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 crypticDeinococcus 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 andresU 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 Deinococcuspromoter was shown to efficiently express LacZ.

[1]  D. Barstow,et al.  The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. , 1988, Gene.

[2]  K. Minton,et al.  In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans , 1994, Journal of bacteriology.

[3]  F. Blattner,et al.  Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen , 1998, Infection and Immunity.

[4]  R. Slater.  Experiments in Molecular Biology , 2014, Humana Press.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  R. Murray,et al.  The Family Deinococcaceae , 1992 .

[7]  D. Cullum-Dugan,et al.  Studies on a radio-resistant micrococcus. I. Isolation, morphology, cultural characteristics, and resistance to gamma radiation , 1956 .

[8]  S Y Xu,et al.  Identification of a thermophilic plasmid origin and its cloning within a new Thermus-E. coli shuttle vector. , 1997, Gene.

[9]  Lawrence P. Wackett,et al.  Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments , 1998, Bio/Technology.

[10]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[11]  D. Chattoraj,et al.  Replication control of plasmid P1 and its host chromosome: the common ground. , 1997, Progress in nucleic acid research and molecular biology.

[12]  K. Nordström,et al.  Mechanisms that contribute to the stable segregation of plasmids. , 1989, Annual review of genetics.

[13]  K. Minton,et al.  Defective transformation of chromosomal markers in DNA polymerase I mutants of the radioresistant bacterium Deinococcus radiodurans. , 1994, Mutation research.

[14]  F. Hutchinson,et al.  Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. , 1977, Journal of molecular biology.

[15]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[16]  I. Lasa,et al.  Characterization of a plasmid replicative origin from an extreme thermophile. , 1998, FEMS microbiology letters.

[17]  V. Mattimore,et al.  Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans , 1994, Journal of bacteriology.

[18]  J. M. Zachara,et al.  Chemical contaminants on DOE lands and selection of contaminant mixtures for subsurface science research , 1992 .

[19]  D. Bastia,et al.  Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Chopin,et al.  Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. , 1988, Biochimie.

[21]  S. Ehrlich,et al.  Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis. , 1991, Plasmid.

[22]  Manuel Espinosa,et al.  Plasmids Replication and Control of Circular Bacterial , 1998 .

[23]  J. Alonso,et al.  Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis. , 1993, Gene.

[24]  A. Devries,et al.  Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  I. Dodd,et al.  Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. , 1990, Nucleic acids research.

[26]  W. J. Brammar,et al.  Transient transcriptional activation of the IncI1 plasmid anti‐restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria , 1999, Molecular microbiology.

[27]  E. Delver,et al.  IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions , 1992, Journal of bacteriology.

[28]  L. Wackett,et al.  Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1 , 1988, Applied and environmental microbiology.

[29]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.

[30]  E. Delver,et al.  Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences , 1993, Journal of bacteriology.

[31]  S. Salzberg,et al.  Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. , 1999, Science.

[32]  S. Ehrlich,et al.  A vector for systematic gene inactivation in Bacillus subtilis. , 1998, Microbiology.

[33]  S. Ehrlich,et al.  Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid‐encoded resolution system , 1997, Molecular microbiology.

[34]  B. Wilkins,et al.  Distribution of the ardA family of antirestriction genes on conjugative plasmids. , 1995, Microbiology.

[35]  S. Cole,et al.  Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis , 1996, Molecular microbiology.

[36]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[37]  D. Summers,et al.  Multicopy plasmid instability: the dimer catastrophe hypothesis , 1993, Molecular microbiology.

[38]  J. Alonso,et al.  Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035. , 1990, Nucleic acids research.

[39]  S. Ehrlich,et al.  The promiscuous plasmids pIP501 and pAM beta 1 from gram-positive bacteria encode complementary resolution functions. , 1994, Plasmid.

[40]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[41]  K. Minton,et al.  Promoter probe and shuttle plasmids for Deinococcus radiodurans. , 1992, Plasmid.

[42]  K. Minton,et al.  Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids. , 1989, Plasmid.

[43]  F. Rojo,et al.  A novel site-specific recombinase encoded by the Streptococcus pyogenes plasmid pSM19035. , 1994, Journal of molecular biology.