Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, bacillus subtilis, and Escherichia coli

Narumi, Issay; Nakayama, Noriyuki; Nakamoto, S.; Kimura, Toshiyuki; Yanagisawa, Tadashi; Kihara, Hiroshi

doi:10.1007/bf00180147

Cited by 19 publications

(14 citation statements)

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“…suggests that the region may aid in the thermostable replication of pSTK1 and pSTE33 by serving as an efficient minus origin, and in fact, no detectable single-stranded DNA was accumulated during pSTE33 replication in B. subtilis, B. stearothermophilus, or E. coli (Narumi et al, 1993). In pSTK1, no significant homology was detected with any plus origin or replication protein, which elements are necessary for rolling circle replication (Gruss and Ehrich, 1989).…”

Section: Discussionmentioning

confidence: 99%

“…DNA manipulations: Plasmid DNA was extracted according to standard protocols (Sambrook et al, 1989) and purified with a QIAGEN column (Diagen GmbH, Hilden, Germany). Plasmid pSTK1 was purified by agarose gel electrophoresis and electroelution, because B. stearothermophilus TK015 was shown to harbor three different plasmids (Narumi et al, 1993). E. coli JMI09 cells were transformed by electroporation (Dower et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

“…The thermostable nature of pSTK1 was inherited by plasmid pSTE33, which is a shuttle vector constructed from pSTK1, plasmid pUC19, and a thermostable kanamycin-resistance marker (Narumi et al, 1993). The thermostability and host range of pSTE33 make it useful for cloning experiments using B. stearothermophilus as a host.…”

Section: Introductionmentioning

confidence: 99%

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Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

et al. 1993

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The complete nucleotide sequence of pSTK1, a cryptic plasmid isolated from B. stearothermophilus TK015, has been determined, pSTK1 has been shown to be 1883 bp in length and contain three open reading frames (ORFs), one of which has a helixturn-helix motif typical of DNA-binding proteins. Also identified was a region that can form an extensive secondary structure, which would show a high degree of similarity to palA, an origin for minus strand elongation in roiling circle replication. INTRODUCTIONThe 1.9-kbp cryptic plasmid pSTK1 was originally isolated from B. stearothermophilus TK015, and it was stably maintained in its host up to 70 °C without changing its copy number. The thermostable nature of pSTK1 was inherited by plasmid pSTE33, which is a shuttle vector constructed from pSTK1, plasmid pUC19, and a thermostable kanamycin-resistance marker (Narumi et al., 1993). The thermostability and host range of pSTE33 make it useful for cloning experiments using B. stearothermophilus as a host.The part of pSTE33 nucleotide sequence corresponding to pUC19 (YanischPerron et al., 1985) and the thermostable kanamycin nucleotidyltransferase gene (Liao et al., 1986) is known. This paper describes 1) the complete nucleotide sequence of pSTK1, which is the key to the thermostable replication of pSTE33 in B. stearothermophilus, and 2) homologies of pSTK1 with other plasmids.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

et al. 1993

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“…As mentioned above, the pGI3 replication protein is homologous to ORF3 of pSTK1. B. stearothermophilus, the host of pSTK1, is a moderate thermophile that can grow at temperatures of up to 67°C without effect on its replication capacity (37). To test the thermostability as well as the segregational stability of pGI3 replication, several pGI3 derivatives were transformed in B. subtilis CU267 and B. thuringiensis HD73 and grown for about 30 generations without antibiotic selection.…”

Section: Vol 179 1997 New Family Of Rolling Circle Replicons 5003mentioning

confidence: 99%

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons

1997

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The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43°C).Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal ␦-endotoxins during sporulation. The chromosome sizes of different B. thuringiensis strains vary from 2.4 to 4.3 Mb, as described for the closely related bacterium B. cereus (10). Since most of the genes present on the small chromosome (2.4 Mb) mapped to one half of the large molecules, it is believed that the genome consists of one constant part and another less stable entity, more easily mobilized into other genetic elements, e.g., plasmids (10). Indeed, plasmid profiles of most B. thuringiensis strains are rather complex, with molecules ranging from 2 to more than 200 kb. So far, however, most of the attention has been focused on the large plasmids because of their capacity to code for the insect-specific endotoxin genes (crystal [cry] genes); very few small (Ͻ15-kb) plasmids have been characterized yet. To our knowledge, 15 cryptic plasmids, including pGI3, have been found in B. thuringiensis. For two of them (pGI1 and pTX14-2), cloning attempts have so far failed (7,13). No cloning has been reported for six others (1, 28), and only five molecules have been partially or fully sequenced.In contrast to the large plasmids that normally display a -type replication mechanism, most of the small gram-positive replicons use the rolling circle replication mode (21). In this case, the replication protein (Rep) introduces a nick at the double-strand origin (dso) from which replication starts. The plus strand is displaced, and polymerization of a new plus strand by 3Ј-OH extension from the nick occurs. Then Rep recognizes a termination sequ...

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“…In Geobacillus spp., only kanamycin, chloramphenicol, and tetracycline resistance genes have been demonstrated to serve as selectable markers. 5,[17][18][19][20][21][22][23] Therefore, there is room to expand new markers for more effective utilization of Geobacillus spp. through complex genetic modifications.…”

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A thiostrepton resistance gene and its mutants serve as selectable markers inGeobacillus kaustophilusHTA426

Wada

Kobayashi

Furukawa

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

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Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, bacillus subtilis, and Escherichia coli

Cited by 19 publications

References 25 publications

Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons

A thiostrepton resistance gene and its mutants serve as selectable markers inGeobacillus kaustophilusHTA426

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