systems.

Amperometric biosensors using Pseudomonas putida cells as a bioelement were developed for 2,4-dichloro phenoxy acetic acid (2,4-D). After the adaptation process of Pseudomonas putida to 2,4-D, cells were immobilized onto the screen printed graphite electrodes (SPG) as well as Clark oxygen probe by gelatin and glutaraldehyde. Optimum pH, temperature, and stability of the biosensor were investigated. Substrate specificities for various phenolic compounds were also searched. In repeatability studies, variation coefficients and standard deviations for both type of systems were calculated; SPG and Clark electrodes were calculated and results are given as a comparison of two systems. Finally, the biosensors were applied to 2,4-D determination in a real herbicide sample.