Keisuke Nakayama

The R‐type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F‐type is related to lambda phage

Pseudomonas aeruginosa produces three types of bacteriocins: R‐, F‐ and S‐type pyocins. The S‐type pyocin is a colicin‐like protein, whereas the R‐type pyocin resembles a contractile but non‐flexible tail structure of bacteriophage, and the F‐type a flexible but non‐contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R‐type pyocin is derived from a common ancestral origin with P2 phage and the F‐type from λ phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.

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 35u2003538u2003bp with 21u2003base 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.

Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (φETA) that encodes ETA and determined the complete nucleotide sequence of the φETA genome. φETA has a head with a hexagonal outline and a non‐contractile and flexible tail. The genome of φETA is a circularly permuted linear double‐stranded DNA, and the genome size is 43u2003081u2003bp. Sixty‐six open reading frames (ORFs) were identified on the φETA genome, including eta, which was found to be located very close to a putative attachment site (attP). φETA converted ETA non‐producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that φETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655.

Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons. Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai strain. To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons. The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains. However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12. In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase‐dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co‐transcribed.

Abstracts from the Japanese Journal of Limnology

The Japanese Journal of Limnology is another official publication of the Japanese Society of Limnology. The original papers in the journal were peer-reviewed by a few authorized referees, and appeared in Japanese with English abstracts.