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

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.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Promoter probe and shuttle plasmids for Deinococcus radiodurans.

Two improved Deinococcus radiodurans-Escherichia coli shuttle vectors have been constructed. pI3 is a 16-kb plasmid that confers chloramphenicol resistance in D. radiodurans (CmR, cat) and ampicillin resistance in E. coli (ApR) and contains a multiple cloning site that does not interrupt sequences necessary for replication or drug resistance in either host. pI304 is a promoter-probe plasmid that is similar to pI3, but lacks the D. radiodurans promoting sequence for the cat gene, while retaining sequences necessary for replication.     

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.     

抗辐射茵(Deinococcus radiopugnance)的质粒pUE30的克隆及序列分析

抗辐射菌(Deinococcus radiopugnance)ATCC 19172株中存在约14.6 kb、8.7 kb、7.0 kb、3.65 kb、及2.45 kb等5种以上的隐秘性质粒,对其中的约2.45 kb的小型质粒pUE30进行了序列测定与分析,该质粒由2 467 bp的碱基对组成,其中包含了267 nt及1 068 nt的2个开放阅读框架(open reading frame,ORF)和一个AT-rich领域。经过与GenBank的数据库分析,其中267 nt的ORF(repC)与豆科根瘤菌(Rhizobium leguminosa-rum)及根癌农杆菌(Agrobacterium tumefaciens)由来的质粒的RepC蛋白质具有一定的同源性;1 068 nt的ORF(repD)与抗辐射菌(Deinococcus radiodurans)Sark株的质粒pUE10的RepU蛋白质、嗜热菌(Thermus sp.)ATCC27737株由来的质粒pMY1的RepA蛋白质具有较高的同源性。研究结果对于利用该小型质粒构建大肠杆菌-抗辐射菌属间的穿梭载体,表明抗辐射菌高效正确的DNA损伤修复机理等具有重要的意义。     

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number.

Plasmids control their copy number by limiting the amount of the initiator for DNA replication. The plasmid pUB110 initiator protein is termed RepU. Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene. The RepU/RepU* protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation.     

Deinococcus radiodurans DNA increases the radiation resistance of Escherichia coli

A genomic DNA library of Deinococcus radiodurans DNA has been prepared using the plasmid vector pBR322. The recombinant plasmid was used to transform a more radiation-sensitive organism, Escherichia coli RR1. Following selection of transformed organisms by their ability to grow on ampicillin, radiation-resistant organisms were selected by irradiation with 137Cs gamma radiation. Increased radiation resistance correlates with the presence of a 3-kb fragment of DNA in these cells which is derived from D. radiodurans.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.     

Expression of recA in Deinococcus radiodurans.

Deinococcus (formerly Micrococcus) radiodurans is remarkable for its extraordinary resistance to ionizing and UV irradiation and many other agents that damage DNA. This organism can repair > 100 double-strand breaks per chromosome induced by ionizing radiation without lethality or mutagenesis. We have previously observed that expression of D. radiodurans recA in Escherichia coli appears lethal. We now find that the RecA protein of D. radiodurans is ot detectable in D. radiodurans except in the setting of DNA damage and that termination of its synthesis is associated with the onset of deinococcal growth. The synthesis of Shigella flexneri RecA (protein sequence identical to that of E. coli RecA) in recA-defective D. radiodurans is described. Despite a large accumulation of the S. flexneri RecA in D. radiodurans, there is no complementation of any D. radiodurans recA phenotype, including DNA damage sensitivity, inhibition of natural transformation, or inability to support a plasmid that requires RecA for replication. To ensure that the cloned S. flexneri recA gene was not inactivated, it was rescued from D. radiodurans and was shown to function normally in E. coli. We conclude that neither D. radiodurans nor S. flexneri RecA is functional in the other species, nor are the kinetics of induction and suppression similar to each other, indicating a difference between these two proteins in their modes of action.     

Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.

Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.     

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Deinococcus radiodurans recovering from the effect of acute dose of gamma (gamma) radiation shows a biphasic mechanism of DNA double strands breaks repair that involves an efficient homologous recombination. However, it shows higher sensitivity to near-UV (NUV) than Escherichia coli and lacks RecBC, a DNA strand break (DSB) repair enzyme in some bacteria. Recombinant Deinococcus expressing the recBC genes of E. coli showed nearly three-fold improvements in near-UV tolerance and nearly 2 log cycle reductions in wild type gamma radiation resistance. RecBC over expression effect on radiation response of D. radiodurans was independent of indigenous RecD. Loss of gamma radiation tolerance was attributed to the enhanced rate of in vivo degradation of radiation damaged DNA and delayed kinetics of DSB repair during post-irradiation recovery. RecBC expressing cells of Deinococcus showed wild type response to Far-UV. These results suggest that the overproduction of RecBC competes with the indigenous mechanism of gamma radiation damaged DNA repair while it supports near-UV tolerance in D. radiodurans.     

Identification and initial characterisation of a pyrimidine dimer UV endonuclease (UV endonuclease beta) from Deinococcus radiodurans; a DNA-repair enzyme that requires manganese ions

An endonuclease that incises lightly ultraviolet-irradiated supercoiled plasmid DNA was identified in cell-free extracts of Deinococcus radiodurans R1 wild-type. The endonuclease was absent from strains mutant in the uvsC, uvsD or uvsE genes identifying it as 'UV endonuclease beta' responsible for the initial incision step of one excision-repair pathway for the removal of pyrimidine dimers from D. radiodurans DNA in vivo. The enzyme was purified free from contaminating nuclease activities and was partially characterised. The enzyme has an apparent molecular weight of 36 000, is ATP-independent, caffeine-insensitive and is inactivated by N-ethylmaleimide. It also has a novel requirement for manganese ions distinguishing it from all other known DNA-repair enzymes.     

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans

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 sigma(70) consensus sequence of E. coli.     

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.