M. A. Titok

Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector.

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.

Genetic Evidence for a Link Between Glycolysis and DNA Replication

Background A challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions. Methodology/Principal Findings We report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature. Full restoration of viability by such alterations was limited to cells with mutations in three elongation factors (the lagging strand DnaE polymerase, the primase and the helicase) out of a large set of thermosensitive mutants affected in most of the replication proteins. Restoration of viability resulted, at least in part, from maintenance of replication protein activity at high temperature. Physiological studies suggested that this restoration depended on the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway and occurred in both glycolytic and gluconeogenic regimens. Restoration took place abruptly over a narrow range of expression of genes in the three-carbon part of glycolysis. However, the absolute value of this range varied greatly with the allele in question. Finally, restoration of cell viability did not appear to be the result of a decrease in growth rate or an induction of major stress responses. Conclusions/Significance Our findings provide the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed. It is proposed that related systems are ubiquitous.

Diversity of IncP-9 plasmids of Pseudomonas

IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an ∼25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7–35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9β cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.

The replication and stable-inheritance functions of IncP-9 plasmid pM3

Little is known of the transfer and maintenance machinery of the IncP-9 plasmids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to streptomycin and tetracycline, was found repeatedly in Pseudomonas species from numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 degrees C and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding Km(R). The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptide products were found to have similarity to previously analysed proteins involved in plasmid replication (rep gene), transfer (mpf; mating-pair formation gene) and stable maintenance (par, mrs genes). The organization of these genes showed similarity to several plasmid systems including the Ti and pSYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the region required for replication, and identified the putative rep gene and putative par promoter region as able to express incompatibility. rep deletion mutants were lost from the cell line, and expression of the rep gene was shown to be controlled by negative autoregulation. A pMT2 derivative with an insertion between the rep and par genes showed very weak, if any, ability to replicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.

Ability of IncP‐9 plasmid pM3 to replicate in Escherichia coli is dependent on both rep and par functions

IncP‐9 plasmids are common in Pseudomonas species and can be transferred to other Gram‐negative eubacteria but tend not to be stably maintained outside their natural host genus. A 1.3 kb ori V‐rep fragment from IncP‐9 plasmid pM3 was sufficient for autonomous replication in Pseudomonas putida but not in Escherichia coli. Replication of ori V‐rep in E. coli was restored when additional rep was provided in trans, suggesting that the replication defect resulted from insufficient rep expression from its natural promoter. A promoter deficiency in E. coli was confirmed by reporter gene assays, transcriptional start point mapping and mutation of the promoter recognition elements. Dissection of the pM3 mini‐replicon, pMT2, showed that this replication deficiency in E. coli is suppressed by additional determinants from its par operon: ParB, which can be supplied in trans, and its target, the par operon promoter, required in cis to ori V‐rep. We propose that ParB binding to its target either changes plasmid DNA and thus promoter conformation or by spreading or looping contacts RNAP at the rep promoter so that rep expression is sufficient to activate ori V.

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.

Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (α-, β-, γ-, δ-, ɛ-, ζ-, η-, and θ-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from gq-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (α-, β-, δ-, ɛ-, and ζ-subgroups) is strict dependence of their inheritance on the temperature factor. At 37°C, the plasmids of δ-, ζ-, and θ-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of η- and γ-subgroups. Inheritance of these plasmids does not depend on temperature. At 28°C and 37°C, the η plasmid is not maintained stably (inheritance stability is 2%), while the γ-plasmid has almost 100% stability.

Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the East European Plain

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.

[Soil strain of Bacillus subtilis harboring a large plasmid that mediates high-frequency conjugal mobilization].

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.

Selection of initiation replication system mutants of IncP-9 plasmid pBS267

A minireplicon containing the rep gene and oriV site of the γ subgroup of the IncP-9 caprolactam pBS267 biodegradation plasmid was cloned for the first time. It was established that a minimized variant of pBS267 plasmid cannot be sustained in E. coli and is inherited in an unstable way in bacteria Pseudomonas. Using in vitro mutagenesis, mutant variants of the minireplicon were produced, characterized by an increased number of copies in cells, the ability to replicate in E. coli, and relatively stable inheritance in P. putida cells. The obtained constructs are the basis for a study of the replication mechanisms of IncP-9 group plasmids, as well as use as vectors for molecular cloning in a wide range of gram-negative bacteria.