Mercury and Organomercurial Resistances Determined by Plasmids in Pseudomonas

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.     

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Polypeptides encoded by the mer operon.

HgCl2-induced polypeptides synthesized by Escherichia coli minicells containing recombinant or natural HgR plasmids were labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All plasmids examined encoded two heavily labeled, HgCl2-inducible polypeptides of 69,000 and 12,000 daltons. Most plasmids also encoded two additional HgCl2-inducible proteins in the 14,000- to 17,000-dalton range. Antiserum prepared against a purified mercuric ion reductase reacts with the 69,000-dalton polypeptide and a minor 66,000-dalton protein seen in several different HgR minicells. Recombinant plasmids constructed from portions of mer DNA from the IncFII plasmid NR1 were also analyzed in the minicell system. Five HgCl2-inducible polypeptides (69,000, 66,000, 15,100, 14,000, and 12,000 daltons) were synthesized in minicells carrying pRR130, a recombinant derivative containing the EcoRI-H and EcoRI-I restriction fragments of NR1. The EcoRI-H fragment of NR1 encodes the three small mer proteins of 15,100, 14,000, and 12,000 daltons and the amino-terminal 40,000 daltons of the mercuric ion reductase monomer.     

Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon

We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor). With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element. Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium. The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype. The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived. Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment. Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.     

Gene copy number effects in the mer operon of plasmid NR1.

The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.     

A deletion analysis of hybrid phage carrying the US region of Herpes simplex virus type 1 (Patton). I. Isolation of deletion derivatives and identification of chi-likes sequences

The EcoRI-H fragment (15.4 kb) of Herpes simplex virus type 1 (HSV-1) has been cloned in lambda gtWES in both orientations. This fragment contains the entire US region and has about 900 bp of terminal redundant sequences derived from the internal and terminal repeats of the S region. 56 independent plaque-forming deletion derivatives of the lambda gt/WES::EcoRI-H hybrid phage were isolated using either EDTA resistance or ability to grow on Escherichia coli(P2) lysogens as selective methods. The endpoints of these deletions were located using nine restriction enzymes that cleave within the EcoRI-H fragment. All of the deletions have at least one endpoint within the cloned fragment. Several unusual features of the lambda hybrids, including heterogeneity of a particular region in the HSV-1 EcoRI-H fragment and the presence of chi-like sequences in the US region of HSV-1, are discussed.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Characterization of a mini ColE1 cloning vector.

Plasmid pHA105 (formerly pAC105), a mini ColE1 plasmid containing one restriction endonuclease EcoRI site, was further characterized using restriction endonuclease analysis thereby revealing its relationship to ColE1. The polypeptides specified by plasmid pHA105 in minicells are of low molecular weight making it a useful plasmid to define cloned polypeptides larger than 16,000 daltons and its use for that purpose was demonstrated. pHA105 was used to clone two different sized fragments of DNA containing the gal operon. pHA105 was also used to reclone a 2 Mdal fragment of DNA that, when expressed, represses the synthesis of capsular polysaccharide. The repression of polysaccharide synthesis was expressed when a plasmid containing one molecule each of pHA105 and the 2 Mdal fragment was prepared (pFM100). In contrast, a plasmid containing two copies of pHA105 and one of the 2 Mdal fragment (pHA138) did not repress polysaccharide synthesis. The results demonstrate that expression of a cloned fragment gene may be prevented in certain arrangements of the vector and cloned fragment. Plasmid pHA105 fails to exhibit relaxation after treatment with sodium dodecyl sulfate in contrast to ColE1 treated in the same way. pHA105 replicates as a dimer form while ColE1 usually does not. A hypothesis that a function of a DNA-protein complex is required for monomeric DNA circle formation is discussed.     

Susceptibility to mercurials of clinical Pseudomonas aeruginosa isolated in México

Susceptibility to inorganic mercuric ions and to organomercurials of 237 Pseudomonas aeruginosa clinical strains isolated in Mexico was determined by agar dilution tests. Resistant strains fell into two classes: i) narrow-spectrum resistant strains (27% of total isolates) resistant only to mercuric ions and to merbromin, and most grouped in pyocin type 1; and ii) broad-spectrum resistant strains (5%) with additional resistances to thimerosal, phenylmercury, methylmercury and p-hydroxymercuribenzoate, that belonged mostly to pyocin type 10. Mercurial resistant isolates showed a higher proportion of resistance to antibiotics and metals than did mercurial sensitive isolates, and broad-spectrum resistant strains had the highest frequency of resistance to antibiotics and to tellurite and arsenate.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.     

Re-examination of the ColE1 DNA regions essential for autonomous replication and their functions

Starting from pAO3, a plasmid consisting of a quarter of colicinogenic factor E1 (ColE1) DNA, various small ColE1 derivatives were constructed by in vitro recombination and their ability to achieve autonomous replication was examined. The 436 base pair HaeIII-C fragment of pAO3 contained information for replication when it was recombined with the non-replicating Amp fragment. However, when it was connected to other DNA fragments, the resulting hybrid molecules were not isolated as plasmids. The present results indicate that the additional region of about 240 base pairs next to the HaeIII-C fragment of ColE1 is also essential for the maintenance of a plasmid state. Moreover, using various small ColE1 derivatives, the DNA region responsible for the interference and incompatibility functions of ColE1 DNAs was located. The results indicate that the interference and incompatibility functions are coded by the same ColE1 DNA segment and are not essential for the maintenance of a plasmid state.     

A deletion analysis of lambda hybrid phage carrying the US region of Herpes virus type 1 (Patton). II. Construction of an SmaI map

The 15.4 kb EcoRI-H fragment of Herpes simplex virus type 1 (HSV-1) strain Patton, which contains the entire short unique (US) region, has been cloned in bacteriophage lambda. The fragment contains a terminal redundancy of about 900 bp that represents the S region terminal-repeat sequences. The restriction enzyme SmaI cleaves the EcoRI-H fragment at more than 30 sites. We have constructed an SmaI map of this fragment using thirteen isolates of lambda gtWES hybrid bacteriophage that carry various deletions of the EcoRI-H fragment.     

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 and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Cloning and expression of the gene(s) for cephalosporinase production of Citrobacter freundii

The chromosomal gene(s) for cephalosporinase production of Citrobacter freundii GN346 has been cloned into vector plasmid pMK1, initially as a 7.3 kb EcoRI fragment. From the substrate profile and the response to anti-GN346 CSase serum of the enzyme produced, it was confirmed that the hybrid plasmid (pTY71) carries the relevant chromosomal cephalosporinase gene from C. freundii GN346. A restriction endonuclease cleavage map of cloned EcoRI fragments was constructed, and the structural gene of the cephalosporinase could be limited in the 1.5 kb BamHI fragment. The cloned gene(s) was expressed at an extremely low level in Escherichia coli. Furthermore, its expression was constitutive in E. coli, although inducible in its own cytoplasm.