Publications

determination of the transcription initiation site and identification of the protein product of the regulatory gene xylr for xyl operons on the tol plasmid.	the xylr gene is a regulatory gene on the tol plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in pseudomonas putida. a dna fragment containing the xylr promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined. the transcription initiation site of the xylr gene was determined in cells of p. putida and escherichia coli by s1 nuclease and reverse transcriptase mapping. two initiation sites were detected which w ...	1985	2993247
evolutionary conservation of genes coding for meta pathway enzymes within tol plasmids pww0 and pww53.	pseudomonas putida mt53 contains a tol plasmid, pww53, that encodes toluene-xylene catabolism. pww53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal tol plasmid pww0. an rp4::pww53 cointegrate plasmid, pww53-4, containing about 35 kbp of pww53 dna, including the entire catabolic pathway genes, was formed, and a restriction map for kpni, hindiii, and bamhi ...	1985	2997136
omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species.	we have used the 2.0-kb dna fragment omega [prentki and krisch, gene 29 (1984) 303-313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the pseudomonas putida tol plasmid pww0. the mutant plasmids were subsequently introduced by conjugal mobilization into a variety of gram-negative bacteria. the omega fragment carries a selectable marker (aada+; spcr/smr), which is expressed in all species tested, as well as flanking transcription and translation te ...	1985	2998930
transposon mutagenesis analysis of meta-cleavage pathway operon genes of the tol plasmid of pseudomonas putida mt-2.	hybrid plasmids containing the regulated meta-cleavage pathway operon of tol plasmid pwwo were mutagenized with transposon tn1000 or tn5. the resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in escherichia coli. the physical locations of the insertions in each of 28 tn1000 and 5 tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. this information permitted the construction of a precise physical and ge ...	1984	6090417
nucleotide sequence of the promoter region of the xyldegf operon on tol plasmid of pseudomonas putida.	the transcription initiation site of the xyldegf operon on the tol plasmid of pseudomonas putida mt-2 was determined in p. putida and in escherichia coli by s1 nuclease and reverse transcriptase mapping. the induced synthesis of mrna started at the same start point in both p. putida and e. coli, although the amount of mrna in e. coli cells was less than that in p. putida. the nucleotide sequence of the region surrounding the start point was also determined. the ribosome-binding site (rbs) comple ...	1984	6092237
transcription of the tol plasmid toluate catabolic pathway operon of pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.	expression of the meta-cleavage pathway operon of tol plasmid pww0 of pseudomonas putida is positively regulated by the xyls gene product. we have sequenced the promoter region of this operon and localized the transcription initiation sites. two overlapping promoters, designated pm1 and pm2, are responsible for the positively regulated expression of the meta-pathway operon. mutants of p. putida were isolated that expressed the meta-cleavage pathway operon constitutively. several plasmid-located ...	1984	6096122
nucleotide sequence surrounding transcription initiation site of xylabc operon on tol plasmid of pseudomonas putida.	the xylabc operon on the tol plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylr. in the study here the nucleotide sequence was determined for the regulatory region of this operon. the in vivo transcription initiation site of the operon was determined by s1 nuclease and reverse transcriptase mapping. rna was prepared from m-methylbenzyl alcohol-induced cells of pseudomonas putida and escherichia coli carrying ptn ...	1984	6324212
characterization of a tol-like plasmid from alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.	alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. dna analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pra1000. hindiii and xhoi restriction endonuclease analysis of pra1000 showed it to be similar to the archetypal tol plasmid, pwwo, differing in the case of hindiii only by the absence of fragments b and ...	1984	6325399
enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by pseudomonas sp. strain b13.	dna fragments containing the xyld and xyll genes of tol plasmid pww0 -161 of pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahg gene of the nah plasmid nah7 , which codes for salicylate hydroxylase, were cloned in pbr322 vector plasmid. deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. the pbr322-based plasmids were ligat ...	1984	6327621
tol plasmid can prevent induction of chemotactic responses to aromatic acids.	growth conditions that elicited positive chemotaxis to benzoate and m-toluate in tol- pseudomonas putida cells failed to elicit taxis to these compounds in tol+ cells. the inability of tol+ cells to respond to these aromatic acids appears to be due to the preferential expression of tol-encoded genes for aromatic degradation over chromosomally encoded genes. expression of chromosomal genes for aromatic degradation is required for cells to form beta-ketoadipate, the inducer of benzoate and m-tolua ...	1984	6501222
complete nucleotide sequence of the metapyrocatechase gene on the toi plasmid of pseudomonas putida mt-2.	metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xyle gene on the tol plasmid of pseudomonas putida mt-2. we have cloned the xyle region in escherichia coli and determined the nucleotide sequence of the dna fragment of 985 base pairs around the gene. the fragment included only one open translational frame of sufficient length to accommodate the enzyme. the predicted amino acid sequence consisted of 307 ...	1983	6826546
molecular cloning of regulatory gene xylr and operator-promoter regions of the xylabc and xyldegf operons of the tol plasmid.	the regulatory gene xylr of the tol plasmid, which functions positively on both xylabc and xyldegf operons in the presence of m-xylene or m-methylbenzyl alcohol, was cloned onto an escherichia coli vector, pacyc177. a fused operon consisting of the operator-promoter region of the xylabc operon and the xyle gene was cloned onto pbr322. the xyle product, catechol 2,3-dioxygenase, was induced by m-xylene or m-methylbenzyl alcohol in the cells containing the fused operon when a 2.8-kilobase segment ...	1983	6885718
chromogenic identification of genetic regulatory signals in bacillus subtilis based on expression of a cloned pseudomonas gene.	a method to isolate fragments of dna that promote gene expression in bacillus subtilis is described. the system is based on production of catechol 2,3-dioxygenase [cato2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), ec 1.13.11.2] encoded by the pseudomonas putida tol plasmid gene xyle. the gene was transferred to ab. subtilis/escherichia coli plasmid vector to construct ptg402. although xyle is functionally expressed in e. coli, cato2ase is not detected in b. subtilis unless a fragment o ...	1983	6405380
localization and functional analysis of transposon mutations in regulatory genes of the tol catabolic pathway.	mutant derivatives of the tol plasmid pww0-161, containing tn5 insertions in the xyls and xylr regulatory genes of the catabolic pathway, have been identified and characterized. the two genes are located together on a 1.5- to 3.0-kilobase segment of tol, just downstream of genes of the enzymes of the meta-cleavage pathway. as predicted by a current model for regulation of the tol catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxyli ...	1983	6188746
characterization by molecular cloning of insertion mutants in tol catabolic functions.	a physical and genetic map of the tol catabolic region of pwwo (tol) was obtained by restriction endonuclease analysis of several dna insertion mutants (xyla, xyla xyls, xyls, and xylr) of r plasmid--tol derivatives. in two cases, the inserted dna was shown from restriction, dna hybridization, or heteroduplex analysis of cloned hind iii fragments to originate from within pwwo fragment hind iii-e. the effect of these dna insertions on tol catabolic activity and on structural alterations to the to ...	1983	6304792
plasmid-encoded regulation of colicin e1 gene expression.	a plasmid-encoded factor that regulates the expression of the colicin e1 gene was found in molecular cloning experiments. the 2,294-base-pair avaii fragment of the colicin e1 plasmid (cole1) carrying the colicin e1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin e1 synthesis as the original cole1 plasmid. an operon fusion was constructed between the 204-bp fragment containing the colicin e1 promoter-operator ...	1983	6313603
