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.     

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.     

Probing the proteome response to toluene exposure in the solvent tolerant Pseudomonas putida S12

To enhance target production from biocatalysts, it is necessary to thoroughly understand the molecular mechanisms involved in production, degradation, and, importantly, adaptation to the required environment. One such bacterium with high potential for biocatalysis is the solvent-tolerant bacteria Pseudomonas putida S12, which, among others, is able to degrade organic solvents. For bioconversion of organic solvents to become a successful industrial process, the understanding of the molecular response upon solvent tolerance is essential. Here we performed a quantitative analysis of the P. putida S12 proteome at different stages of adaptation to toluene. Using a stable isotope dimethylation labeling approach we monitored the differential expression of 528 proteins, including often hard-to-detect membrane associate proteins, such as multiple RND-family transporters and ABC transporters of nutrients. Our quantitative proteomics approach revealed the remarkable ability of P. putida S12 to severely change its protein expression profile upon toluene exposure. This proteome response entails a significant increase in energy metabolism and expression of the solvent efflux pump SrpABC, confirming its role in solvent tolerance. Other proteins strongly up-regulated in the presence of toluene include the multidrug efflux membrane protein PP1272 and the cation/acetate symporter ActP and may form interesting alternative targets for improving solvent tolerance.     

Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents.

Pseudomonas putida S12 could adapt to grow on styrene in a two-phase styrene-water system. Acetate was toxic for P. putida S12, but cells were similarly able to adapt to higher acetate concentrations. Only by using these acetate-adapted cells was growth observed in the presence of supersaturating concentrations of toxic nonmetabolizable solvents such as toluene.     

Active efflux of toluene in a solvent-resistant bacterium.

We investigated the mechanisms behind the organic-solvent resistance of the solvent-tolerant strain Pseudomonas putida S12. By use of 14C-labeled toluene, we obtained evidence that an energy-dependent export system may be responsible for this resistance to toluene.     

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Evolution of a degradative bacterial consortium during the enrichment of naphtha solvent

A microbial mixed culture able to degrade naphtha solvent, a model of hydrocarbon aromatic mixture, was isolated from a hydrocarbon-polluted soil. Composition of the population was monitored by phenotypic and molecular methods applied on soil DNA, on whole enrichment culture DNA, and on 85 isolated strains. Strains were characterized for their 16S rDNA restriction profiles and for their random amplified polymorphic DNA profiles. Catabolic capabilities were monitored by phenotypic traits and by PCR assays for the presence of the catabolic genes methyl mono-oxygenase ( xylA, M), catechol 2,3 dioxygenase (xylE) and toluene dioxygenase (todC1) of TOL and TOD pathways. Different haplotypes belonging to Pseudomonas putida, Ps. aureofaciens and Ps. aeruginosa were found to degrade aromatic compounds and naphtha solvent. The intrinsic catabolic activity of the microbial population of the polluted site was detected by PCR amplification of the xylE gene directly from soil DNA.     

Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.     

Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.     

Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.

As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community.     

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.     

Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil

Bioremediation is often used for in situ remediation of petroleum-contaminated sites. The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation. Diesel. toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates. The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA DNA colony hybridization. The diesel DNA-extract possessed both the xy/E catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation. The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene. Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973. A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene. 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene. These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site. Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present.     

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons.

Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P(ow) (octanol/water partition coefficient) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg2+ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene. Although the strain also thrived in high concentrations of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.     

Metabolism of Chlorotoluenes by Burkholderia sp. Strain PS12 and Toluene Dioxygenase of Pseudomonas putida F1: Evidence for Monooxygenation by Toluene and Chlorobenzene Dioxygenases

The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes were formed as minor products from 2- and 3-chlorotoluene, whereas a chloro- and methyl-substituted cyclohexadiene was the only product formed from 4-chlorotoluene. The toluene dioxygenase of P. putida F1 and chlorobenzene dioxygenase from Burkholderia sp. strain PS12 are the first enzymes described that efficiently catalyze the oxidation of 2-chlorotoluene.     

Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida.

Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway.     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.