Quantitative Measurement of Pseudomonas Syringae Tailocins and Analysis of their Receptors

Abstract

One of the most revolutionary scientific breakthroughs was the discovery of penicillin in 1928, which led to an antibiotic revolution. In the years following, a plethora of antibiotics readily became available to combat prominent and detrimental pathogens in both agriculture and medicine. However, over the years, these bacteria developed resistance to commonly used antibiotics due to overuse. With that, an arms-race between new antibiotic development and bacterial evolution began and continues to this day. Current antibiotics are constantly modified, and new ones are vehemently searched for, however many researchers are investigating alternatives to antibiotics altogether. One such alternative that shows promise is bacteriocins.Bacteriocins are antimicrobials that target closely related species to the producer strain to out-compete them for a similar niche. Some of the most well studied bacteriocins are those from Pseudomonads, which are derived from the tails of bacteriophage, earning them the moniker: tailocins. There are two types of tailocins: F and R-types, which differ in the type of bacteriophage they came from. R-types are derived from myoviridae phage and possess a rigid tail that contracts while F-types are derived from siphoviridae phage and have a flexible, non-contractile tail. Most studies have focused on R-types, which will be the focus of this report. R-type tailocins produced by Pseudomonads bind to lipopolysaccharide (LPS), which is a lipid embedded in the outer membrane of gram-negative bacteria. Sugars are built on top of the lipid portion of LPS and stick out into the environment. This structure of sugars varies significantly between bacteria, from composition and order of sugars as well as side chains and modifications. This difference in LPS presumably affects what tailocins can bind and thus kill the bacteria. Modifications in the LPS composition can result in resistance to previously susceptible tailocins as well as susceptibility to previously ineffective tailocins. A key part of being able to manipulate and study something is being able to effectively measure it. Current methods used to measure killing activity of tailocins are purely qualitative. The method currently used to test whether a bacterium is sensitive to a tailocin or not, is the soft-agar overlay assay. In this assay, the target strain is plating onto a petri dish and an aliquot of tailocin is spotted onto a lawn of bacteria. If the tailocin kills the bacteria, a zone of killing will appear, however if it no killing zone appears, the bacteria is resistant. However certain tailocins do not kill their targets as effectively as others, resulting in a hazy zone of killing, rather than a crips, clean clearing. The first chapter looks at a quantitative way to measure tailocin killing using a 96-well plate and reading the growth of the bacteria after treatment with tailocins. Luminescence was documented to see how effective each tailocin was at killing the bacteria (reduction in growth/less luminescence). In the second chapter, the plate assay was further evaluated to see what other applications it could be used for. This assay was used to verify that tailocins could be concentrated using dialysis and aquacide. This is relevant, as being able to concentrate proteins is important for commercial applications. Furthermore, binding between tailocins and their receptors was investigated using the plate assay. By mixing tailocins with various sugars before adding them to the bacteria wells, it could be determined whether the sugar bound to the tailocin or not; if the tailocin didn’t kill the bacteria in the wells, it was inhibited by the sugar, whereas if it maintained killing activity, it did not bind to the sugar. Similarly, the same was done with LPS from target and non-target strains: tailocins were mixed with several types of LPS to see if they inhibited the tailocin or not. From here, the relationship between tailocin and receptor was further probed by testing tailocins against Pseudomonas syringae strains that have had their LPS characterized. The outer region of LPS of Pseudomonas syringae is a homopolymer, consisting of either D- or L- rhamnose. Thus, it was observed that there was a distinct divide in what tailocins could bind to which bacteria: either those with D-rhamnose LPS or those with L-rhamnose LPS. This was first assessed using the soft agar assay and later verified quantitatively using the plate assay. In the last chapter, a unique way to extract and purify bacteriocins was sought. The protocol currently in place is tedious and yields end solutions with various tailocin concentrations, thus a method that will give higher concentrations of tailocins and is easier for commercial production was investigated. One potential approach to this was tagging different bacteriocins with a his-tag (aptatacin and the sheath protein of USA011 tailocin). The tagged bacteriocins were purified using a his-column, then visualized using a Western blot, however results were inconclusive. This dissertation illustrates various methodical improvements in measuring tailocins and their binding to receptors as well as a different approach to purifying bacteriocins. Furthermore, different tailocins were looked at against characterized LPS strains to show a distinct split between whether tailocins bind D vs L-rhamnose. These findings can help make bacteriocins more usable in industrial settings as well as help elucidate the complexity between tailocins and their interactions with LPS.