Evaluation of two xenobiotic reductases from Pseudomonas putida for their suitability for magnetic nanoparticle-directed enzyme prodrug therapy as a novel approach to cancer treatment.

Directed enzyme prodrug therapy (DEPT) is a cancer chemotherapy strategy in which bacterial enzymes are delivered to a cancer site before prodrug administration, resulting in prodrug activation at the cancer site and more localized treatment. A major limitation to DEPT is the poor effectiveness of the most studied enzyme for the CB1954 prodrug, NfnB from Escherichia coli, at concentrations suitable for human use. Much research into finding alternative enzymes to NfnB has resulted in the identification of the Xenobiotic reductases, XenA and XenB, which have been shown in the literature to reduce environmentally polluting nitro-compounds. In this study, they were assessed for their potential use in cancer prodrug therapy strategies. Both proteins were cloned into the pET28a+ expression vector to give the genetically modified proteins XenA-his and XenB-his, of which only XenB-his was active when tested with CB1954. XenB-his was further modified to include a cysteine-tag to facilitate direct immobilization on to a gold surface for future magnetic nanoparticle DEPT (MNDEPT) treatments and was named XenB-cys. When tested using high-performance liquid chromatography (HPLC), XenB-his and XenB-cys both demonstrated a preference for reducing CB1954 at the 4-nitro position. Furthermore, XenB-his and XenB-cys successfully induced cell death in SK-OV-3 cells when combined with CB1954. This led to XenB-cys being identified as a promising candidate for use in future MNDEPT treatments.

Structural characterization of pyoverdines produced by Pseudomonas putida KT2440 and Pseudomonas taiwanensis VLB120.

The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.

Influence of bacterial extracellular polymeric substances on the sorption of Zn on γ-alumina: A combination of FTIR and EXAFS studies.

Extracellular polymeric substances (EPS) isolated from bacteria, are abound of functional groups which can react with metals and consequently influence the immobilization of metals. In this study, we combined with Zn K-edge Extended X-ray Absorption Fine Structure (EXAFS), Fourier Transform Infrared (FTIR) spectroscopy, and High-Resolution Transmission Electron Microscopy (HRTEM) techniques to study the effects of EPS isolated from Bacillus subtilis and Pseudomonas putida on Zn sorption on γ-alumina. The results revealed that Zn sorption on aluminum oxide was pH-dependent and significantly influenced by bacterial EPS. At pH 7.5, Zn sorbed on γ-alumina was in the form of Zn-Al layered doubled hydroxide (LDH) precipitates, whereas at pH 5.5, Zn sorbed on γ-alumina was as a Zn-Al bidentate mononuclear surface complex. The amount of sorbed Zn at pH 7.5 was 1.3-3.7 times higher than that at pH 5.5. However, in the presence of 2 g L-1 EPS, regardless of pH conditions and EPS source, Zn + EPS + γ-alumina ternary complex was formed on the surface of γ-alumina, which resulted in decreased Zn sorption (reduced by 8.4-67.8%) at pH 7.5 and enhanced Zn sorption (increased by 10.0-124.7%) at pH 5.5. The FTIR and EXAFS spectra demonstrated that both the carboxyl and phosphoryl moieties of EPS were crucial in this process. These findings highlight EPS effects on Zn interacts with γ-alumina.

Unusual poly(3-hydroxyalkanoate) (PHA) biosynthesis behavior of Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002 isolated from palm oil mill effluent.

Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002, isolated from palm oil mill effluent, accumulated poly(3-hydroxyalkanoates) (PHAs) when grown on aliphatic fatty acids, sugars, and glycerol. The substrates were supplied at 20:1 C/N mole ratio. Among C-even n-alkanoic acids, myristic acid gave the highest PHA content 26 and 28 wt% in P. putida and D. tsuruhatensis, respectively. Among C-odd n-alkanoic acids, undecanoic gave the highest PHA content at 40 wt% in P. putida and 46 wt% in D. tsuruhatensis on pentadecanoic acid. Sugar and glycerol gave <10 wt% of PHA content for both bacteria. Interestingly, D. tsuruhatensis accumulated both short- and medium-chain length PHA when supplied with n-alkanoic acids ranging from octanoic to lauric, sucrose, and glycerol with 3-hydroxybutyrate as the major monomer unit. In P. putida, the major hydroxyalkanoates unit was 3-hydroxyoctanoate and 3-hydroxydecanoate when grown on C-even acids. Conversely, 3-hydroxyheptanoate, 3-hydrxoynonanoate, and 3-hydroxyundecanoate were accumulated with C-odd acids. Weight-averaged molecular weight (Mw ) was in the range of 53-81 kDa and 107-415 kDa for P. putida and D. tsuruhatensis, respectively. Calorimetric analyses indicated that both bacteria synthesized semicrystalline polymer with good thermal stability with degradation temperature (Td ) ranging from 178 to 282 °C.

U(VI) biosorption by bi-functionalized Pseudomonas putida @ chitosan bead: Modeling and optimization using RSM.

