Makoto Kageyama

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 sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO.

SummaryThe recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA+-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R′ plasmids.

A novel transposon-like structure carries the genes for pyocin AP41, a Pseudomonas aeruginosa bacteriocin with a DNase domain homology to E2 group colicins

SummaryThe genetic determinant for pyocin AP41, a bacteriocin produced by Pseudomonas aeruginosa, has been cloned. The determinant is located on the chromosome flanked by a pair of inverted repeats, forming a transposon-like structure (TnAP41). TnAP41 possesses some features characteristic of the Tn3 family of transposons. Based on a comparison with the structure of the corresponding region of the chromosome of a nonproducer strain, we propose that P. aeruginosa has acquired pyocinogeny by the transposition of TnAP41 into the chromosome. The determinant comprises two ORFs encoding the protein subunits responsible for the killing action (the large component) and immunity (the small component). Amino acid sequences of the C-terminus of the large component (the deoxyribonuclease domain) and the immunity protein show remarkable homology to those of E2 group colicins, suggesting that these bacteriocins, which are produced by distantly related species, have originated from a common ancestor.

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. II. Physical characterization of pyocin R2 genes using R-prime plasmids constructed from R68.45.

SummaryThe chromosome segment which contains the genes responsible for production of pyocin R2 in P. aeruginosa PAO was defined physically using R-prime plasmids constructed in vivo from R68.45. The previous conclusion from genetic mapping that the cluster of pyocin R2 genes is located in between trpC and trpE genes was confirmed by deletion mapping of various R prime plasmids bearing the trpC gene. The pyocin R2 gene cluster was further localized on two contiguous HinDIII fragments of 16 kb and 8.0 kb. PML14 strain, in which R-type pyocin genes were completely deleted, had only one 11 kb HindIII fragment instead. Heteroduplexes between this 11 kb fragment with the two HindIII fragments of PAO revealed that the cluster of pyocin R2 genes was an insertion 13 kb long.

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO

SummaryThirty-seven mutants defective in pyocin R2 production in the P. aeruginosa PAO strain were subjected to fine mapping of pyocin R2 genes by transduction with phage F116L. Sixteen complementation groups (designated prtA through prtP) involved in pyocin R2 production were tentatively identified by complementation tests using phage F116L. Their linkages to trpC and trpE markers and fine mapping by three point crosses demonstrated that most of the mutations (prtA through prtN) were located in between trpC and trpE, and that the prtP mutation was localized outside this major prt cluster but in the proximity of the rifA and strA region.

Construction and Characterization of Chimeric Proteins Between Pyocins and Colicin E3

Pyocins S1, S2 and AP41 are the most frequently found protease-sensitive bacteriocins inPseudomonas aeruginosa. They are classified by the different receptor specificities. A peculiar feature of these bacteriocins is that their genetic determinants are all located at definite sites on the the chromosome. This is in sharp contrast to the case of other bacteriocins, such as colicins, which are of plasmid origin. We cloned these pyocin genes on appropriate plasmids and determined their DNA base sequences, and purified their proteins.