Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin R1: change in permeability of the cell envelope

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, caused an increase in binding of fluorescent label, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), to sensitive cells. In pyocin R1-treated cells, cytoplasmic soluble proteins and crude ribosomes as well as cell envelopes were labeled by dansyl chloride. The amount of bound dye was proportional to the multiplicity of pyocin R1 and reached a maximal level at high multiplicity. In addition, pyocin R1 rapidly caused an increase in fluorescence intensity of the hydrophobic probes N-phenyl-1-naphthylamine, pyrene, and perylene, which were mixed with cells. These results show that pyocin R1 damages locally a cell envelope barrier to hydrophobic solutes and allows dyes to penetrate into the intracellular space across the barrier.     

Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.     

Uptake of Pyocin S3 Occurs through the Outer Membrane Ferripyoverdine Type II Receptor of Pseudomonas aeruginosa

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.     

Purification of the pyocin S2 complex from Pseudomonas aeruginosa PAO1: analysis of DNase activity

Pyocin S2 purified from mitomycin C-induced lysates of Pseudomonas aeruginosa strain PAO1 has been shown to consist of a complex of two proteins. Further analysis of the purified S2 complex revealed that the 74 kd S2 pyocin demonstrates DNase activity which can be blocked by S2-specific antisera. Chromosomal DNA from pyocin sensitive cells treated with the pyocin S2 complex in vitro did not show any degradation, suggesting that the 10 kd protein inhibits the DNase activity of the S2 protein. These results suggest an alternative mechanism for the toxicity associated with the S2 pyocin.     

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.     

The inherent DNase of pyocin AP41 causes breakdown of chromosomal DNA.

Pyocin AP41 degrades the chromosomal DNA in sensitive strains of Pseudomonas aeruginosa but has little effect on RNA, protein, and lipid syntheses. In vitro experiments showed that the carboxyl-terminal part of the large subunit of pyocin AP41 carries an inherent DNase that is responsible for its killing action.     

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.     

Interaction with lectins and differential wheat germ agglutinin binding of pyocin 103-sensitive and -resistant Neisseria gonorrhoeae

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.     

Cytotoxicity of pyocin S2 to tumor and normal cells and its interaction with cell surfaces

Cytotoxicity and adsorption of pyocin S2 produced by Pseudomonas aeruginosa M47 (PAO 3047) to virally transformed mammalian cells, human malignant cells and normal cells in the same species were studied. Pyocin S2 inhibited the growth of not only tumor cells (XC, TSV-5, mKS-A TU-7, HeLa-S3 and AS-II cells) but also normal cells (BALB/3T3 and BHK 21 cells). The inhibitory effects on the cells increased with an increase of pyocin S2 activity. On the other hand, there were some tumor cells (155-4 T2 and HGC-27 cells) and normal cells (normal rat kidney and human embryo lung cells) which were resistant to pyocin S2. The pyosin S2 activity was neutralized by the cell membrane preparations from pyosin S2-sensitive cells, but not by those from pyocin-resistant cells. This neutralization ability was inhibited by high concentrations of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine and}\text{N-}\text{acetyl}$ neuraminic acid and completely destroyed by periodate and neuraminidase. The inhibition by the saccharides was concentration dependent. These results suggest that the toxicity of pyocin S2 to several mammalian cells is due to the presence of the binding site for pyocin S2 in the cell membrane and further, that the carbohydrate moiety, especially of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine}$ and sialic acid, may play an important role as an initial binding site for pyocin S2.     

