Characteristics and restriction analysis of the 4-chlorobiphenyl catabolic plasmid, pSS50.

The plasmid pSS50 is a 53-kilobase self-transmissible plasmid of broad host range that has been isolated from several Alcaligenes and Acinetobacter species. This plasmid has previously been shown to mediate the mineralization of 4-chlorobiphenyl to carbon dioxide and water. Physical characterization of this plasmid by restriction analysis indicates that most hexanucleotide cleavage sites are clustered in a 5-kilobase region, leaving large regions without restriction sites. The paucity of restriction sites is not due to DNA methylation.     

The complete sequence and analysis of the large virulence plasmid pSS of Shigella sonnei

The complete sequence of pSS, which is the large virulence plasmid of Shigella sonnei, was determined. The 214-kb plasmid is composed of segments of virulence-associated genes, the O-antigen gene clusters, a range of replication and maintenance genes, and large numbers of insertion sequence (IS) elements. Two hundred and forty-one open reading frames (ORFs) were identified, of which 117 are highly homologous to IS elements or transposases, 57 are homologous to known pathogenesis-associated proteins, and 30 are related to replication, plasmid maintenance, or other metabolic functions. Thirty-seven ORFs have no similarity to proteins with a known function, including two with no significant similarity to any hypothetical proteins. Interestingly, 10 ORFs encoding O-antigen gene clusters were identified on the plasmid and this is markedly different from most other Shigella spp. virulent plasmids. A novel toxin-antitoxin system, a series of stbDE homologs, was found on the plasmid immediately downstream of the replication region; the sole segregation stability system may be responsible for the instability of pSS. The pSS plasmid is a mixture of genes with different origins and functions. The sequence suggests a remarkable history of IS-mediated recombination and acquisition of DNA across a range of bacterial species.     

Genetic and molecular analysis of a regulatory region of the herbicide 2,4-dichlorophenoxyacetate catabolic plasmid pJP4

In Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba) degradation plus mercury resistance. The plasmid genes specifying 2,4-D and 3-Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF. Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed. Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592bp. Similar stem-loop structures were observed among other 2,4-D plasmids. The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32kDa. The deduced amino acid sequence of tfdR indicates that it is a member of the LysR-type activators. Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4-D negative, but 3-Cba positive. In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4-D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.     

Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain.     

Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50.

The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate. Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2. This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp. strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate. The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70.     

Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50.

The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate. Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2. This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp. strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate. The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70.     

Molecular and population analyses of a recombination event in the catabolic plasmid pJP4

Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.     

Isolation and restriction endonuclease analysis of a mycoplasma plasmid

A 1.7-kb plasmid was isolated from a large colony type strain of Mycoplasma mycoides subsp. mycoides. It was cloned into the Escherichia coli vector M13mp19, and DNA from the resultant recombinant was used to construct a restriction map of the plasmid. Because mycoplasmas show deviation from normal coding patterns, the availability of a mycoplasma plasmid could be useful in the development of a cloning vector for them.     

Cloning and characterization of tfdS, the repressor-activator gene of tfdB, from the 2,4-dichlorophenoxyacetic acid catabolic plasmid pJP4.

Plasmid pRO101, a derivative of plasmid pJP4 which contains Tn1721 inserted into a nonessential region, is inducible for 2,4-dichlorophenol hydroxylase (DCPH) encoded by tfdB. Plasmid pRO103, which has a deletion in the BamHI-F--BamHI-E region of plasmid pRO101, has elevated basal levels of DCPH but is uninducible. The regulatory gene for tfdB, designated tfdS, was cloned as an 8.3-kilobase-pair EcoRI-E fragment. When the cloned tfdS gene was in trans with plasmid pRO103, the baseline DCPH levels were repressed to normal uninduced levels and were fully induced when this strain was grown in the presence of 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol, or 4-chlorocatechol. However, when tfdS was in trans with tfdB in the absence of tfdCDEF, tfdB was repressed but could not be induced. When tfdS and tfdC1, which encodes chlorocatechol 1,2-dioxygenase, are in trans with tfdB, tfdB remained uninduced, indicating that a downstream metabolite of chloro-cis,cis-muconate, either 2-cis-chlorodiene lactone or chloromaleylacetic acid, is the effector. Collectively, these data demonstrate that the gene product of tfdS acts as a repressor of tfdB in the absence of an effector and as an activator of tfdB when an effector is present.     

