New derivatives of TOL plasmid pWW0

Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWW0mut1, isolated from strain PPW1-1. Additional deletions were observed in pWW0mut1 after 'benzoate-curing' (plasmids pWW0mut15, pWW0mut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.     

Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of a P. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.     

Use of Recombinant Bioluminescent Bacteria for On-Site Monitoring of Toluene Analogs at Petroleum Contaminated Sites

This paper describes the application of a genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, to monitor toluene analogs in groundwater collected from petroleum hydrocarbon contaminated sites. KG1206 contains the intact TOL plasmid and a second plasmid with the Pm-lux gene allowing it to produce bioluminescence in the presence of toluene analogs and their derivatives such as toluene, xylenes, and m-toluate. The simple bioluminescence assay consisted of mixing one volume of groundwater sample with four volumes of broth culture followed by bioluminescence measurement after 30?min. The maximum bioluminescent response with pure chemicals followed the order: m-methyl benzyl alchohol>m-toluate>toluene>m-xylene>benzoate>p-xylene>o-xylene. The bioluminescence production was well correlated to the m-toluate concentrations (R2>0.97) in field samples, with concentrations predicted from the bioassay falling within 75–158% of true concentration. However, no strong correlation was observed between the bioluminescence intensity and the total inducer concentration in the groundwater. Results from this study demonstrate the potential of using recombinant bioluminescent bacteria as a rapid and simple tool for monitoring specific pollutants at contaminated sites.     

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)     

ntn genes determining the early steps in the divergent catabolism of 4-nitrotoluene and toluene in Pseudomonas sp. strain TW3

Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMAB genes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the beta-ketoadipate pathway.     

XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.     

Utilization of quinate and p-hydroxybenzoate by actinomycetes: key enzymes and taxonomic relevance

474 strains of the actinomycete genera Streptomyces (including species of the former genera Chainia and Streptoverticillium), Pseudonocardia and Micromonospora were examined for their ability to degrade quinate (Q) and p-hydroxybenzoate (pHB); selected strains were also tested for their capacity to catabolize benzoate (B). Whereas in the case of Q (5-10 g/l of a mineral salts agar medium) the growth response signalizes assimilation, pHB has to be supplied in lower concentration (routinely 0.3 g/l together with small amounts of peptone and yeast extract in liquid broth), and its degradation has to be determined spectrophotometrically. 27% of the streptomycete strains were able to grow with Q, and 57% with pHB. The three strains of "Chainia" that were tested metabolized Q and pHB, but none of the fourty species of "Streptoverticillium" showed this ability. 80% of the 30 strains of Psn. autotrophica grew with Q, and 100% degraded pHB and B. Two of the five Micromonospora strains gave a positive response with pHB, but not with Q.-Toluene treated cells (preincubated with Q, pHB or B, respectively) gave a positive Rothera reaction with protocatechuate or catechol respectively, thus demonstrating that these organisms employed the beta-ketoadipate pathway (orthofission) for the degradation of Q, pHB and B. The assay of five relevant enzymes in cell-free extracts of nine selected organisms showed that in nocardioform actinomycetes (Pseudonocardia, Rhodococcus) all enzymes of the protocatechuate branch of the ketoadipate pathway seem to be induced by beta-ketoadipate as demonstrated here for protocatechuate-3,4-dioxygenase. In contrast, in Streptomyces this enzyme appears to be induced by its substrate, protocatechuate, whereas the regulation of the other enzymes of this pathway remains to be elucidated.     

Mutations affecting the alpha subunit of Bordetella pertussis RNA polymerase suppress growth inhibition conferred by short C-terminal deletions of the response regulator BvgA

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg- phenotype. When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the alpha subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the alpha subunit of B. pertussis RNA polymerase were also isolated. Two of these resulted in increased levels of the alpha subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced.     

The comparative characteristics of the Salmonella serotypes rarely encountered in the Maritime Territory

The results of the comparative study of the phenotypical properties and the plasmid profile of 63 strains of salmonellae, belonging to 44 serotypes of groups B, C1, C2, C3, D, E1, E4, F. The study revealed that strains of different serotypes had their individual plasmid profile. Strains of the same serotype of salmonellae isolated from similar sources had an identical plasmid profile, while strains isolated from different sources differed in their plasmid profiles, though they might have a similar phenotype. Plasmid analysis was shown to be an effective method for the intraspecific typing of rarely isolated Salmonella serotypes and suitable for use as the basis of the microbiological monitoring of salmonellae.     

A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1

Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 18O2 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen. Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these effectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.     

In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column

A diesel fuel-contaminated aquifer was bioremediated in situ by the injection of oxidants (O2 and NO3-) and nutrients in order to stimulate microbial activity. After 3.5 years of remediation, an aquifer sample was excavated and the material was used (i) to isolate bacterial strains able to grow on selected hydrocarbons under denitrifying conditions and (ii) to construct a laboratory aquifer column in order to simulate the aerobic and denitrifying remediation processes. Five bacterial strains isolated from the aquifer sample were able to grow on toluene (strains T2 to T4, T6, and T10), and nine bacterial strains grew on toluene and m-xylene (strains M3 to M7 and M9 to M12). Strains T2 to T4, T6, and T10 were cocci, and strains M3 to M7 and M9 to M12 were rods. The morphological and physiological differences were also reflected in small sequence variabilities in domain III of the 23S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S rRNA of one isolate (T3 and M3) of each group revealed a close phylogenetic relationship for both groups of isolates to organisms of the genus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcus tolulyticus group, and Azoarcus evansii were used to investigate the significance of hydrocarbon-degrading Azoarcus spp. in the laboratory aquifer column. The number of bacteria in the column determined after DAPI (4',6-diamidino-2-phenylindole) staining was 5.8 x 10(8) to 1.1 x 10(9) cells g of aquifer material-1. About 1% (in the anaerobic zone of the column) to 2% (in the aerobic zone of the column) of these bacteria were detectable by using a combination of probes Azo644 and Azo1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are significant members of the indigenous microbiota. More than 90% of the total number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the beta subdivision of the class Proteobacteria (beta-Proteobacteria), and 10 to 16% belonged to the gamma-Proteobacteria. Bacteria of the alpha-Proteobacteria were present in high numbers (10%) only in the aerobic zone of the column.     

The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation

The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.     

Chondrocyte implantation in the repair of chondral lesions of the knee: economics and quality of life

Exposure to low levels of chemicals indoors is often to a mixture of volatile organic compounds (VOCs). It is of interest to determine if the symptomatic and sensory responses can be attributed to a single chemical or to a mixture of chemicals. To determine if sensory or symptomatic responses differ with exposure to single or mixed VOCs, 100 female subjects participated in a 6-hr exposure study. Subjects were exposed to one of six equimolar concentrations equivalent to 24 mg/m3 toluene, control, m-xylene, n-butyl acetate, m-xylene plus n-butyl acetate, a mixture of 21 chemicals including n-butyl acetate and m-xylene, and to the same mixture of chemicals without n-butyl acetate and m-xylene (19 chemicals). The results indicated that there was no difference in reporting of symptoms or sensory responses between the exposures. When the control group was added, some variables, primarily odor intensity and nasal irritation, attained significance.     

Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.     

Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways

Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.     

Colicin U, a novel colicin produced by Shigella boydii

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)