tol plasmid pww0 in constructed halobenzoate-degrading pseudomonas strains: enzyme regulation and dna structure.	wr211 and wr216 are derivatives of halobenzoate-degrading pseudomonas sp. strain b13 into which the 117-kilobase tol degradative plasmid pww0 has been transferred from pseudomonas putida mt-2. wr211 has lost the ability to grow on the tol-specific substrate m-xylene but retains the ability to grow on its metabolite, m-toluate. an analysis of the induction of enzymes was consistent with wr211 carrying a nonfunctional regulatory gene, xy1r, wr216 is a spontaneous derivative of wr211 which grows on ...	1982	7061391
excision and integration of degradative pathway genes from tol plasmid pww0.	wr211 is a transconjugant resulting from transfer of the 117-kilobase (kb) tol degradative plasmid pww0 into pseudomonas sp. strain b13. the plasmid of this strain, pww01211, is 78 kb long, having suffered a deletion of 39 kb. we show that wr211 contains the 39 kb that is missing from its plasmid, together with at least an additional 17 kb of pww0 dna integrated in another part of the genome, probably the chromosome. the ability of wr211 to grow on the tol-specific substrate m-toluate is the res ...	1982	7061392
tol plasmid pww0 in constructed halobenzoate-degrading pseudomonas strains: prevention of meta pathway.	the hybrid pathway for chlorobenzoate metabolism was studied in wr211 and wr216, which were derived from pseudomonas sp. b13 by acquisition of tol plasmid pww0 from pseudomonas putida mt-2. chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. when wr211 utilizes 3-chlorobenzoate (3cb), the expression of catechol 2,3-dioxygenase (c23o) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivati ...	1982	7061393
plasmid gene organization: naphthalene/salicylate oxidation.	genes for naphthalene metabolism are localized on nah7, an 83-kilobase (kb) plasmid, in two gene clusters under salicylate control. polar mutations formed by insertion of the transposon tn5 permit detection of the transcription direction and the gene organization within two approximately 10-kb dna segments separated by a approximately 7-kb regulatory gene region. the gene cluster specifying conversion of naphthalene to salicylate lies near the left initiation of a 25-kb dna fragment a released b ...	1982	6278499
spontaneous deletions in the tol plasmid pww20 which give rise to the b3 regulatory mutants of pseudomonas putida mt20.	the size of the tol plasmid pww20 from pseudomonas putida mt20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270-280 kilobase pairs (kb). during growth on benzoate, mt20 segregates strains carrying mutations in the plasmid regulatory gene xyls; these so-called b3 strains retain the ability to grow on m-xylene (mxy+) but do not grow on its metabolite m-toluate (mtol-) and have also lost the ability to transfer the plasmid (tra-). analysis ...	1982	6288840
identification of chromosomally integrated tol dna in cured derivatives of pseudomonas putida paw1.	some plasmid-free tol- strains derived from pseudomonas putida paw1 (which carries the tol plasmid pww0) have a segment of tol dna located chromosomally. of three independently isolated strains, paw86 had an integrated tol segment of 16 kilobases and paw85 had two copies of this segment in different chromosomal locations, whereas the chromosomal dna of paw82 showed no homology with the tol plasmid. in cultures of the parental strain, it appears that a 56-kilobase tol dna segment is located chrom ...	1982	6290457
the tol plasmid is naturally derepressed for transfer.	pseudomonas putida mt-2, formerly known as pseudomonas arvilla mt-2, which carries the wild-type tol plasmid, and p. putida strain ac37 carrying tol, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages pr4 and prd1. pseudomonas putida strain pps388, also harbouring the plasmid, was not lysed. in a p. putida mt-2 host, tol transferred 18-fold better on a surface (2.5 x 10(-1) transconjugants per donor h-1) than in liquid; when p. putida pps388 was the host, however, a fre ...	1982	6134782
[tol-plasmid-specific mrna study].		1982	6179726
isolation and characterization of spontaneously occurring tol plasmid mutants of pseudomonas putida hs1.	a strain of pseudomonas (p. putida hs1) was found to resemble p. putida (arvilla) mt-2 in its ability to degrade toluene, m- and p-xylene, 1,2,4-trimethylbenzene (pseudocumene), and 3-ethyltoluene via oxidation of a methyl substituent and reactions of the meta-fission pathway. the ability to degrade these substrates by p. putida hs1 (ppc1) was shown to be encoded by a tol (pdk1) plasmid as evidenced by: (i) spontaneous loss of the tol-related phenotype after growth with benzoate, (ii) transfer o ...	1981	7240090
