Features of the plasmid pMV158-encoded MobM, a protein involved in its mobilization

The streptococcal promiscuous plasmid pMV158 can be mobilized between a number of bacterial species by means of three elements: (i) the plasmid-encoded nicking-closing protein MobM, involved in the initiation and termination of the conjugative transfer; (ii) the DNA sequence where the MobM-mediated nick takes place (the oriT(pMV158)); and (iii) the function(s) provided by auxiliary plasmids. MobM belongs to the Pre/Mob family of plasmid-encoded DNA-relaxing proteins (relaxases). Purified MobM protein has been used to assay cleavage conditions on plasmid supercoiled DNA. Some structural features of MobM have been addressed by analytical ultracentrifugation, circular dichroism, thermal denaturation, and fluorescence emission. The protein behaved as a dimer of identical subunits with an ellipsoidal shape. MobM showed a high (about 60%) alpha-helical content and a midpoint denaturation of about 40 degrees C. Cell fractionation assays showed that MobM was associated to the cell membrane. This association was abolished when a great alteration was introduced within a putative coiled-coil located at the C-terminal region of the protein. Emission fluorescence suggested that the three Trp residues of MobM are located within a hydrophobic environment. A molecular model of MobM on the known structure of colicin Ia has been built.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region.

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro. Received: 12 October 1998 / Accepted: 28 February 1999     

Region of the streptococcal plasmid pMV158 required for conjugative mobilization.

The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUB110. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.     

The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding

Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB_V superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn²⁺ or Mg²⁺ cations in a dosage-dependent manner. However, whereas Mn²⁺ strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg²⁺. Furthermore, MobMN199 exhibited a high affinity binding for Mn²⁺ but not for Mg²⁺. We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein–DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn²⁺ during pMV158 transfer.     

Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.     

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

Construction of the mobilizable plasmid pMV158GFP,a derivative of pMV158 that carries the gene encoding the green fluorescent protein

Plasmid pMV158 has been employed to construct cloning non-mobilizable vectors for various Gram-positive organisms. Here we report the construction of a mobilizable pMV158-based plasmid that harbors the gene encoding the green fluorescent protein under the control of a promoter inducible by maltose. The plasmid was mobilized between strains of Streptococcus pneumoniae as well as from S. pneumoniae to Lactococcus lactis or Enterococcus faecalis at the same frequency as its parental. Transconjugant that received the GFP-tagged plasmid could be detected by their fluorescence, which was especially high in E. faecalis cells.     

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.     

Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.     

Specific DNA binding of the TraM protein to the oriT region of plasmid R100.

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.     

Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae

This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae--a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling-circle mechanism, and sequence comparisons place it in the pMV158 family of rolling-circle replicons. The plasmid contains predicted rep, cop, and rnaII genes for control of replication initiating at a predicted double-strand origin. The plasmid has an ssoT single-strand origin, which is efficient enough to allow only very small amounts of the single-stranded DNA intermediate to accumulate. The overall efficiency of replication is sufficient to render the plasmid segregationally stable without selection in P. larvae and in Bacillus megaterium, but not in Escherichia coli. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements.     

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.