In this work, Pseudomonas putida cells immobilized into chitosan beads (PICB) were synthesized to investigate the impact of microorganism entrapment on biosorption capacity of prepared biosorbent for U(VI) biosorption from aqueous solutions. Response Surface Methodology (RSM) based on Central Composite Design (CCD) was utilized to evaluate the performance of the PICB in comparison with chitosan beads (CB) under batch mode. Performing experiments under optimal condition sets viz. pH 5, initial U(VI) concentration 500mg/L, biosorbent dosage 0.4g/L and 20wt.% bacterial cells showed that the observed biosorption capacity enhanced by 1.27 times from 398mg/g (CB) to 504mg/g (PICB) that confirmed the effectiveness of cells immobilization process. FTIR and potentiometric titration were then utilized to characterize the prepared biosorbents. While the dominant functional group in the binding process was NH3(+) (4.78meq/g) in the CB, the functional groups of NH3(+), NH2, OH, COOH (6.00meq/g) were responsible for the PICB. The equilibrium and kinetic studies revealed that the Langmuir isotherm model and the pseudo-second-order kinetic model were in better fitness with the CB and PICB experimental data. In conclusion, the present study indicated that the PICB could be a suitable biosorbent for uranium (VI) biosorption from aqueous solutions.

Crude oil biodegradation aided by biosurfactants from Pseudozyma sp. NII 08165 or its culture broth.

The aim of this work was to evaluate the biosurfactants produced by the yeast Pseudozyma sp. NII 08165 for enhancing the degradation of crude oil by a model hydrocarbon degrading strain, Pseudomonas putida MTCC 1194. Pseudozyma biosurfactants were supplemented at various concentrations to the P. putida culture medium containing crude oil as sole carbon source. Supplementation of the biosurfactants enhanced the degradation of crude oil by P. putida; the maximum degradation of hydrocarbons was observed with a 2.5 mg L(-1) supplementation of biosurfactants. Growth inhibition constant of the Pseudozyma biosurfactants was 11.07 mg L(-1). It was interesting to note that Pseudozyma sp. NII 08165 alone could also degrade diesel and kerosene. Culture broth of Pseudozyma containing biosurfactants resulted up to ∼46% improvement in degradation of C10-C24 alkanes by P. putida. The enhancement in degradation efficiency of the bacterium with the culture broth supplementation was even more pronounced than that with relatively purer biosurfactants.

ATR-FTIR spectroscopic evidence for biomolecular phosphorus and carboxyl groups facilitating bacterial adhesion to iron oxides.

Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy has been used to probe the binding of bacteria to hematite (α-Fe2O3) and goethite (α-FeOOH). In situ ATR-FTIR experiments with bacteria (Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli), mixed amino acids, polypeptide extracts, deoxyribonucleic acid (DNA), and a suite of model compounds were conducted. These compounds represent carboxyl, catecholate, amide, and phosphate groups present in siderophores, amino acids, polysaccharides, phospholipids, and DNA. Due in part to the ubiquitous presence of carboxyl groups in biomolecules, numerous IR peaks corresponding to outer-sphere or unbound (1400 cm(-1)) and inner-sphere (1310-1320 cm(-1)) coordinated carboxyl groups are noted following reaction of bacteria and biomolecules with α-Fe2O3 and α-FeOOH. However, the data also reveal that the presence of low-level amounts (i.e., 0.45-0.79%) of biomolecular phosphorous groups result in strong IR bands at ∼1043 cm(-1), corresponding to inner-sphere Fe-O-P bonds, underscoring the importance of bacteria associated P-containing groups in biomolecule and cell adhesion. Spectral comparisons also reveal slightly greater P-O-Fe contributions for bacteria (Pseudomonad, E. coli) deposited on α-FeOOH, as compared to α-Fe2O3. This data demonstrates that slight differences in bacterial adhesion to Fe oxides can be attributed to bacterial species and Fe-oxide minerals. However, more importantly, the strong binding affinity of phosphate in all bacteria samples to both Fe-oxides results in the formation of inner-sphere Fe-O-P bonds, signifying the critical role of biomolecular P in the initiation of bacterial adhesion.

Construction and optimization of a Pseudomonas putida whole-cell bioreporter for detection of bioavailable copper.

A Pseudomonas putida whole-cell bioreporter for detecting bioavailable copper was constructed by inserting a CueR-regulated sensor element upstream of a promoterless green fluorescent protein (GFP) reporter gene. The constructed bioreporter cells expressed GFP only in response to Cu and Ag when cultivated in different metal salt solutions. M9 supplemented medium provided higher sensitivity compared with LB medium. The optimal test condition was cell suspension with an OD600 of 0.4-0.5 incubated at 30 °C. The detection range of Cu is 1-70 mg/l under optimal test condition in M9 supplemented medium.

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.

An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1.

Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K (m) value of 0.2 mM.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria.

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Multivariate analysis of microarray data by principal component discriminant analysis: prioritizing relevant transcripts linked to the degradation of different carbohydrates in Pseudomonas putida S12.

