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...

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the ؊10 and ؊35 regions of these promoters resembled the 70 consensus sequence of E. coli.Its extraordinary tolerance to extremely high doses of ionizing radiation has made Deinococcus radiodurans the focus of growing scientific interest. This non-spore-forming bacterium is able to survive up to 4,000 times the lethal radiation dose for humans without mutation or loss of viability (2, 9). D. radiodurans is also of interest as a representative of a deeply branching family within the domain Bacteria (10). The sequence of the D. radiodurans R1 genome was recently published and shown to consist of two chromosomes, a megaplasmid, and one plasmid (17).Despite the interest in D. radiodurans, little is known concerning basic gene expression and promoters. Earlier studies showed that Deinococcus promoter regions are poorly recognized in Escherichia coli, and E. coli promoters that were tested were not recognized in D. radiodurans (7,14), suggesting that deinococcal promoters might be different from the classical E. coli 70 type. However, no transcriptional analysis of deinococcal promoters has been carried out. Analysis of the recently published genome sequence revealed only three putative sigma factors, one classing with vegetative 70 (rpoD) sequences, and two classing with extracytoplasmic alternative transcription factors (annotated as rpoE and DR0804 [17]). Surprisingly, orthologs of the nitrogen-starvation, general starvation, and heat shock sigma factors (rpoN, rpoS, and rpoH, respectively) were not found.One reason for the lack of information on promoters in deinococci is the lack of convenient genetic tools for studying promoters. A promoter cloning vector has been described (7), but it involves an antibiotic resistance reporter and is a large plasmid with limited cloning sites. Therefore, we developed a suite of integrative promoter-screening vectors that allow the screening and assessment of promoter regions in D. radiodurans based on lacZ and xylE as reporters. These vectors were used to isolate and analyze promoter regions, and promoter regions were further defined by transcripti...