An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress

The novel insertion sequence ISS12 plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress. Under normal culturing conditions the P. putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequency of cells in this variant P. putida S12 population was 1000 times higher than in "normal" P. putida S12 populations. Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene. srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal level of srp promoter activity in the P. putida S12 variant was seven times higher than in wild-type P. putida S12. Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock. These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions.     

Effect of Environmental Factors on the trans/cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12

The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30(deg)C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.     

Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes.

Pseudomonas putida S12 was more tolerant to ethanol when preadapted to supersaturating concentrations of toluene. Cellular reactions at the membrane level to the toxicities of both compounds were different. In growing cells of P. putida S12, sublethal concentrations of toluene resulted in an increase in the degree of saturation of the membrane fatty acids, whereas toxically equivalent concentrations of ethanol led to a decrease in this value. Contrary to this, cells also reacted to both substances with a strong increase of the trans unsaturated fatty acids and a corresponding decrease of the cis unsaturated fatty acids under conditions where growth and other cellular membrane reactions were totally inhibited. While the isomerization of cis to trans unsaturated fatty acids compensates for the fluidizing effect caused by ethanol, a decrease in the degree of saturation is antagonistic with respect to the chemo-physical properties of the membrane. Consequently, the results support the hypothesis that the decrease in the degree of saturation induced by ethanol is not an adaptation mechanism but is caused by an inhibitory effect of the compound on the biosynthesis of saturated fatty acids.     

Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria.

Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.     

Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.     

Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12.

The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.     

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.     

Indigo formation by microorganisms expressing styrene monooxygenase activity.

The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole.     

Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.     

Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12

The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach ensures constant growth conditions, both in the presence and absence of toluene. A considerable negative effect of toluene on the cell yield was found. The need for energy in the defence against toluene was reflected by differentially expressed proteins for cell energy management. In toluene-stressed cells the balance between proton motive force (PMF) enforcing and dissipating systems was shifted. NAD(P)H generating systems were upregulated whereas the major proton-driven system, ATP synthase, was downregulated. Other differentially expressed proteins were identified: outer membrane proteins, transport proteins, stress-related proteins and translation-related proteins. In addition, a protein with no assigned function was found. This study yielded a more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 and several novel leads have been obtained for further targeted investigations.     

Actinide and metal toxicity to prospective bioremediation bacteria

Bacteria may be beneficial for alleviating actinide contaminant migration through processes such as bioaccumulation or metal reduction. However, sites with radioactive contamination often contain multiple additional contaminants, including metals and organic chelators. Bacteria-based bioremediation requires that the microorganism functions in the presence of the target contaminant, as well as other contaminants. Here, we evaluate the toxicity of actinides, metals and chelators to two different bacteria proposed for use in radionuclide bioremediation, Deinococcus radiodurans and Pseudomonas putida, and the toxicity of Pu(VI) to Shewanella putrefaciens. Growth of D. radiodurans was inhibited at metal concentrations ranging from 1.8 microM Cd(II) to 32 mM Fe(III). Growth of P. putida was inhibited at metal concentrations ranging from 50 microM Ni(II) to 240 mM Fe(III). Actinides inhibited growth at mM concentrations: chelated Pu(IV), U(VI) and Np(V) inhibit D. radiodurans growth at 5.2, 2.5 and 2.1 mM respectively. Chelated U(VI) inhibits P. putida growth at 1.7 mM, while 3.6 mM chelated Pu(IV) inhibits growth only slightly. Pu(VI) inhibits S. putrefaciens growth at 6 mM. These results indicate that actinide toxicity is primarily chemical (not radiological), and that radiation resistance does not ensure radionuclide tolerance. This study also shows that Pu is less toxic than U and that actinides are less toxic than other types of metals, which suggests that actinide toxicity will not impede bioremediation using naturally occurring bacteria.     

Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion of l-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-molC-mol(-1)) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9mM (1.8gl(-1)) was obtained, at a yield of 8.5% (C-molC-mol(-1)). A 2-fold increase of the specific p-hydroxybenzoate production rate (q(p)) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that l-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, q(p) increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient l-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified.     

Extracytoplasmic storage as the nickel resistance mechanism in a natural isolate of Pseudomonas putida S4

Metal resistances in microbes are important to study not only to understand metal homeostasis but also to use such organisms further in environmental bioremediation. Nickel (Ni2+) is an important micronutrient, which at higher concentration becomes toxic. Many Ni2+-resistant organisms are known, which resist metal by active efflux. Pseudomonas putida S4, a natural isolate from India, is reported to show a multi-metal resistance profile. In the present study, the Ni2+-resistance mechanism in strain S4 was examined. Wild-type cells gradually accumulated Ni2+ but kept it preferentially in the periplasmic space in a bound form. In Ni2+-sensitive mutants, periplasmic storage was disturbed and more metal accumulated cytoplasmically, producing toxicity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of periplasmic proteins revealed a band of approximately 18 kDa, which appeared only in Ni2+-exposed wild-type cells, and which was absent from cells not exposed to Ni2+ as well as from Ni2+-sensitive mutants. On the basis of these observations, we propose a Ni2+-resistance mechanism in P. putida S4 based on sequestration of metal in the periplasmic space. This is the first study of sequestration-based Ni2+ resistance.     

Energetics and surface properties of Pseudomonas putida DOT-T1E in a two-phase fermentation system with 1-decanol as second phase

The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl2. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.     

Indigo formation by aromatic hydrocarbon-degrading bacteria

A variety of aromatic hydrocarbon-degrading bacteria expressing different oxygenases were tested for their ability to produce indigo from indole. Styrene-grown cells of Pseudomonas putida S12 and CA-3 expressing styrene mono-oxygenase produced indigo at rates of 4–8 nmol min–1 mg dry wt–1. Toluene-grown cells of P. putida F1 and naphthalene-grown cells of P. putida PpG7 expressing dioxygenases formed indigo at rates of 1.5 and 2.5 nmol min–1 mg dry wt–1, respectively. © Rapid Science Ltd. 1998