The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. RNA isolated from these samples was analysed in duplicate on an anonymous clone-based array to avoid bias during data analysis. The relevant transcripts were identified by analysing the loadings of the principal components (PC) and discriminants (D) in PCA and PCDA, respectively. Even more specifically, the relevant transcripts for a specific phenotype could also be ranked from the loadings under an angle (biplot) obtained after PCDA analysis. The leads identified in this way were compared with those identified using the commonly applied fold-difference and hierarchical clustering approaches. The different data analysis methods gave different results. The methods used were complementary and together resulted in a comprehensive picture of the processes important for the different carbon sources studied. For the more subtle, regulatory processes in a cell, the PCDA approach seemed to be the most effective. Except for glucose and gluconate dehydrogenase, all genes involved in the degradation of glucose, gluconate and fructose were identified. Moreover, the transcriptomics approach resulted in potential new insights into the physiology of the degradation of these carbon sources. Indications of iron limitation were observed with cells grown on glucose, gluconate or succinate but not with fructose-grown cells. Moreover, several cytochrome- or quinone-associated genes seemed to be specifically up- or downregulated, indicating that the composition of the electron-transport chain in P. putida S12 might change significantly in fructose-grown cells compared to glucose-, gluconate- or succinate-grown cells.

Construction and evaluation of nagR-nagAa::lux fusion strains in biosensing for salicylic acid derivatives.

The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia coli strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 microM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.

Temporal dynamics and degradation activity of an bacterial inoculum for treating waste metal-working fluid.

In order for established bioreactors to be effective for treating chemically mixed wastes such as metal working fluids (MWF) it is essential that they harbour microbial populations that can maintain sufficient active biomass and degrade each of the chemical constituents present. In this study we investigated the effectiveness of a bacterial consortium composed of four species (Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis and Pseudomonas putida), assembled on the basis of their apparent ubiquity in waste MWF, degradation ability and tolerance to fluctuating chemistry of the waste. The temporal dynamics of the inoculum and its effects on the fate of individual chemical components of the waste were studied, by regular sampling, over 400 h. Using a complementary approach of culture with chemotaxonomic (FAME) analysis and applying group specific probes (FISH), the inoculum was found to represent a significant component of the community in bioreactors with and without presence of indigenous MWF populations. In addition, the reduction in the COD by the consortium was approximately 85% of the total pollution load, and 30-40% more effectively than any other treatment (indigenous MWF community alone or activated sludge). Furthermore, all the chemical constituents, including the biocide (a formaldehyde release agent) demonstrated > 60% reduction. Many chemical components of the MWF proved to be recalcitrant in the other treatments. The results of this study confirm that assemblage of an inoculum, based on a comprehensive knowledge of the indigenous microbial community, in the target habitat, is a highly effective way of selecting microbial populations for bioaugmentation of bioreactors.

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants.

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.

An efficient screening approach for anti-Microcystis compounds based on knowledge of aquatic microbial ecosystem.

To improve the efficiency of screening for anti-Microcystis compounds, we planned to use algae-lysing bacteria that kill the organisms of water blooms. A two step-screening process was carried out, i.e., the screening of algae-lysing bacteria and the selection of anti-Microcystis producers from the bacteria. Sources for the isolation of the bacteria were a co-cultivated fluid of a water sample with axenic Microcystis viridis, a water sample collected in a water bloom season, and a water bloom sample. The water bloom sample was the best source for the isolation of the algae-lysing bacteria and such bacteria were shown to exhibit potent activity. Seventeen strains out of 20 isolated algae-lysing bacteria produced anti-Microcystis activities, and one of the principles was the previously reported argimicin A. These results indicate that algae-lysing bacteria in water blooms may be good sources for potent and selective anticyanobacterial compounds.

A novel cell surface polysaccharide in Pseudomonas putida WCS358, which shares characteristics with Escherichia coli K antigens, is not involved in root colonization.

Previously we have shown that flagella and the O-specific polysaccharide of lipopolysaccharide play a role in colonization of the potato root by plant growth-promoting Pseudomonas strains WCS374 and WCS358. In this paper, we describe a novel cell surface-exposed structure in Pseudomonas putida WCS358 examined with a specific monoclonal antibody. This cell surface structure appeared to be a polysaccharide, which was accessible to the monoclonal antibody at the outer cell surface. Further study revealed that it does not contain 2-keto-3-deoxyoctonate, heptose, or lipid A, indicating that it is not a second type of lipopolysaccharide. Instead, the polysaccharide shared some characteristics with K antigen described for Escherichia coli. From a series of 49 different soil bacteria tested, only one other potato plant growth-promoting Pseudomonas strain reacted positively with the monoclonal antibody. Mutant cells lacking the novel antigen were efficiently isolated by an enrichment method involving magnetic antibodies. Mutant strains defective in the novel antigen contained normal lipopolysaccharide. One of these mutants was affected in neither its ability to adhere to sterile potato root pieces nor its ability to colonize potato roots. We conclude that the bacterial cell surface of P. putida WCS358 contains at least two different polysaccharide structures. These are the O-specific polysaccharide of lipopolysaccharide, which is relevant for potato root colonization, and the novel polysaccharide, which is not involved in adhesion to or colonization of the potato root.

Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain.