Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl₂), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd^r or Ni^r density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni^r transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.     

Retromobilization of heavy metal resistance genes in unpolluted and heavy metal polluted soil

Abstract: Retromobilization of the nonconjugative (Tra⁻Mob⁺) IncQ vector, pMOL155, and the non-mobilizable (Tra⁻Mob⁻) vector, pMOL149, by means of the IncP plasmids RP4 and pULB113 (RP4::Mu3A), was studied in plate matings and in soil microcosms, and compared with direct and triparental mobilization. Both vectors harbour the czc genes, originating from Alcaligenes eutrophus , which code for resistance to Co, Zn, and Cd. The donor of the czc genes was Escherichia coli which did not express these genes. The recipient, Alcaligenes eutrophus , expressed the czc genes very well. Retromobilization, direct and triparental mobilization of pMOL155 was observed in sterile soil. Both the addition of nutrients and heavy metals significantly enhanced the number of (retro)transconjugants. Retromobilization was also detected in nutrient amended nonsterile soil, but the presence of the autochthonous soil biota strongly reduced the number of retrotransconjugants and also prevented their increase upon application of heavy metals to the soil. Retromobilization of the czc genes, cloned in pMOL149, by using pULB113 was also observed, yet only in sterile, nutrient amended, heavy metal polluted soil.     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples.

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.     

CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus.

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).     

Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co^r, Zn^r, and Cd^r), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.     

Comparative study of non-conjugative R-plasmids from enterobacteria and Pseudomonas aeruginosa

Nonconjugative R-plasmids pBS76 and pBS94 (Sm Su), pBS95 and pBS96 (Sm Su Ap) isolated from clinical strains of Pseudomonas aeruginosa and plasmids pKMR281-pKMN284 (Sm Su), pKMR285-pKMR286 (Sm Su Tc) isolated from clinical strains of enterobacteria have been studied. Restriction maps of these plasmids are presented in the paper with some of plasmid genes for antibiotic resistance localized on them. The resistance determinants of plasmids pBS95 and pBS96 are shown to be included in transposon Tn3612 analogous to Tn3. Plasmids pBS76, pBS94-96 are of the wide host range and belong to incompatibility group P4 (IncQ). Plasmids pKMR281-pKMR286 are mutually incompatible and share the conspicuous DNA homology. They are inherited only by enterobacteria and are compatible with IncQ plasmids but in contrast to them are mobilized by RP4 plasmid with lower frequency.     

Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5alpha by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1

The gram-negative anaerobe Dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. We report here the characterization of a novel native plasmid, pDN1, from D. nodosus. Sequence analysis has revealed that pDN1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to Escherichia coli incompatibility group Q. However, in contrast to these plasmids, pDN1 encodes no antibiotic resistance determinants, lacks genes E and F, and hence is smaller than all previously reported IncQ plasmids. In addition, pDN1 belongs to a different incompatibility group than the IncQ plasmids to which it is related. However, pDN1 does contain the replication and mobilization genes that are responsible for the extremely broad host range characteristic of IncQ plasmids, and derivatives of pDN1 replicate in E. coli. In addition, the mobilization determinants of pDN1 are functional, since derivatives of pDN1 are mobilized by the IncPalpha plasmid RP4 in E. coli.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.     

Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.     

Retrotransfer of IncP1-like plasmids from aquatic bacteria

The first observation of plasmid retrotransfer by plasmids isolated from environmental sources is reported. A high incidence of retrotransferring ability amongst plasmids isolated from epilithic bacteria was found; some of these plasmids retrotransferred an IncQ plasmid at very high frequencies. Despite the broad host-range of the majority of the plasmids, only five out of 12 could be assigned to an incompatibility group by DNA hybridization. All five were designated IncP1; this revealed a limitation of probes derived from clinical sources for use with environmental isolates. Incompatibility testing by plate mating suggested that four additional plasmids displayed varying, albeit lower, degrees of incompatibility to the IncP1 plasmid RP1.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.     

Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A.

The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment. By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre. The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34. ncc is not expressed in Escherichia coli. The nre locus causes low-level nickel resistance in both Alcaligenes and E. coli strains. The nucleotide sequence of the ncc locus revealed seven open reading frames designated nccYXHCBAN. The corresponding predicted proteins share strong similarities with proteins encoded by the metal resistance loci cnr (cnrYXHCBA) and czc (czcRCBAD) of A. eutrophus CH34. When different DNA fragments carrying ncc genes were heterologously expressed under the control of the bacteriophage T7 promoter, five protein bands representing NccA (116 kDa), NccB (40 kDa), NccC (46 kDa), NccN (23.5 kDa), and NccX (16.5 kDa) were detected.