铜绿假单胞菌中S型绿脓杆菌素与荧光嗜铁素的功能协同性分析

在铜绿假单胞菌(Pseudomonas aeruginosa)中,S型绿脓杆菌素S2和S4与细菌中的铁载体荧光嗜铁素(pyoverdine)使用相同的摄取通道,表明二者之间存在某些联系。本研究表征了细菌中3个S型绿脓杆菌素(Pys2、PA3866、PyoS5)的单细菌基因表达分布,并研究了S2型绿脓杆菌素对细菌摄取荧光嗜铁素的影响。结果表明,在DNA损伤压力下,S型绿脓杆菌素基因的表达在细菌种群中呈现出高度分化,外源加入S2型绿脓杆菌素会减少细菌对荧光嗜铁素的摄取,因此S2型绿脓杆菌素的存在会阻止不合成荧光嗜铁素的“欺骗者”摄取环境中荧光嗜铁素,进而减弱其对活性氧(reactive oxygen species,ROS)压力的抵抗能力。另外我们发现,在细菌中过表达SOS响应(SOS response)调节因子PrtN时,荧光嗜铁素相关合成基因的表达量显著降低,进而导致荧光嗜铁素的总合成量和外分泌量显著降低。以上结果表明细菌中SOS压力响应系统与铁摄取系统的功能是存在相互联系的。     

In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

Epidemiological Fingerprinting of Pseudomonas aeruginosa by the Production of and Sensitivity to Pyocin and Bacteriophage

A new method has been devised to trace cross-infection by Pseudomonas aeruginosa. Unknown strains growing logarithmically in liquid media were treated with mitomycin C to induce the liberation of pyocin and phage. The lysates were then tested against 27 selected indicator strains, and the zones of clearing were differentiated as to killing by pyocin or lysis by phage. Twenty-four standard pyocin-phage lysates were then applied to each of the unknowns, and the sensitivity pattern was recorded. Thus, an “epidemiological fingerprint” consisting of 51 operational characteristics was established for each isolate. Organisms from the same source had identical or similar fingerprints, but organisms from different origins could easily be distinguished. Pyocin production, pyocin sensitivity, and phage production were found to be stable genetic characters; however, spontaneous mutations in phage sensitivity were frequently encountered. The epidemiological fingerprint has proven to be a sensitive tool in establishing the identity or dissimilarity of unknown strains. This method has been of great value in tracing the epidemiology of P. aeruginosa in the hospital environment. Each of the 157 P. aeruginosa strains tested has been typable by this method.     

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

The 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.     

Physicochemical properties of pyocin F1.

The physiochemical properties of pyocin F1 were studied. Pyocin F1 consists of flexuous rod-like particles homogenous in size. Each particle was composed of rod and fiber parts. The rod part was 105.5 +/- 9.5 nm long and 10.0 +/- 1.4 nm wide, and showed regular striations amounting to 23 layers. The fiber part was composed of several filaments; the length of the longest filament was 43.0 +/- 12.0 nm. The amino acid composition, the partial specific volume (0.720 ml/g), the sedimentation coefficient (S020,W = 35.1S), and the translational diffusion constant (0.94 +/- 0.01 x 10(-7) cm2/s) were determined. The particle weight was calculated to be 3.23 x 10(6) daltons.     

Effect of pyocin R1 on the glucose metabolism of sensitive cells of Pseudomonas aeruginosa.

The effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment. (Formula: see text). The possible mechanism of this change is discussed.     

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.     

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.     

Effect of iron concentration in the growth medium on the sensitivity of Pseudomonas aeruginosa to pyocin S2

The iron concentration in the growth medium was found to affect the susceptibility of Pseudomonas aeruginosa PML1550 to pyocin S2, a bacteriocin. The efficiency of killing by pyocin S2 was very low when the indicator cells were grown in an iron-rich medium. The capacity of these cells to adsorb pyocin S2 was reduced. Cultivation under limitation of iron (1 microM or less) was necessary to produce a fully sensitive cell population. The growth under iron limitation was accompanied by the appearance of four protein components in the outer membrane of the cells. Nine mutants resistant to pyocin S2 were isolated and their outer membranes were analyzed. They all lacked one component (Fe-b protein) as well as the adsorption capacity for pyocin S2. These findings suggest a possible role of this protein as the receptor for pyocin S2.     

Biological Cost of Pyocin Production during the SOS Response in Pseudomonas aeruginosa

LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.