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.     

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.     

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.     

Phenotypic mixing of pyocin R2 and bacteriophage PS17 in Pseudomonas aeruginosa PAO.

Previous results indicate that a group of bacteriocins in Pseudomonas aeruginosa, named R-type pyocins, have a structure resembling bacteriophage tails and share some serological homology with certain bacteriophages. This paper presents genetic evidence which strongly suggests that components of pyocin R2, an R-type pyocin of P. aeruginosa PAO, and tail components of bacteriophage PS17 are interchangeable. Complementation tests with pyocin R2-deficient mutants of PAO and ts mutants of PS17 revealed that various phenotypic interactions occur between the pyocin and bacteriophage in PAO cells lysogenized or infected with PS17. (i) Certain pyocin R2-deficient mutations were phenotypically suppressed in cells carrying PS17 prophage. (ii) A temperature-sensitive mutant of PS17, tsQ31, was phenotypically suppressed in PAO cells treated with mitomycin C. (iii) Phenotypically mixed phages with receptor and serological specificities of pyocin R2 were formed in PS17 lysogens of certain pyocin R2-deficient mutants.     

R-type pyocin is required for competitive growth advantage between Pseudomonas aeruginosa strains

R-type pyocin is a bacteriophage tail-shaped bacteriocin produced by Pseudomonas aeruginosa, but its physiological roles are relatively unknown. Here we describe a role of R-type pyocin in the competitive growth advantages between P. aeruginosa strains. Partial purification and gene disruption revealed that the major killing activity from the culture supernatant of PA14 is attributed to R-type pyocin, neither F-type nor S-type pyocins. These findings may provide insight into the forces governing P. aeruginosa population dynamics to promote and maintain its biodiversity.     

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.     

Comparative study on fibers isolated from four R-type pyocins, phage-tail-like bacteriocins of Pseudomonas aeruginosa

Five R-type pyocins have been reported which are almost identical with one another in their morphology and subunit composition, though distinct in receptor-binding specificity. We isolated fibers from pyocin R2, R3, and R4 by essentially the same procedure as used in our previous isolation of pyocin R1 fiber with unimpaired receptor-binding ability. All the isolated fibers including R1 fiber were indistinguishable from one another, in terms of electron microscopic observation and subunit composition analysis by SDS gel electrophoresis. They consisted mainly of Subunit No. 2 (Mw 71,000) and No. 9 (31,000) proteins. Although Subunit No. 9 protein in every fiber was susceptible to trypsin and afforded a fragment with the same molecular weight (about 19,000) detectable in the SDS gel, Subunit No. 2 protein was cleaved with trypsin only after the fiber had been treated with an organomercurial, 4-(p-sulfophenylazo)-2-mercuriphenol. The cleavage of Subunit No. 2 protein proceeded to give several fragments with molecular weights ranging from 64,000 to 34,000, and the fragmentation patterns were electrophoretically distinct at least among R1 fiber, R3 fiber, and others (R2 and R4 fibers). The results indicate that Subunit No. 2 proteins of these fibers are different from one another in the structure surrounding trypsin-susceptible peptide bonds. Immunological investigations with anti-R1 fiber antibodies provided some additional information on the difference among R-type pyocins at the fiber level.     

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.     

Isolation and Characterization of a Bacteriophage Tail-Like Bacteriocin from a Strain of Rhizobium

Bacterial strain 16-3 spontaneously produces a bacteriocin which inhibits the growth of closely related strain 16-2. Both strains were newly isolated from root nodules of lupines and probably belong to the species Rhizobium lupini. Production of infectious progeny of newly isolated virulent phage 16-2-4 in strain 16-2 is inhibited completely if complexes are bacteriocin-treated during the first half of the latent period. Treatment begun during the second half leads to premature lysis of complexes and inactivates only those progeny phages which were not yet fully matured at the moment of the particle-induced lysis. Examination by electron microscope of the bacteriocin enrichment revealed the presence of particles 123 nm in length which resemble the tails of T-even bacteriophages. Since the particles sediment together with the bactericidal activity in the sucrose gradient and adsorb specifically to bacteriocin-sensitive cells, it is concluded that they are identical with the bactericidal agent. The particles are not found attached to phage heads and cannot self-propagate; they are regarded as incomplete and are named INCO particles. INCO particles consist of a core enveloped by a contractile sheath. One end of the sheath is connected to a baseplate to which six fibers, each 32 nm in length, are attached. These connect the baseplate of an adsorbing particle to the cell surface. Since INCo cores are probably empty, it is concluded that specific adsorption of the particles to the bacterial surface is sufficient to inactive sensitive cells irreversibly.     

Morphological studies on relaxed and contracted forms of purified pyocin particles

The bacteriocin from Pseudomonas aeruginosa, pyocin, consists of a contractile sheath and inner core reminiscent of T-even coliphage tails. Contraction of the outer sheath was found to be promoted by 0.5 m magnesium chloride, 1% Formalin, low pH, sonic treatment, and freezing or thawing or both. The contraction caused by 0.5 m magnesium chloride, however, was found to be reversible and occurred upon reduction of the salt concentration from 0.5 to 0.02 m. In addition, direct assay showed that pyocin activity was nearly proportional to the percentage of only uncontracted forms. Initial studies suggested that the adsorption of purified pyocin onto cell wall fragments from the sensitive indicator strain of P. aeruginosa occurs with the relaxed particle only and not with the contracted form. However, after adsorption, contraction occurred. Various morphological structures, such as tail fibers and base-platelike appendages, were also observed. Upon contraction, six tail fibers were observed on many particles, four of which appeared to originate from the sheath and two from the inner core. Polysheaths and polycores several hundred nanometers in length were also occasionally observed.     

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.     

Biological properties of the phage-tail-like particles from Myxococcus coralloides D

Abstract Electron microscopy of preparations of the Myxocococcus coralloides D autolytic supernatants revealed the presence of phage-tail-like particles. The particles consisted of a core, a contractile sheath and a baseplate with fibers. The induction of the defective prophage occurred when the cultures reached the stationary phase, but the events occurring during induction did not lead to cell lysis. Particles nerver appeared during exponential growth. They were not observed when M. coralloides D grew on solid media, either. Purified samples of phage tails were able to inhibit most of the myxobacterial strains which were tested when the particles were in the extended state, but they were unable to multiply on the sensitive strains.     

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.     

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.     

Structure and folding of bacteriophage T4 gene product 9 triggering infection. I. Production and properties of recombinant protein

Gene product 9 (gp9, 288 amino acid residues per monomer, molecular weight 30.7 kD) of bacteriophage T4 triggers the baseplate reorganization and the sheath contraction after interaction of the long tail fibers with the receptors of the bacterial cell. In this work we have produced the recombinant protein and determined that gp9 is a stable homotrimer and active in in vitro complementation assay completing the defective phage particles which lack gp9. According to CD-spectroscopy data, the gp9 polypeptide chain contains 65-73% beta-structure and 11-16% alpha-helical segments, this being in good agreement with secondary structure prediction results. Additionally, we have constructed a set of plasmid vectors for expression of gp9 deletion mutants. The fragments with consecutive truncations of the N-terminus of the molecule, as well as the full-length protein, are trimers resistant to SDS treatment and decrease infective phage particle formation in in vitro complementation assay with native gp9. The deletion of the molecule C-terminal region results in failure of trimerization and decreases the stability of the protein.     

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.     

A circular dichroism study of sheath contraction in pyocin R1

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.     

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.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. I. Localization of the pyocin R2 gene cluster between the trpCD and trpE genes

Thirty-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.