Purification and characterization of pyocins frompseudomonas aeruginosa

Three types of pyocins were found in Pseudomonas aeruginosa strain 986 and named pyocin type P25, P50, and P70. Production of these types was inducible by UV irradiation. Their molar mass was estimated. The pyocins obtained were different from the known pyocins R, S, and F in their chemical and physical properties. No immunological cross reaction was observed among these pyocins.     

Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes.

Most strains of Pseudomonas aeruginosa produce various types of bacteriocins (pyocins), namely, R-, F-, and S-type pyocins. The production of all types of pyocins was shown to be regulated by positive (prtN) and negative (prtR) regulatory genes. The prtN gene activates the expression of various pyocin genes, probably by the interaction of its product with the DNA sequences conserved in the 5' noncoding regions of the pyocin genes. The prtR gene represses the expression of the prtN gene, and its product, predicted from the nucleotide sequence, has a structure characteristic of phage repressors and seems to be inactivated by the RecA protein activated by DNA damage. A model for the regulation of the pyocin genes is proposed.     

Characterization of a bacteriophage related to R-type pyocins.

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.     

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 lambda 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 pyocins of Pseudomonas aeruginosa

Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C. Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5). It interacts with the small component (immunity protein). The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN. R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively. The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.     

Synergistic activity of gentamicin plus carbenicillin upon Pseudomonas aeruginosa: its relationship with pyocin and antibiotic susceptibility.

The synergistic effect of combinations of gentamicin and carbenicillin, as well as the type or subtype of the pyocins produced, were investigated in 170 strains of Pseudomonas aeruginosa isolated from clinical specimens. A high proportion of strains were synergistically inhibited (73.5%), but among strains producing pyocins 7, 14 and 31, synergy was infrequent or absent. The synergistic effect was more frequent upon gentamicin- or carbenicillin-susceptible strains. However, among untypable strains, synergy was more frequent among gentamicin-resistant strains. Susceptibility to both gentamicin and carbenicillin must be considered if antibiotic susceptibility is to be related to synergy.     

Retargeting R-type pyocins to generate novel bactericidal protein complexes

R-type pyocins are high-molecular-weight bacteriocins that resemble bacteriophage tail structures and are produced by some Pseudomonas aeruginosa strains. R-type pyocins kill by dissipating the bacterial membrane potential after binding. The high-potency, single-hit bactericidal kinetics of R-type pyocins suggest that they could be effective antimicrobials. However, the limited antibacterial spectra of natural R-type pyocins would ultimately compromise their clinical utility. The spectra of these protein complexes are determined in large part by their tail fibers. By replacing the pyocin tail fibers with tail fibers of Pseudomonas phage PS17, we changed the bactericidal specificity of R2 pyocin particles to a different subset of P. aeruginosa strains, including some resistant to PS17 phage. We further extended this idea by fusing parts of R2 tail fibers with parts of tail fibers from phages that infect other bacteria, including Escherichia coli and Yersinia pestis, changing the killing spectrum of pyocins from P. aeruginosa to the bacterial genus, species, or strain that serves as a host for the donor phage. The assembly of active R-type pyocins requires chaperones specific for the C-terminal portion of the tail fiber. Natural and retargeted R-type pyocins exhibit narrow bactericidal spectra and thus can be expected to cause little collateral damage to the healthy microbiotae and not to promote the horizontal spread of multidrug resistance among bacteria. Engineered R-type pyocins may offer a novel alternative to traditional antibiotics in some infections.     

Isolation and characterization of a new bacteriophage, KF1, immunologically cross-reactive with F-type pyocins

Pseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively. The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCl and the sedimentation coefficient of the phage were 1.54 g/ml and 392S, respectively. Some other biochemical properties were described. The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4 x 10(7). The density of the DNA in CsCl was 1.719 g/ml, the melting temperature was 95.4 degrees C. The guanine plus cytosine content was calculated to be 60 to 64%.     

Pyocin typing of Pseudomonas aeruginosa strains

The possibility of using the typing of P. aeruginosa strains by their pyocins as one of the epidemiological markers in the study of P. aeruginosa hospital infections has been established. As this method of typing is characterized by certain variability, the authors propose that the method of the "cross analysis" of pyocins produced by P. aeruginosa strains be used simultaneously. This method is based on the following phenomenon: if the cultures to be compared are different, the pyocin produced by one strain suppresses the growth of the other one, and if the cultures are identical, no suppression of their growth by pyocins is observed.     

Bacteriocin-mediated competition in cystic fibrosis lung infections

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations, especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that (i) isolates from later infection stages inhibited earlier infecting strains less, but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage over time; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.     

Genetic comparison of bacteriophage PS17 and Pseudomonas aeruginosa R-type pyocin.

