Identification of the Rts 1 DNA fragment responsible for temperature sensitive growth of host cells harboring a drug resistance factor Rts 1

Okawa, Noriyuki; Tanaka, Masahiko; Finver, Sheldon N; Kaji, Akira

doi:10.1016/0006-291x(87)91526-9

Cited by 6 publications

(8 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To elucidate the mechanism of action of the Tsg phenotype of Rtsl, we initially identified a 3.65-kb SalI fragment containing the kanamycin resistance gene thought to be responsible for this phenotype (18,26). However, further examination of the HincII-S fragment ascribed the Tsg phenotype to the 146-bp fragment.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, partial nucleotide sequences of Rtsl segments have been reported, including regions involved in DNA replication (10,12), incompatibility (13), kanamycin resistance (8,15), and temperature-dependent instability of plasmid (19,22). In a previous publication (18), we reported that the 3.65-kb Sall fragment of Rtsl conferred the Tsg phenotype and that this fragment contained the kanamycin resistance gene. In this study, we narrowed down the responsible fragments for Tsg and sequenced them.…”

mentioning

confidence: 92%

“…It has the following temperature-dependent phenotypes: (i) T4 bacteriophage restriction at 32°C but not at 42°C (9); (ii) temperature-sensitive formation of covalently closed circular DNA (Tsc) (27); and (iii) temperature-dependent instability (Tdi) of plasmid (19,22). In addition, it causes temperaturesensitive growth (Tsg) of host bacteria (4,18,23). Bacteria harboring this plasmid grow very slowly at 42°C but normally at 32°C.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria

et al. 1991

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 92%

mentioning

confidence: 99%

See 1 more Smart Citation

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria

et al. 1991

View full text Add to dashboard Cite

show abstract

“…Since then, two loci responsible for this phenotype have been identified, tsg (43,47) and hig (60)(61)(62). Since the tsg locus contains no open reading frame (ORF), the AT-rich DNA segment itself is thought to be responsible for the thermosensitivity of host cell growth.…”

mentioning

confidence: 99%

Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes

et al. 2002

Self Cite

View full text Add to dashboard Cite

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.Rts1 is a low-copy-number kanamycin resistance plasmid originally isolated from a clinical strain of Proteus vulgaris (56). Its molecular mass was originally estimated to be about 140 MDa (29). This large plasmid is the prototype for the T incompatibility group (9) and expresses pleiotropic thermosensitive phenotypes in autonomous replication (13, 58), conjugative transfer (56), host cell growth (12, 57), and restriction of T-even phages (28,32,66).As in many large plasmids of gram-negative bacteria, Rts1 requires two elements for its autonomous replication, a replication initiation protein encoded by the plasmid (repA) and a short segment containing the replication origin, ori (30,34,59). Because the replication of this mini-Rts1 plasmid is stable at 37°C but is inhibited at 42°C in Escherichia coli (30), as observed for Rts1 (56, 58), the replication machinery of Rts1 itself is thermosensitive. A locus termed tdi (temperature-dependent instability) has also been identified as another locus responsible for the temperature sensitivity (48, 55), but its real physiological role in the replication (or maintenance) of Rts1 is unknown.As for the temperature-sensitive effect on host cell growth, Terawaki et al. (57) first reported that Rts1 inhibits its host cell growth at 42°C but not at 37°C. Since then, two loci responsible for this phenotype have been identified, tsg (43, 47) and hig (60-62). Since the tsg locus contains no open reading frame (ORF), the AT-rich DNA segment itself is thought to be responsible for the thermosensitivity of host cell growth. On the other hand, the hig locus encodes a system belonging to the proteic killer family, where the HigB and HigA proteins function as a toxin and an antitoxin, respectively. The temperaturesensitive host cell growth conferred by the hig system is mediated by selectively killing the host that has lost all copies of Rts1 at a nonpermissive temperature (postsegregation killing).Conjugation of Rts1 is also thermosensitive, but the temperature range for conjugation differs from that for replication, efficient at 25°C but not at 37°C (56). Thus, the thermosensitivity of ...

