Variation in chlorobenzoate catabolism by Pseudomonas putida P111 as a consequence of genetic alterations.

Pseudomonas putida P111 is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates. The involvement of two separate dioxygenases was noted from data on plasmid profiles and DNA hybridization. The benzoate dioxygenase, which converts 3-chlorobenzoate (3-CB), 4-CB, and benzoate to the corresponding catechols via reduction of a dihydrodiol, was shown to be chromosomally coded. The chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to the corresponding catechols without the need of a functional dioldehydrogenase was shown to be encoded on plasmid pPB111 (75 kb). Cured strains were unable to utilize ortho-chlorobenzoates for growth. DNA hybridization data indicated that catabolism of the corresponding chlorocatechols was coded on chromosomal genes. Maintenance of plasmid pPB111 was dependent on the presence of ortho-chlorobenzoates in the growth media. A unique variant of P111 (P111D), able to grow on 3,5-dichlorobenzoate (3,5-DCB), was obtained by continuous subculturing from media containing progressively lower and higher concentrations of 3-CB and 3,5-DCB, respectively. The low frequency of segregants able to grow on 2,5-DCB, 2,3-DCB, and 2,3, 5-trichlorobenzoate was evident by lag periods greater than 200 h. Continued subculture on 3,5-DCB resulted in the formation of new plasmid pPH111 (120 kb), which was homologous to pPB111. A probe from the clc operon, which encodes for the chlorocatechol pathway, hybridized to plasmid pPH111 and to the chromosome of the wild-type strain P111 but not to its plasmid pPB111 nor to the chromosome of strain P111A, which had lost the ability to utilize chlorobenzoates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.     

Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture

A hybrid metabolic pathway through which benzene, toluene, and p-xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146-1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad-host-range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt-2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3-, 3.7-, and 1.4-fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. (c) 1995 John Wiley & Sons, Inc.     

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.     

Autecological properties of 3-chlorobenzoate-degrading bacteria and their population dynamics when introduced into sediments

Ecologically significant properties of wild-type and genetically engineered bacteria capable of degrading 3-chlorobenzoate (3-CB) were compared in the laboratory, and isolates were introduced into streambed sediments in microcosms to observe their population dynamics. 3-CB metabolism, growth on algal extract, temperature optima, and ingestion by protozoa were ecological properties considered relevant to the persistence of these bacteria if introduced into nature. Cell-specific Vmax for 3-CB metabolism and cell-specific mineralization rates each spanned approximately 2 orders of magnitude, but isolates did not rank consistently. The Ks for 3-CB metabolism for Alcaligenes sp. BR60 was approximately 40-fold lower than the mean value for the other isolates, which differed only approximately 4-fold among themselves. All isolates grew on an algal extract nearly as well as on tryptone-yeast extract, implying potential for survival on natural metabolic substrates in situ. Most isolates had temperature optima that were 3-15 degrees C higher than maximum stream water temperature (22 degrees C). Ciliates preferentially ingested P. acidovorans M3GY, and either P. putida RC-4(pSI30) or its parent strain were least preferred, but microflagellates did not exhibit consistent preferences. Fluorescent antibodies were prepared against isolates to permit detection of target cells in natural communities. In three different microcosm experiments the cell densities of introduced isolates declined over a period of days. In one experiment, 3-CB additions (100 mg/L) led to increases of P. alcaligenes C-0 and P. acidovorans M3GY cell densities within 1 day, although P. putida RC-4(pSI30) took 4 days. In a second experiment, the persistence of P. putida RC-4(pSI30) and its parent strain P. putida RC-4 were compared and rates of initial population decline were not statistically different. 3-CB addition stimulated the growth of other organisms while densities of the P. putida strains further declined. In a third experiment exposure to 100 mg 3-CB/L slowed the rate of decline of P. acidovorans M3GY densities compared to a 10 mg/L concentration or unamended control. Competition with the native flora was a significant factor affecting the persistence of introduced 3-CB degraders.     

The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT-T1E is located on a large self-transmissible plasmid

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967-3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A-like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10(-8) per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations.     

Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential of Pseudomonas putida F1

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and P. putida F1).     

Chlorobenzoate-Degrading Bacteria in Similar Pristine Soils Exhibit Different Community Structures and Population Dynamics in Response to Anthropogenic 2-, 3-, and 4-Chlorobenzoate Levels

A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 g of 2-, 3-, or 4-CB g^–1 soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 10⁷ CFU g^–1 of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 g 3-CB g^–1, 3-CB degradation occurred, suggesting that 500 g 3-CB g^–1 was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.     

The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.     

Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium

A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism. The TOL plasmid pWWO of the mesophile P. putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P. putida Q5, as shown by plasmid analysis by agarose gel electrophoresis. Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P. putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C. Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow. The production and demonstrated functioning of P. putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.     

Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.     

Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements.