Plasmid-mediated mineralization of 4-chlorobiphenyl.

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB.     

Effect of vitamins on the aerobic degradation of 2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl

The effect of vitamins on the aerobic degradation and dechlorination of 2-chlorophenol and 4-chlorophenol by Pseudomonas picketti, strain LD1, and 4-chlorobiphenyl by Pseudomonas sp. strain CPE1 was determined. These microorganisms are capable of using the target compounds as the sole carbon and energy source, but do not need vitamins to metabolize them. The addition to the culture medium of a vitamin solution containing biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, niacin, pantothenic acid, cyanocobalamin, p-aminobenzoic acid, and thioctic acid (total final concentration: 600 ppb) resulted in a 7%–16% increase in the amount of target compounds degraded over the incubation period required for the concentration of the compound in the cultures to drop to approximately zero. A corresponding increase in the amount of chloride ion produced was also detected during the same period, indicating active (and often stoichiometric) dechlorination of the target compounds.     

Biodegradation of 4-chlorobiphenyl by Micrococcus species

A Micrococcus sp., isolated by enrichment culture, grew on 4-chlorobiphenyl at 2 g/l as sole carbon source and produced 4-chlorobenzoic acid in the culture medium as a dead-end metabolite. The organism degraded 4-chlorobiphenyl by 2,3-dihydroxylation followed by meta-ring cleavage to yield 4-chlorobenzoate and carbon fragments for cell growth.     

The metabolism of 4-chlorobiphenyl in the pig.

1-Chlorobiphenyl was administered retrocarotidly in a saline oil emulsion to anesthetized pigs. The urine obtained from the pigs 2 h after injection of the 4-chlorobiphenyl was analyzed for metabolites. Mass spectrometric analysis indicated the presence of a monohydroxylated species (M+ 204) and a dihydroxylated compound (M+ 220) and these were identified as 4'-chloro-4-biphenylol and 4'-chloro-3,4-biphenyldiol, respectively. Examination of the urine extracts from pigs 0.5, 1, and 2 h after administration of the 4-chlorobiphenyl indicated an increase in both urinary metabolites with time, however, neither compound was detected in the bile. Blood and organ samples were examined also for metabolites and the results clearly showed the presence of 4'-chloro-4-biphenylol in the blood, kidney, and liver and absence in the lung, brain, and heart. The diol was not observed in any of these samples.     

Localization of SsoII restriction endonuclease and methylase genes on the map of the P4 plasmid

The restrictional mapping of naturally occurring plasmid P4 from Shigella sonnei 47 strain coding for the SsoII restriction endonuclease and methylase genes has been made. Using the genetic engineering approach the locations of the SsoII host cell specificity system enzymes genes have been determined.     

Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls

The biodegradation products of 4-chlorobiphenyl were analyzed in an Achromobacter sp. strain and a Bacillus brevis strain. Both strains generated the same metabolites, with 4-chlorobenzoic acid as the major metabolic product. Our results corroborate previous observations whereby most bacterial strains degrade the chlorobiphenyls via a major pathway which proceeds by an hydroxylation in position 2,3 and a meta-1,2 fission. However, we also detected several metabolites whose structure suggests the existence of other routes for the degradation of chlorinated biphenyls.     

Molecular cloning and mapping of SphI restriction fragments of plasmid RP4

A combined physical and functional map of plasmid RP4 is presented including the sites for 18 restriction endonucleases. Several cleavage sites of SphI, BalI, and ApaI are suitable for the dissection of the transfer gene regions. Recombinant plasmids containing RP4 SphI fragments were constructed to assist in localizing sites relative to each other and to assign functions conferred by RP4 to the host.     

Regulation of naphthalene catabolic genes of plasmid NAH7.

Tn5 insertion mutations defining a regulatory gene, nahR, of the naphthalene catabolic pathway encoded by the NAH7 plasmid were mapped within a small NAH7 region only a few hundred bases upstream of the nahG gene, the most promoter-proximal gene of the nahGHIJK operon. The nahR mutations blocked the induction of both the nahABCDEF and nahGHIJK operons, and the defect was completely corrected in the presence of the wild-type allele in a trans position. The pleiotropic, recessive, and negative nature of these mutations indicates that the nahR gene specifies a regulatory element which is required to activate both nah operons.