Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

Abstract

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.Ever since its discovery in 1956 (3), Deinococcus radiodurans and other members of the Deinococcaceae have become the paradigm of natural resistance to high-level ionizing and UV radiation. D. radiodurans cells are able to survive levels of gamma radiation 1,000 times higher than the lethal dose for humans and were shown to survive and accurately repair massive DNA damage. At 1.5 megarads, up to 130 double-strand breaks occur per cell, which are repaired without mutagenesis or loss of viability (13). In contrast, the presence of a mere two double-strand breaks is lethal to Escherichia coli (21). As a consequence, this pink-pigmented, non-spore-forming bacterium has received considerable attention from the scientific community, from both a fundamental and an applied point of view. Because of their radioresistance, Deinococcus species hold great potential for bioremediation of complex waste mixtures containing organic solvents, heavy metals, and radioisotopes. Recently, Lange et al. (23) have demonstrated toluene dioxygenase (TDO) activity in engineered strains of D. radiodurans R1. TDO is a broad-spectrum dioxygenase capable of degrading trichloroethylene (42), a common organopollutant at many Department of Energy waste sites (32). In these strains, TDO activity was not inhibi...

Endogenous nitric oxide regulates the recovery of the radiation-resistant bacterium Deinococcus radiodurans from exposure to UV light

“…2A). Expression of recombinant proteins in Dr has only been achieved in a few cases due to the difficult of maintaining exogenous plasmids in the bacterium (30,31). It should also be noted that selection with antibiotics following irradiation retards growth of uninduced Dr ⌬nos:pNOS compared ⌬nos.…”

Involvement of Two Putative Alternative Sigma Factors in Stress Response of the Radioresistant Bacterium Deinococcus radiodurans

“…The pRADZ1 empty vector, which encodes low ␤-galactosidase activity that is not induced during heat shock, was transformed into all three strains as a control. The pRADZ1 construct is present at about the same copy number as the chromosomes in D. radiodurans (24). Transformants were grown to mid-exponential phase at 30°C and subsequently shifted to heat shock at 40°C during midexponential phase to induce promoter-reporter activity.…”

Characterization of a Cryptic Plasmid, pSM103mini, from Polyethylene-Glycol DegradingSphingopyxis macrogoltabidaStrain 103

“…Plasmid-borne resolvases are reported to be involved in plasmid stabilization by reducing the number of plasmid multimers resulting from homologous recombination. 16,17) Hense ORF3 was tentatively identified as resU, encoding a resolvase important in plasmid stabilization.…”

“…The resolvase gene found in pUE10 in Deinococcus radiodurans SARK was reported directly to affect its stability under non-selective conditions, but not its transformation efficiency. 16) But in the cases of pSM19035 in Streptococcus pyogenes 19) and pAM1 in Enterococcus faecalis, 20) the recombinases are required also for plasmid replication. Thus it is necessary to investigate further the necessity of resU in pSM103mini replication and maintenance.…”