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.     

Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.     

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa .     

Mutational analysis of a bifunctional ferrisiderophore receptor and signal-transducing protein from Pseudomonas aeruginosa

The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.     

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.     

FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.     

Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor,FpvI

A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida. Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression. A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.     

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.     

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases.

Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.     

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.     

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.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.     

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.     

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。     

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.     

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.     

Purification and characterization of pyocins R fromPseudomonas aeruginosa

Five types of pyocins were found in Pseudomonas aeruginosa strain 112. Production of these types was induced by UV irradiation. The pyocin activity was found to be resistant to trypsin treatment. Their molar mass was found to be 282, 251, 112, 89.1 and 54.9 kg/mol, respectively. The pyocins obtained were different from any known type (such as R, S, F) in their chemical and physical properties.     

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.     

Temperature and pyoverdine-mediated iron acquisition control surface motility of Pseudomonas putida

Pseudomonas putida KT2440 is unable to swarm at its common temperature of growth in the laboratory (30 degrees C) but exhibits surface motility similar to swarming patterns in other Pseudomonas between 18 degrees C and 28 degrees C. These motile cells show differentiation, consisting on elongation and the presence of surface appendages. Analysis of a collection of mutants to define the molecular determinants of this type of surface movement in KT2440 shows that while type IV pili and lipopolysaccharide O-antigen are requisites flagella are not. Although surface motility of flagellar mutants was macroscopically undistinguishable from that of the wild type, microscopy analysis revealed that these mutants move using a distinct mechanism to that of the wild-type strain. Mutants either in the siderophore pyoverdine (ppsD) or in the FpvA siderophore receptor were also unable to spread on surfaces. Motility in the ppsD strain was totally restored with pyoverdine and partially with the wild-type ppsD allele. Phenotype of the fpvA strain was not complemented by this siderophore. We discuss that iron influences surface motility and that it can be an environmental cue for swarming-like movement in P. putida. This study constitutes the first report assigning an important role to pyoverdine iron acquisition in en masse bacterial surface movement.