Yoshiro Terawaki

The complete nucleotide sequence of φCTX, a cytotoxin‐converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages

φCTX is a cytotoxin‐converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the φCTX phage genome. The precise genome size was 35 538 bp with 21 base 5′‐extruding cohesive ends. Forty‐seven open reading frames (ORFs) were identified on the φCTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the φCTX genome was an extensive homology with the coliphage P2 and P2‐related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9–65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G+C content and codon usage of most φCTX genes were similar to those of the host P. aeruginosa chromosome. In addition, φCTX was found to share several common features with P2, including the morphology, non‐inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca2+‐dependent receptor binding. These findings indicate that φCTX is a P2‐like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of φCTX, P2 and other P2 relatives revealed the presence of several hot‐spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.

Isolation and Characterization of Shewanella alga from Human Clinical Specimens and Emendation of the Description of S. alga Simidu et al., 1990, 335

Genetic and phenotypic studies on the strains biochemically identified as Shewanella putrefaciens, which had a G+C content ranging from 52 to 54 mol% were conducted. The moles percent G+C of the type strain of S. putrefaciens is 46. Surprisingly, DNA homology experiments revealed that all these strains are genetically related to Shewanella alga (which was reported to produce tetrodotoxin), not to the type strain of S. putrefaciens. In this study, we reidentified clinical strains of S. putrefaciens which have a high range of moles percent G+C, as does S. alga. We also characterized the reidentified strains and found that the original description of S. alga (U. Simidu, K. Kita-Tsukamoto, T. Yasumoto, and M. Yotsu, Int. J. Syst. Bacteriol. 40:331-336, 1990) is insufficient to identify this strain. An emended description of S. alga is given.

Molecular analysis of a cytotoxin‐converting phage, φCTX, of Pseudomonas aeruginosa: structure of the attP–cos–ctx region and integration into the serine tRNA gene

The Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage φCTX. The genome of φCTX is a 35.5 kb double‐stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of φCTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP–cos–ctx region. The termini of φCTX are 21‐base 5′ extended‐single‐stranded DNAs. The ctxgene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP–cos–ctx region contains six kinds of repeats and integration host factor‐binding sequences and showed sequence‐directed static bends, suggesting its potential to form a highly ordered structure. In addition, φCTX was found to integrate into the serine tRNA gene which was mapped to the 43–45 min region on the P. aeruginosachromosome.

Phage‐conversion of cytotoxin production in Pseudomonas aeruginosa

We isolated a temperate phage which carried the cytotoxin gene (ctx) from a cytotoxin (CTX)‐producing Pseudomonas aeruginosa strain, PA158. The phage, φCTX, had a head with a hexagonal outline and a contractile tail with tail fibres. The phage genome was a linear double‐stranded 35.5kb DNA with single‐stranded cohesive ends (cos). The attP, cos and ctx genes were all located very close to one another within a 2.3kb segment on the phage genome in the order given (in the circular form). φCTX converted CTX non‐producing P. aeruginosa strains into CTX producers. A single copy of φCTX DNA was integrated at the same site on the host chromosome (attB) in every lysogen, including PA15B. However, the amount of CTX produced in these lysogens varied from strain to strain and was less than that in PA158.

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

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.

Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of activation of the protoxin

The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non‐producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host‐range vector plasmid.

Chromosomal Location of Genes Participating in the Degradation of Purines in Pseudomonas aeruginosa

SummaryGenetic mapping of the genes (puu) that encode the enzymes catalysing degradation of purines in Pseudomonas aeruginosa strain PAO has been carried out. Mutants that are deficient in adenine deaminase (puuA), guanine deaminase (puuB), xanthine dehydrogenase (puuC), uricase (puuD), allantoinase (puuE), and/or allantoicase (puuF) were isolated and used for the genetic study. Conjugation by FP5 factor and generalized transduction by phage G101 gave the following map locations of these six genes on the chromosome: hisI-puuB-hisII; trpA,B-puuA-ilv202; met9011-catA1-tyu-nar9011-(puuC, puuD, puuE)-puuF. A close linkage among the puuC, puuD and puuE was demonstrated by the transduction.

Temperature Sensitivity of Cell Growth in Escherichia coli associated with the Temperature Sensitive R(KM) Factor

IT was shown earlier1 that an R factor determining resistance to kanamycin is spontaneously eliminated at 42° C, and its conjugal transfer is inhibited at this temperature. This temperature sensitivity was ascribed to temperature sensitive replication of the R factor. We present here evidence that the growth of cells is also inhibited at higher temperatures as a result of the presence of the temperature sensitive R factor.

Chromosomal locations of catA, pobA, pcaA, dcu and chu genes in Pseudomonas aeruginosa

Eleven catabolic markers have been located on the chromosome of Pseudomonas aeruginosa PAO using FPS-mediated conjugation and G101 transduction. Most of these markers are located in the region 20–35 min, and the remainder in the region later than 60 min. Four chu genes concerned in the sequential degradation of choline to glycine are closely linked.

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.