Yoshiyuki Kamio

Cupric ion resistance as a new genetic marker of a temperature sensitive R plasmid, Rtsl in Escherichiacoli

Abstract A temperature sensitive kanamycin (Km) resistant R plasmid, Rtsl, was found to confer cupric ion (Cu 2+ ) resistance on its hosts in Escherichia coli . At conjugal transfer, two kinds of segregants were obtained from Rtsl, i.e. Cu 2+ resistant, Km sensitive and Km resistant, Cu 2+ sensitive plasmids. Protein T existed in E. coli cells harboring Rtsl or the Cu r Km s -plasmid. The inhibitory effect on the host cell growth at 43°C was observed with Rtsl + or the Km r Cu s -plasmid + cells. A relationship between these Rtsl derivatives and Rtsl in Proteus mirabilis which has been studied was discussed.

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

Abstract A replication region, consisting of a 1.1-megadalton (Md) Eco RI/ Hin dIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with Acc I endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc + region within mini-Rts1 were not detected.

Molecular cloning and mapping of a deletion derivative of the plasmid Rts1

Abstract The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with Eco RI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned Eco RI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A Sal I fragment (1.5 Md) in E1 (the largest Eco RI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a Bam HI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest Eco RI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md Hin dIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.

A temperature sensitive protein in outer membrane of Escherichia coli K-12 harbouring a temperature sensitive R plasmid, Rts1.

Summary A temperature sensitive protein (protein T) of molecular weight 80,000 was found to exist in the outer membrane of Escherichia coli K-12 strains harbouring a temperature sensitive R plasmid R ts 1, by the two dimensional acrylamide gel electrophoresis. Protein T was not detected either in R − , pTW2 (a mutant of Rtauthor ) cells or in XR27 cells in which R ts 1 is integrated into the host chromosome. Protein T was detected only when the R ts 1 + cells were grown at the lower temperature, and it disappeared from the outer membrane within 90 min by growing the cells at 43°C. The critical temperature at which protein T disappeared was approximately 39°C.

Nucleotide sequence and copy control function of the extension of the incI region (incI-b) of Rts1

An Rts1 derivative, pTW20, contains three incompatibility (inc) regions, incI-a (incI in previous studies), incII, and newly determined incI-b loci. By restriction analysis, we have located the incI-b adjacent to the incI-a region on the pTW20 map. Nucleotide sequence analysis of the minimal incI-b region revealed the presence of four repeated sequences, each consisting of 18 bp, which is similar to the incI-a and incII repeats existing on mini-Rts1. All four repeating units were required for expression of a strong incompatibility. In addition, RepA protein, essential for the replication of Rts1, bound specifically to the repeated sequences, suggesting that the repeats would titrate out RepA protein as do incI-a and IncII. Insertion of the incI-b to a mini-Rts1 plasmid in a natural arrangement decreases the copy number of mini-Rts1 to the same level as that of mini-F. The incI-a and incI-b might be a single constituent in incompatibility and copy number control of Rts1.

An Rtsl—Derivative plasmid conferring UV sensitivity on Escherichia coli host

A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on Escherichiacoli host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, puv, being responsible for the UV sensitivity was located on the kanamycin resistance gene containing BamH1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of puv.

Alteration of flagella by a temperature sensitive R plasmid Rtsl in Escherichia coli K-12

Summary The presence of Rtsl and its mutants plasmids in Escherichia coli K-12 strains inhibited the motility of the host cells at 30°C. Aberrant flagella were observed with the plasmid+ cells. Flagella protein (flagellin) obtained from the plasmid+ cells did not co-electrophorese in polyacrylamide gel with that from plasmid− cells. Flagellin isolated from the cells harboring a mutant of Rtsl, pTW10, gave two bands on polyacrylamide gel electrophoresis.