R-type Zx Spectrum Download
Luminosa Renzullo
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.
Actually remember one of my friends later( maybe a year) turned up with a Philips computer, which he always had problems getting games for, compared with the spectrum. Not sure if it was a 8235 or could have been their version of the MSX as well...
The best-characterized, and perhaps best-known, means to alter microbial community composition involves the use of broad spectrum antibiotics targeting physiological processes conserved across diverse bacterial taxa. However, lowered efficacy of broad spectrum antibiotics due to the evolution of resistance coupled with the realization that their use can lead to detrimental off-target effects on beneficial microbes has created new research momentum to identify and characterize new types of intermicrobial interactions that possess higher specificity [8,9,10,11]. Bacteriocins are molecules produced by bacterial cells that are largely considered to specifically target different strains of the same species or closely related species [6, 9]. In contrast to relatively indiscriminate activity of some broad spectrum antibiotics, bacteriocin targeting is mediated through a requirement for interactions with particular receptors on target cells before antimicrobial functions take place [6, 12]. The precision of bacteriocin killing activity also suggests that they are used in nature as a means to outcompete microbes that occupy similar niches, and thus characterization of bacteriocin activity and sensitivity from a diverse collection of strains could inform previously unrecognized ecological patterns [13]. Perhaps most relevant for their therapeutic use, the relatively specific killing spectrum of bacteriocins could provide a powerful means to precisely engineer microbiomes while avoiding off-target effects seen with other treatments [4, 9].
As an additional step to characterize killing spectra, we used hierarchical clustering to group strains based on activity consistent with R-type syringacin killing activity (Fig. 2). According to groupings arising from this analysis, screened strains can be sorted into at least two deeply branching main clusters (which we term clusters I and II) and at least five total groups. The two main clusters are dominated by strains with relatively broad killing spectrum (an average of 19 and 16 strains targeted per cluster) but with little overlap between the strains targeted by each. A third group is nested within cluster II according to our analysis and contained killing activities that were relatively more selective (average of 7). The fourth group clusters closely with the third group mentioned above likely due to the relatively low number of targets but contains strains that have a minimal number (one or two) of targets. The fifth cluster is composed of strains with no observed killing activity.
There are multiple cases where sensitivity dramatically differs between closely related strains. For instance, strain CC457 from sensitivity group B is closely related to both Paf and Ptt (which are both sensitivity group A). Likewise, USA007 is classified in sensitivity group A but is closely related to CC1544 and CC1416 from sensitivity group B. There are also numerous cases where relatively divergent strains have convergently evolved to have very similar sensitivities to syringacins. Only a small subset of strains screened herein appear to have the exact same sensitivity spectrum, and there are multiple instances where there are a small number of differences between strains with similar sensitivity spectrums.
To test whether recombination of the Rbp and chaperone alone are sufficient to retarget syringacins, we utilized a mutant strain of P. syringae pv. syringae B728a (PsyB728a) in which both the Rbp and chaperone were previously deleted [14]. We have already demonstrated that we can complement phage-derived syringacin production and killing in this strain background by ectopically expressing the PsyB728a genes from a plasmid [14]. However, instead of complementing this strain with its native Rbp and chaperone, we instead chose to test whether killing activity of this strain could be complemented by both loci from a strain with a distinctly different killing spectrum (P. syringae pv. japonica, Pja). As shown in Fig. 4, not only can the Rbp and chaperone from Pja complement deletion of these genes from PsyB728a but the killing spectrum of this complemented strain is also consistent with the spectrum from Pja instead of PsyB728a. Therefore, horizontal transfer of both the Rbp and chaperone is sufficient to retarget the P. syringae R-type syringacin.
Ectopic expression of the Rbp and chaperone is sufficient to retarget R-type syringacins. The results of overlays of six different strains and five different PEG-selected supernatants are shown. Overlay strains are shown on the y axis, whereas supernatants are shown on the x axis. Three strains are sensitive to killing by R-type syringacins produced by PsyB728a and not Pja (Psy301D, Pan, USA007), while three strains are sensitive to R-type syringacins produced by Pja and not PsyB728a (PtoDC3000, USA011, Por1_6). Killing activity is abolished in a mutant of PsyB728a where both the receptor-binding protein (Rbp) and chaperone from the R-type syringacin has been deleted, and this mutant can be complemented by the expression of both these genes ectopically from a plasmid. Ectopic expression of the Rbp and chaperone from Pja in this PsyB728a ΔRbp/chaperone background retargets this strain so that the killing spectrum matches that of Pja
Weak killing activity in any of the assays is challenging to score, and it is possible that very weak activity (due to a low number of killing particles or due to overgrowth by the overlay strain) could be scored as a null in our assays. To counter this, we have repeated all inductions, PEG selections, and overlays at least twice independently and have reported results where only one of the two assays showed activity. We have found that tailocin particles are concentrated during PEG precipitation, and thus it is unlikely that lack of observed activity after PEG selection is the product of loss of tailocin particles in the far majority of cases. We also note that, as with any selection, recovery of killing particles after PEG treatment is based upon assumptions of the biophysical properties of the particles themselves. For instance, strain CC1543 appears to have a killing spectrum largely consistent with tailocin activity from other strains, but this activity does not precipitate with PEG. It is possible that this strain does harbor an active tailocin, but for currently unknown reasons (i.e., perhaps a shorter tail), this activity does not precipitate with PEG.
Although many R-type pyocin genes share common ancestry with bacteriophage genes, pyocins are not bacteriophages; they do not contain nucleic acid and cannot replicate. As protein particles, they do not have a genome. They kill by a mechanism unlike phage-mediated lysis, even though the initial event toward killing by both R-type pyocins and phages is binding to an accessible receptor on the target bacterium. In fact, as observed in these studies, R-type pyocins with tail fibers identical to those of a phage exhibited a killing spectrum broader than the host range of the phage from which their specificity-determining tail fibers had been derived. This phenomenon is likely due to DNA restriction or immunity systems of the host that can prevent phage infection or to other host-encoded functions required for phage replication and lysis of the host but not required for pyocin-mediated killing. The killing of target bacteria by R-type pyocins also does not result in the immediate release of endotoxins, as does lytic killing by phages and by many bactericidal antibiotics (13, 16, 44).
Tail fibers from the enormous number and diversity of bacteriophages in the environment provide a rich source of binding specificities for engineering pyocins. Thus, we anticipate being able to engineer R-type pyocins to target a broad range of bacterial pathogens, targeting many diverse surface structures. The application of phage display techniques may provide even greater diversity of binding specificities than exists naturally on phages. Given this dependency on specific surface receptors, R-type pyocins probably cannot be engineered to provide broad-spectrum antibacterial activity. However, these approaches based on fusing phage tail fibers to pyocin tail fibers could lead to an almost unlimited number of narrow-spectrum bactericidal agents. As for nearly any antibacterial agent, pyocin-resistant bacteria are likely to arise. In the case of R-type pyocins, this resistance most likely would be due to loss of a surface receptor (5, 10, 29, 34), a trait not known to be transmitted horizontally by the acquisition of a pathogenicity island or plasmid, a phenomenon that has proven to be troublesome with traditional antibiotics (1, 21). In addition, if the surface receptor molecule targeted by the agent is a virulence factor or fitness factor (lipopolysaccharide often is), then the inevitable emergence of resistance might often genetically compromise the pathogen's virulence (39).
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