metabolism of allylglycine and cis-crotylglycine by pseudomonas putida (arvilla) mt-2 harboring a tol plasmid.	spontaneous mutants which acquired the ability to utilize d-allylglycine (d-2-amino-4-pentenoic acid) and dl-cis-crotylglycine (dl-2-amino-cis-4-hexenoic acid) but not l-allylglycine or dl-trans-crotylglycine could be readily isolated from pseudomonas putida mt-2 (pam1). derivative strains of pam1 putatively cured of the tol (pwwo) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the tol (pwwo ...	1981	7287632
excision of the 40kb segment of the tol plasmid from pseudomonas putida mt-2 involves direct repeats.		1981	6950198
molecular and functional analysis of the tol plasmid pwwo from pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway.	the genetic organization of the pseudomonas putida plasmid pwwo-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase dna segment. the physical or ...	1981	6950388
molecular cloning of tol genes xylb and xyle in escherichia coli.	the xylb and xyle genes in the tol plasmid of pseudomonas putida mt-2, which code for benzyl alcohol dehydrogenase and catechol 2,3-oxygenase, respectively, were cloned onto plasmid pbr322 in escherichia coli for detailed mapping. the xylb gene was mapped in a 2.9-kilobase region within the bamhi bc fragment of ptn2, an in vivo rp4-tol recombinant, whereas the xyle gene was mapped in a 1.8-kilobase region within the bamhi bd fragment. the directions of transcription of these genes were deduced f ...	1981	7009570
molecular cloning of gene xyls of the tol plasmid: evidence for positive regulation of the xyldegf operon by xyls.	the xyldegf operon and the regulatory gene xyls of the tol plasmid found in pseudomonas putida mt-2 were cloned onto escherichia coli vector plasmids. a 9.5-kilobase fragment, derived from the tol segment of ptn2 deoxyribonucleic acid, carried the xyl genes d, e, g, and f, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. the enzymes were noninducible unless a 3-kilobase psti fragment, d ...	1981	6271729
hybrid pathway for chlorobenzoate metabolism in pseudomonas sp. b13 derivatives.	derivatives of pseudomonas sp. b13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the tol plasmid of pseudomonas putida mt-2 were studied. the utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. during growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deo ...	1980	7380800
physical and functional mapping of rp4-tol plasmid recombinants: analysis of insertion and deletion mutants.	cleavage sites for the restriction endonucleases xhoi, bamhi, hindiii, and ecori were mapped on the ptn2 plasmid, a recombinant of tol and rp4, which specifies the toluene-degrading enzymes in the same way as the wild-type tol plasmid. the ptn2 plasmid, purified from a strain of escherichia coli, contained the entire length of the rp4 plasmid (about 54 kilobase pairs [kb]) and the tol segment (about 56 kb). the tol segment is inserted at about 12 and 5 kb away from the ecori and bamhi cleavage s ...	1980	6252192
construction of a partial diploid for the degradative pathway encoded by the tol plasmid (pwwo) from pseudomonas putida mt-2: evidence for the positive nature of the regulation by the xyir gene.		1980	6929031
a cleavage map of the tol plasmid of pseudomonas putida mt-2.	a cleavage map of the tol plasmid pwwo has been determined for the restriction endonucleases hindiii and xhoi. a number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified xhoi fragments to southern blots of hindiii digest products and (iii) analysis of a number of deletion mutants.	1979	231727
an endonuclease cleavage map of the plasmid pwwo-8, a derivative of the tol plasmid of pseudomonas putida mt-2.	cleavage sites on the pwwo-8 plasmid were determined for the restriction endonucleases hindiii and xhoi. terminal labelling using dna polymerase i was particularly useful both for the characterisation of the smaller cleavage products and for confirmation of the order of fragments in the intact plasmid.	1979	285316
transposition of plasmid dna segments specifying hydrocarbon degradation and their expression in various microorganisms.	the conjugative tol plasmid (75 mdal), specifying biodegradation of xylenes, toluene, and trimethylbenzene derivatives, undergoes dissociation in pseudomonas aeruginosa pao to a nonconjugative tol() plasmid (28 mdal) and a transfer plasmid termed toldelta (48 mdal). the tol() plasmid is rendered transmissible through introduction of a number of conjugative plasmids such as factor k, cam, and toldelta but not by the fp2 derivative pr0271. transfer of tol(*) via factor k or toldelta is mediated ...	1978	277912
