Promoter Cloning in the Radioresistant Bacterium Deinococcus radiodurans

Abstract

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...

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

“…One of the difficulties for gene disruption in this strain has been the high natural resistance of D. radiodurans R1 to kanamycin, up to 4 g of kanamycin per ml (R. Meima, unpublished data) and the low-level kanamycin resistance obtained with existing cassettes, 8 g of kanamycin per ml (25). The result is a high percentage of colonies with no insert due to spontaneous resistance.…”

“…The result is a high percentage of colonies with no insert due to spontaneous resistance. Therefore, a new kanamycin cassette was constructed for D. radiodurans, with a kanamycin resistance marker driven by the previously characterized D. radiodurans groESL minimal promoter region, which contains only the 70 -controlled major start site (25). This new cassette allowed screening at concentrations of kanamycin of up to 25 g/ml, which greatly reduced the number of colonies arising from spontaneous resistance.…”

“…sig1 and sig2 PCR products were cloned into pCR2.1-TOPO (Invitrogen) and then transferred as a KpnI-XhoI fragment to pMTL23 (5), yielding pSIG123 and pSIG223, respectively. To construct the kanamycin resistance (Km r ) cassette, the BamHI-BglII groESL promoter fragment from pGROES4Z (25) was cloned into the unique BglII site of pCR2.1 upstream of the promoterless Km r marker. The groESL-Km r fusion was then PCR amplified with blunt-ended, restriction-tagged primers and cloned into pCR2.1-TOPO to generate pPgroK.…”

Oxidative Stress Resistance inDeinococcus radiodurans

“…The efficiency of transformation is higher with double-stranded DNA (dsDNA) than with single-stranded DNA (ssDNA) (444) and with plasmid DNA than with linearized DNA (448). Transformation with nonmethylated donor DNA passed through a dam dcm E. coli strain significantly increases the transformation efficiency (404). UVirradiated transforming DNA has a much higher transforming ability in D. radiodurans than in Haemophilus influenzae due to the efficiency of D. radiodurans at repairing irradiated transforming DNA in the same manner as its own irradiated DNA (448).…”

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

“…The pPolX : : cat plasmid was constructed by inserting the cat cassette, expressing in Deinococcus, at the SacII site in pETpolX. In brief, the cat gene was PCR amplified from pRAD1 (Meima et al, 2001) using CatF and CatR primers and ligated at the blunt-ended SacII site in pETpolX to yield pPolX : : cat ( Supplementary Fig. S1).…”