Miroslawa Wlodarczyk

Molecular characterization of functional modules of plasmid pWKS1 of Paracoccus pantotrophus DSM 11072

The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44.1 and 37.8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several theta-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6.5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.

Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits

The replicon of the pTAV3 megaplasmid (approx. 400 kb) of Paracoccus versutus has been localized to a 4center dot3 kb EcoRI restriction fragment and its entire nucleotide sequence determined. The G+C content of the entire sequence is 66 mol%, which is within the range (62-66 mol%) previously determined for P. versutus total DNA. ORF1 encodes a replication initiation protein Rep (47.2 kDa), which shares substantial similarity with putative proteins of the Coxiella burnetii plasmids QpH1 and QpDV, and the replication protein of Pseudomonas syringae plasmid pPS10. ORF2, located in the opposite transcriptional orientation to ORF1, encodes a putative protein that shares similarity to a subfamily of ATPases involved in plasmid partitioning. The highest similarity was observed with homologous proteins (RepA) encoded by the repABC family of replicons found in several plasmids of Agrobacterium, Rhizobium and Paracoccus spp. The predicted product of ORF3 was similar to AcoR, Nif and NtrC transcriptional activators. A strong incompatibility determinant (inc) was localized between ORF1 (rep) and ORF2 (parA). The origin of replication of pTAV400 contains a short A+T-rich region and several imperfect palindromic sequences. Curing experiments demonstrated that the megaplasmid bears genes required for growth in minimal media and can therefore be referred to as a mini-chromosome. Megaplasmids pTAV3 of P. versutus UW1 and pKLW2 of Paracoccus pantotrophus DSM 11073 were found to carry closely related, incompatible replicons. It has been shown that plasmid pORI6 (containing oriV of pTAV3 cloned into plasmid pABW1, which does not replicate in Paracoccus spp.) can be trans activated not only by pTAV3, but also by pKLW2. Using pORI6, it was demonstrated that replication systems related to pTAV3 are also present in the replicons of Paracoccus alcaliphilus JCM 7364, Paracoccus thiocyanatus IAM 12816 and Paracoccus methylutens DM 12.

Comparative characterization of repABC-type replicons of Paracoccus pantotrophus composite plasmids.

The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus. We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus. We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus. A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons. Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1). Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.

Plasmids of carotenoid-producing Paracoccus spp. (Alphaproteobacteria) - structure, diversity and evolution.

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

Conjugal transfer of plasmid and chromosomal markers between strains of Thiobacillus versutus

In experiments on conjugation in Thiobacillus versutus with the use of pTAV1-less strains as recipients, we have proved that the derivative of the wild-type T. versutus cryptic plasmid pTAV1 (107 kb) marked with Tn1721 (Tcr) transposon demonstrates Tra- phenotype but can be mobilized for transfer by pSa Tra+ broad-host-range helper plasmid at a low frequency. The possibility of chromosomal gene exchange between different auxotrophic and drug-resistant T. versutus mutants has been confirmed. The previously assumed participation of plasmid pTAV1 in the above process must be excluded because conjugal transfer of chromosomal markers can be observed even when two pTAV1-free strains are mated. Formation of some classes of transconjugants can be reasonably explained only when two-directional chromosomal DNA transfer (retrotransfer) is considered. At this stage of our studies we can not propose any hypothesis on the mechanism of chromosomal gene transfer. The possible role of the megaplasmids discovered in T. versutus in chromosome mobilization needs to be elucidated.

Genetic organization of the basic replicon of plasmid pMTH4 of a facultatively methylotrophic bacterium Paracoccus methylutens DM12.

Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA). In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus). The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA. The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci. The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli. Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.