show abstract

“…The temperature-dependent phenotypes of Rtsl include growth inhibition of the host cell (34), restriction of T4 bacteriophage growth (18), formation of covalently closed circular (ccc) forms (45), and temperaturedependent instability (Tdi) of the plasmid (19,35). Recently, partial nucleotide sequences of Rtsl segments have been reported, including regions involved in DNA replication (20), incompatibility (21), and kanamycin resistance (30).…”

mentioning

confidence: 99%

Nucleotide sequence of an Rts1 fragment causing temperature-dependent instability

et al. 1988

View full text Add to dashboard Cite

Rtsl is a multiphenotypic drug resistance plasmid which is eliminated from host bacteria at 42°C but not at 32°C. This phenotype has been called temperature-dependent instability (Tdi). We determined the nucleotide sequence of the Rtsl DNA b' segment which causes this phenotype. Within this 786-base-pair segment, several open reading frames (ORFs) were found, including one which encodes a protein with a molecular weight of 16,000. A protein approximately corresponding to this protein is expressed in Escherichia coli minicells harboring plasmids containing the b' segment. In addition, we found the chi sequence at 112 bases proximal to this ORF. Temperature-dependent elimination due to this segment was not observed in the RecA strain of E. coli, but the RecB protein was not required for expression of this phenotype. We constructed various deletion derivatives and found that three portions, the region containing the chi (nucleotides 1 to 24), ORF (nucleotides 25 to 546), and tail (nucleotides 631 to 786) sequences are necessary for Tdi activity. Site-directed mutagenesis studies indicated that ORF I is required for Tdi expression.Rtsl is a multiphenotypic temperature-sensitive, highmolecular-weight plasmid (10) conferring kanamycin resistance on its host. The temperature-dependent phenotypes of Rtsl include growth inhibition of the host cell (34), restriction of T4 bacteriophage growth (18), formation of covalently closed circular (ccc) forms (45), and temperaturedependent instability (Tdi) of the plasmid (19,35). Recently, partial nucleotide sequences of Rtsl segments have been reported, including regions involved in DNA replication (20), incompatibility (21), and kanamycin resistance (30). In a previous publication (35), we identified a segment of approximately 800 base pairs (bp) (0.5 megadaltons) as the region responsible for the Tdi phenotype. This segment was called the b' fragment.To further understand the mechanism of Tdi expression, we have now sequenced this b' fragment. Among several open reading frames (ORFs) with the Shine-Dalgarno sequence and transcriptional initiation signals, ORF I encodes a 16,000-Mr protein. This ORF is preceded by the chi sequence (38). Evidence has been obtained which indicates that the ORF I protein and the chi sequence play important roles in expression of the Tdi phenotype. In addition, pBR322, containing the b' fragment, failed to exhibit the Tdi phenotype in recombination-deficient bacteria missing the RecA mutation. MATERIALS AND METHODSBacterial strains and plasmids. The Escherichia coli strains, phage, and plasmids used in this study are listed in Table 1. M13mp8 and mp9 (27) and the M13 sequencing kit were purchased from New England BioLabs, Inc.Media. Trypticase soy broth (from BBL Microbiology Systems) and L broth (LB broth base, from GIBCO Laboratories, for transformation experiments) were used for liquid media. Yeast extract and Tryptone, obtained from Difco Laboratories, and M9 minimal agar were used for M13 cloning. MacConkey agar base (Difco) without lactose an...

show abstract

Identification of the Rts 1 DNA fragment responsible for temperature sensitive growth of host cells harboring a drug resistance factor Rts 1

Cited by 6 publications

References 17 publications

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria

Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes

Nucleotide sequence of an Rts1 fragment causing temperature-dependent instability

Contact Info

Product

Resources

About