Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

Spread and survival of promiscuous IncP-1 plasmids

Plasmids classified to the IncP-1 incompatibility group belong to the most stably maintained mobile elements among low copy number plasmids known to date. The remarkable persistence is achieved by various tightly controlled stability mechanisms like active partitioning, efficient conjugative transfer system, killing of plasmid-free segregants and multimer resolution. The unique feature of IncP-1 plasmids is the central control operon coding for global regulators which control the expression of genes involved in vegetative replication, stable maintenance and conjugative transfer. The multivalent regulatory network provides means for coordinated expression of all plasmid functions. The current state of knowledge about two fully sequenced plasmids RK2 and R751, representatives of the IncP-1alpha and IncP-1beta subgroups, is presented.     

Plasmids for naphthalene biodegradation incompatible with IncP-2 and IncP-7 group plasmids

Fourteen conjugative naphthalene degradative plasmids have been classified by incompatibility. It is shown that the plasmids of IncP-9 group are characterized by the minor entry exclusion, with respect to the R plasmids belonging to IncP-2 or IncP-7 groups. On the other hand, the naphthalene degradative plasmids of incompatibility group P-7 exhibit a markedly pronounced entry exclusion, with respect to the R plasmids of the same incompatibility groups. Two naphthalene degradative plasmids reveal incompatibility with the reference plasmids of two Inc groups (P-2 and P-7). These plasmids control also resistance of bacterial cells to potassium tellurite, which is characteristic of the IncP-2 plasmids. Two other naphthalene degradative plasmids are capable of stable coexistence with the IncP-2, P-7 or P-9 reference plasmids.     

Reconsideration of the previously classified incompatibility groups of plasmids,IncP-1 and IncP-11

This study presents the reassessment of earlier published data with reference to the article published in Environmental Microbiology entitled ‘IncP-type plasmids carrying genes for antibiotic resistance or aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains’ by Lo et al. This correspondence clarifies misperceptions of plasmids classified under incompatibility (Inc) groups IncP-1 and IncP-11.     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

The p lambda CM system: phage immunity-specific incompatibility with IncP-1 plasmids

Summary A pCM2 replicon derived by an N^– deletion from ::Tn9 which carries the imm434 immunity region is incompatible with some (but not all) IncP-1 plasmids. The imm pCM1 replicon does not show the same incompatibility behavior.     

Evidence for past integration of IncP-1 plasmids into bacterial chromosomes

Plasmids of the IncP-1 incompatibility group are self-transmissible between and stably maintained in a very broad range of Gram-negative bacteria. A characteristic feature of IncP-1 genomes is the existence of multiple binding sites (OB) for the KorB protein which plays a dual role in active partitioning of plasmid and coordinate regulation of expression of genes for replication, maintenance and transfer. A search of the available bacterial genome sequences revealed a significant number (70 out of 322) with one or more putative KorB binding sites. Binding of KorB to such a site was demonstrated by chromatin immunoprecipitation (ChIP) for Pseudomonas putida KT2440. While such a site may arise by chance, this is unlikely for Pseudomonas aeruginosa UCBPP-PA14 whose genome sequence contains four clustered OB sites and several regions have more than 80% nucleotide identity to traJ, trbJ and trbL of IncP-1 plasmids. A number of other bacterial genomes also contain integrated partial IncP-1 genomes or their remnants. These data provide evidence for multiple past integration events of IncP-1 plasmids into bacterial chromosomes and provide new evidence for IncP-1 plasmids being important elements in gene mobility.     

Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

Utilization of IncP-1 plasmids as vectors for transposon mutagenesis in myxobacteria

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.     

Detection of conjugative plasmids and antibiotic resistance genes in anthropogenic soils from Germany and India

PCR typing methods were used to assess the presence of plasmids of the incompatibility (Inc) groups IncP, IncN, IncW, IncQ and rolling circle plasmids of the pMV158 type in total DNA extracts from anthropogenic soils from India and Germany. Ten different soils from two different locations in Germany, the urban park Berlin Tiergarten and the abandoned sewage field Berlin-Buch, and from four different locations in India were analysed. PCR amplification of the total DNA extracts revealed the prevalence of IncP-specific sequences in Berlin Buch and Indian soil samples. The detected IncP plasmids contained at least one transfer function, the origin of transfer, oriT. In contrast to IncP-specific sequences, IncQ, IncN, IncW and pMV158-specific sequences were never detected. The presence of ampC, tet (O), ermB, SHV-5, mecA, and vanA antibiotic resistance genes was also tested. Three Indian soil samples irrigated with wastewater contained the ampC gene, whereas the other resistance genes were not found in any of the samples. Detection of IncP trfA2 and oriT sequences by PCR amplification and hybridization is a clear indication that IncP plasmids are prevalent in these habitats. Exogenous plasmid isolation revealed conjugative plasmids belonging to the IncPbeta group encoding resistance to ampicillin.     

Structural instability and stabilization of IncP-1 plasmids integrated into the chromosome of Myxococcus xanthus

IncP-1 plasmids are self transmissible to Myxococcus xanthus and maintained integrated into the host chromosome where they are liable to structural instability: deletions can span through the integrated plasmid; the frequency of these events depends on the recipient strain and on the localization of the insertion on the chromosome, but not on the structure of the plasmid.It is possible to isolate stabilized insertion, even from one of the most unstable strains, by growing immobilized cells in carrageenan beads in continuous nonselective culture. The remaining resistant cells are stabilized. Both the structural instability and the possible stabilization of the insertion can be useful when IncP-1 plasmids are used as cloning vectors in Myxococcus xanthus.     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751

The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well. Both KorA(RK2) (IncP-1 alpha) and KorA(R751) (IncP-1 beta) recognise the 5'-GTTTAGCTAAAC-3' palindrome. Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter. Class I (high affinity) operators for KorA(RK2) are flanked by an A-A-A/T sequence in the upstream half; the T base was shown to greatly influence strong repression. A C-A-G triplet was present in the same region in the R751 O(A) sequences and the G base was accordingly found to be important for strong KorA(R751) repression. An obvious difference between the two KorA proteins is a histidine to serine change at the C-proximal end of the putative recognition helix of the HTH motif (aa 56). An IncP-1 alpha KorAH56S mutant protein had higher affinity for all operators but had improved more on R751 operators than on RK2 operators. This indicates that KorA of RK2 is not maximised for DNA binding activity and that the aa difference at position 56 may play a role in differentiation between alpha and beta KorA operators.     

New molecular variants of epsilon and beta IncP-1 plasmids are present in estuarine waters

In this work the presence of broad-host-plasmids in an estuary in Portugal has been investigated. Pseudomonas putida KT2442 was used as model recipient bacteria in biparental matings with tetracycline and mercury to select for resistance phenotypes. As a result, 7 transconjugants were shown to carry broad-host-plasmids from the IncP-1 group, as seen by PCR amplification of the trfA gene. Sequence analysis confirmed the isolation of 4 plasmids from β-1 subgroup and 3 assigned to the recently described ε subgroup. To our knowledge this is the first report concerning the detection and isolation of IncP-1β and ε plasmids in estuarine waters. Moreover it is shown that, even though the retrieved plasmids are phylogenetically close to previously characterized plasmids, such as pB10 and pKJK5, respectively, they constitute new molecular variants.     

Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.