PS17 is a bacteriophage of Pseudomonas aeruginosa that is serologically cross-reactive with phage tail-like bacteriocins called R-type pyocins. In addition to having immunological cross-reactivity, certain genes are functionally complementable between PS17 and R-type pyocins. To compare the genetic structures of PS17 and R-type pyocins, a physical map of PS17 genes was constructed by cloning phage DNA fragments on RSF1010-derived vector plasmids. The head and tail gene clusters were tandemly arrayed and together occupied about half of the 41-kilobase-pair PS17 chromosome. With use of these phage clones, the following results were obtained with respect to the genetic relationship between PS17 and R-type pyocins: (i) serological cross-reaction between PS17 and pyocin occurred for the major sheath protein and two components of the fiber, (ii) a certain pyocin mutation was complemented by cloned phage fragments, and (iii) the phage DNA fragment carrying sheath and core tube genes was shown to hybridize to the DNA fragment carrying the pyocin R2 genes.     

Functional domains of S-type pyocins deduced from chimeric molecules.

Functional domain structures of pyocins AP41, S1, and S2 were assigned by examining the functions of chimeric pyocins and deletion derivatives. Pyocins AP41, S1, and S2 are essentially composed of three domains, the receptor-binding domain, the translocation domain, and the DNase domain, in that order from the N terminus to the C terminus. The alignment of these domains is distinct from that in E2-group colicins with functions similar to those of these pyocins. Pyocins AP41 and S2 have a fourth domain between the receptor-binding and the translocation domains, which is dispensable for their killing functions.     

Molecular structures and functions of pyocins S1 and S2 in Pseudomonas aeruginosa.

Pyocins S1 and S2 are S-type bacteriocins of Pseudomonas aeruginosa with different receptor recognition specificities. The genetic determinants of these pyocins have been cloned from the chromosomes of P. aeruginosa NIH-H and PAO, respectively. Each determinant constitutes an operon encoding two proteins of molecular weights 65,600 and 10,000 (pyocin S1) or 74,000 and 10,000 (pyocin S2) with a characteristic sequence (P box), a possible regulatory element involved in the induction of pyocin production, in the 5' upstream region. These pyocins have almost identical primary sequences; only the amino-terminal portions of the large proteins are substantially different. The sequence homology suggests that pyocins S1 and S2, like pyocin AP41, originated from a common ancestor of the E2 group colicins. Purified pyocins S1 and S2 make up a complex of the two proteins. Both pyocins cause breakdown of chromosomal DNA as well as complete inhibition of lipid synthesis in sensitive cells. The large protein, but not the pyocin complex, shows in vitro DNase activity. This activity is inhibited by the small protein of either pyocin. Putative domain structures of these pyocins and their killing mechanism are discussed.     

Pyocin production by bacillus pyocyaneus and sensitivity of strains of different origin to them

Using the method proposed by Gillies and Govan and their indicator strains, 342 P. aeruginosa strains isolated from the patients were studied in respect to their pyocinogenicity and typed according to the production of different types of pyocins. Besides, in 206 cultures the pyocin sensitivity of 16 standard P. aeruginosa strains (5 strains obtained from Govan and 11 strains provided by the authors) was determined. All the tested cultures fell into 23 pyocin types; of these, types I and X occured most frequently, 56 strains identified by means of indicators could not be typed due to the fact that the corresponding pyocin types were absent in Govan's scheme. The cultures isolated from the patients and the environmental objects during the outbreak of P. aeruginosa in a hospital were proved to belong to the same pyocin type (III). The double typing of the cultures, according to pyocin production and pyocin sensitivity, allowed to determine individual characteristics of 75% of the tested cultures.     

Purification and properties of an S-type pyocin, pyocin AP41

Pyocin AP41, a protease-sensitive bacteriocin produced by Pseudomonas aeruginosa PAF41, was purified to a homogeneous state and characterized. The molecular weight of this pyocin was about 95,000 as determined by the combination of gel filtration and sedimentation velocity analysis. This pyocin was a complex of two kinds of polypeptides. Highly purified preparations showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their apparent molecular weights were 90,000 and 6,000 to 7,000, respectively. Two proteins could be separated by gel filtration in the presence of 6 M urea. Amino acid compositions of these components were determined. The large component had pyocin activity similar to the complex, whereas the small component did not. Sensitive cells were killed by this pyocin only under growing conditions and with single-hit kinetics. The pyocin-treated cells lysed in about 30 min with concomitant production of their resident pyocins or phages. The induced production of resident pyocins caused by pyocin AP41 depended on a recA gene function.     

Pyocin S2 (Sa) kills Pseudomonas aeruginosa strains via the FpvA type I ferripyoverdine receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.     

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.     

Construction and characterization of pyocin-colicin chimeric proteins.

Chimeric proteins were constructed from pyocin S1 or S2 and colicin E3 or E2, and their characteristics were investigated with special reference to the domain structure. The nuclease domains were interchangeable between two bacteriocins so that a new kind of pyocin, with RNase activity, was created. A bacteriocin which can kill both Pseudomonas aeruginosa and Escherichia coli was also constructed. Investigations with various chimeric proteins indicate that the translocation domain as well as the receptor-binding domain is species specific. Inhibition of lipid synthesis, which is characteristic of pyocins, was also observed with chimeric pyocins carrying the DNase domain of colicin E2 but not with those carrying the RNase domain of E3. Thus, the DNase domain is responsible for the inhibition of lipid synthesis.     

In vitro PYOCIN activity ofPseudomonas aeruginosa strains pretreated with antibiotics

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.