tol plasmid in pseudomonas aeruginosa pao: thermosensitivity of self-maintenance and inhibition of host cell growth.	the tol plasmid originally isolated in pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of pseudomonas, i.e., p. putida, p. fluorescens, and p. aeruginosa, except for a strain of p. aeruginosa, strain pao. the same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. in addition, the transmissibility of the tol plasmid from strain pao to p. putida was low when the ...	1978	415040
isolation of tol and rp4 recombinants by integrative suppression.	we obtained genetic and molecular evidence of non-thermosensitive recombinants of rp4 (kmr tcr cbr/apr) and the thermosensitive tol plasmid. as first isolated in pseudomonas aeruginosa pao, the recombinant plasmid ptn1 specified noninducible synthesis of tol enzymes and was transmissible to escherichia coli on selection for the transfer of kanamycin resistance. the phenotypic expression of tol genes of ptn1 in e. coli was low and also noninducible. a spontaneous segregant, ptn2, appearing from p ...	1978	418059
regulation of the degradative pathway enzymes coded for by the tol plasmid (pwwo) from pseudomonas putida mt-2.	pseudomonas putida mt-2 carries a plasmid (tol, pwwo) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m ...	1978	659369
tol is a broad-host-range plasmid.	we readily isolated insertions of the carbenicillin resistance element tn401 into the tol plasmid in pseudomonas putida. hybrid tol::tn401 plasmids stably express the cbr phenotype in pseudomonas aeruginosa and escherichia coli. whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the pseudomonas species.	1978	97271
isolation of a mutant tol plasmid with increased activity and transmissibility from pseudomonas putida (arvilla) mt-2.	strains with greater ability to dissimilate m-toluate were obtained from the wild-type pseudomonas putida (arvilla) mt-2 that harbors the tol plasmid. increased growth of a mutant strain on aromatic substrates was coupled with simultaneous increase in the activity of metapyrocatechase, an enzyme coded by the tol plasmid, without changing its catalytic properties. in the mutant and the wild-type strains, the inducer specificity and the induction kinetics of metapyrocatechase synthesis were the sa ...	1977	830645
a comparative study of the nah and tol catabolic plasmids in pseudomonas putida.	a comparative study of the nah and tol catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. the plasmids were studied in a strain of p. putida and its mutant derivatives. the nah and tol plasmids were found to be incompatible. under the conditions used in these experiments the tol plasmid transferred into some strains into which nah was unable to transfer. the use of mutants to remove certain catabolic activities enco ...	1977	603460
apparent fusion of the tol plasmid with the r91 drug resistance plasmid in pseudomonas aeruginosa.	the tol catabolic plasmid was shown to be compatible with the r91 drug resistance plasmid. however, the tol plasmid was extremely unstable in mutant pa03 of p. aeruginosa. by selecting for stabilization of the tol plasmid in pa03 harbouring r91, it was possible to isolate a strain in which markers from both r91 and tol appeared to exist in a single recombinant plasmid. this plasmid, pnd3, encoded resistance to carbenicillin, was able to transfer at the same frequency as the r91 plasmid and encod ...	1977	414707
ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new tol plasmids.	thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium. eight of these were classified as pseudomonas putida, one as a fluorescent pseudomonas sp., and four as nonfluorescent pseudomonas sp. all 13 strains appeared to carry tol plasmids superficially similar to that previously described in p. putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon ...	1976	1254555
metabolism of toluene and xylenes by pseudomonas (putida (arvilla) mt-2: evidence for a new function of the tol plasmid.	pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, tol. these compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the tol plasmid. the specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three ...	1975	1176436
metabolism of benzoate and the methylbenzoates by pseudomonas putida (arvilla) mt-2: evidence for the existence of a tol plasmid.	mutant strains of pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin c. the mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase a ...	1974	4418209