Alcaligenes eutrophus NH9 was isolated from soil. This strain can utilize 3-chlorobenzoate (3-CB) as a sole source of carbon and energy. Most of the 3-CB-negative segregants had lost one of the plasmids present in the parent strain. The genes for catabolism of 3-CB were located within a 9.2-kb SacI fragment of this plasmid (pENH91). The genes were found to hybridize with genes for components of the modified ortho cleavage pathway from Pseudomonas putida. In one of the 3-CB-negative segregants, the plasmid had undergone the deletion of a segment with a size of about 12.5 kb that covered the catabolic genes. The deletion event seemed to be the result of reciprocal recombination between two highly homologous sequences with sizes of 2.5 kb that were present as a direct repeat at the two ends of the region that included the catabolic genes. Nucleotide sequence analysis of homologous fragments revealed a structure that resembled an insertion sequence and relatedness to IS21. During repeated subculturing of NH9 on liquid media with 3-CB, the culture was taken over by a derivative strain (designated NH9A) in which the degradative plasmid carried a duplicate copy of the 12.5-kb region that contained the catabolic genes. The duplication of these genes seemed again to have been mediated by recombination between the direct repeat sequences.     

Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit

The possibility of the accidental or deliberate release of genetically engineered microorganisms into the environment has accentuated the need to study their survival in, and effect on, natural habitats. In this study, Pseudomonas putida UWC1 harboring a non-self-transmissible plasmid, pD10, encoding the breakdown of 3-chlorobenzoate was shown to survive in a fully functioning laboratory-scale activated-sludge unit (ASU) for more than 8 weeks. The ASU maintained a healthy, diverse protozoal population throughout the experiment, and the introduced strain did not adversely affect the functioning of the unit. Although plasmid pD10 was stably maintained in the host bacterium, the introduced strain did not enhance the degradation of 3-chlorobenzoate in the ASU. When reisolated from the ASU, derivatives of strain UWC1 (pD10) were identified which were able to transfer plasmid pD10 to a recipient strain, P. putida PaW340, indicating the in situ transfer of mobilizing plasmids from the indigenous population to the introduced strain. Results from plate filter matings showed that bacteria present in the activated-sludge population could act as recipients for plasmid pD10 and actively expressed genes carried on the plasmid. Some of these activated-sludge transconjugants gave higher rates of 3-chlorobenzoate breakdown than did strain UWC1(pD10) in batch culture.     

Plasmid-mediated bioaugmentation of activated sludge bacteria in a sequencing batch moving bed reactor using pNB2

AIMS: The applicability of plasmid pNB2 for bioaugmentation of bacteria in model wastewater treatment reactors receiving 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: A setup of three biofilm reactors was studied, all initially inoculated with bacteria from activated sludge. Reactor PB received a Pseudomonas putida pNB2 donor strain not able to degrade 3-CA. Positive control reactor P received a 3-CA degrading Comamonas testosteroni pNB2-transconjugant. The negative control reactor N remained unchanged. Reactor P showed 3-CA degradation from the beginning of the experiment whereas in reactor PB, degradation started after an initial lag period. No degradation was observed in reactor N. PCR analysis showed that the P. putida donor abundance dropped in reactor PB, whereas the plasmid abundance did not, indicating transfer to other bacteria. A number of different 3-CA degrading C. testosteroni strains carrying pNB2 could be isolated from reactor PB. CONCLUSIONS: A successful plasmid-mediated bioaugmentation was achieved with C. testosteroni being the dominant 3-CA degrading pNB2 transconjugant species active in reactor PB. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of gene transfer to contribute to establishment and spread of genetic information in general, particularly emphasizing the spread of xenobiotic degrading potential by dissemination of catabolic genes.     

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).     

Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil

One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68?g?l(-1). In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.     

Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium.

A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism. The TOL plasmid pWWO of the mesophile P. putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P. putida Q5, as shown by plasmid analysis by agarose gel electrophoresis. Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P. putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C. Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow. The production and demonstrated functioning of P. putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.     

Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain

We describe an operon, copABCD, that encodes copper-binding and sequestering proteins for copper homeostasis in the copper-sensitive strain Pseudomonas putida PNL-MK25. This is the second operon characterized as being involved in copper homeostasis, in addition to a P1-type ATPase encoded by cueAR, which was previously shown to be active in the same strain. In this study, 3 copper-responsive mutants were obtained through mini-Tn5::gfp mutagenesis and were found to exhibit reduced tolerance to copper. Sequencing analysis of the transposon-tagged region in the 3 mutants revealed insertions in 2 genes of an operon homologous to the copABCD of P. syringae and pcoABCD of Escherichia coli. Gene expression studies demonstrated that the P. putida copABCD is inducible starting from 3 micromol/L copper levels. Copper-sensitivity studies revealed that the tolerance of the mutant strains was reduced only marginally (only 0.16-fold) in comparison to a 6-fold reduced tolerance of the cueAR mutant. Thus, the cop operon in this strain has a minimal role when compared with its role both in other copper-resistant strains, such as P. syringae pv. syringae, and in the cueAR operon of the same strain. We propose that the reduced function of the copABCD operon is likely to be due to the presence of fewer metal-binding domains in the encoded proteins.     

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.