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The Role of the S-type Pyocins in Pseudomonas Aeruginosa Biolfilm Dynamics

EPA Grant Number: F5D20722
Title: The Role of the S-type Pyocins in Pseudomonas Aeruginosa Biolfilm Dynamics
Investigators: Proctor, Diana
Institution: Smith College
EPA Project Officer: Boddie, Georgette
Project Period: September 1, 2005 through August 6, 2005
Project Amount: $74,344
RFA: STAR Graduate Fellowships (2005)
Research Category: Academic Fellowships

Description:

Objective:

Most bacteria in nature are found in biofilms – surface-bound, single and multi-species aggregations encapsulated in a complex polysaccharide matrix. Many models describe biofilms as climax communities, static, steady-state systems shaped largely by developmental mutualism. We prefer a more dynamic model of biofilm development in which bacteria compete for limited resources, including substrate space. We envision that bacteria competing for space in biofilms use bacteriocins, a class of toxic proteins produced by 99% of bacteria, as a mechanism for invasion into established biofilms. The bacteriocins of Pseudomonas include the S-type pyocins S2 and AR41, which, like many bacteriocins, consist of two functional domains, one exhibiting DNase activity (“killing domain”) and one conferring specific immunity to the producer strain (“immunity domain”). Pyocins S2 and AR41 inhibit phospholipid synthesis and degrade the DNA of susceptible strains, thus creating open ecological space for invasion by the producer strain. We wish to explore the possibility that the pyocins evolved as toxins to allow producer strains to invade or defend ecological space in the biofilm setting.

We contend that the pyocins evolved primarily in the context of biofilm communities as toxins that allow bacteria to invade or defend ecological niches. To evaluate this hypothesis we explore the extent to which standing populations of sensitive P. aeruginosa can be invaded by pyocin producing conspecifics. This study aims to

  1. assess the killing efficiency of pyocin AR41 compared to traditional small molecule antibiotics;
  2. and to examine the population and competition dynamics of mixed producer (AP41) and sensitive (PAO1) Pseudomonas strains.

Approach:

In objective 1, we examine the killing efficiency of pyocin AR41 using susceptibility and killing kinetics assays and visualize, using scanning confocal laser microscopy (SCLM), the pattern of S-type pyocin AR41 gene expression in developing and mature biofilms. In objective 2, we examine the population dynamics of mixed P. aeruginosa populations, employing simultaneous and sequential competition assays. Using SCLM and differential plating (using a tetracycline resistant PAO1 strain), we quantitatively and qualitatively assess the invasive pattern of pyocin producer (AP41) into both developing and established biofilms of sensitive strains (PAO1).

Expected Results:

By studying the toxicity of the S-type pyocin AR41, we hope to learn

  1. if pyocin sensitivity depends on growth context, as is true of most traditional antimicrobials,
  2. and if pyocin sensitivity is nutrient dependent. By gauging sensitivity to pyocin AR41, and by directly observing colonization and biofilm maturation as pyocin producers compete directly with a sensitive strain for limited substrate space, we hope to elucidate the role pyocins play in competition dynamics in natural environments.

This study will not only shed light on the fundamental regulation of biofilm dynamics, but will also have important and immediate application. Specifically, the demonstration that pyocin-producing strains readily invade established biofilms, suggests that engineered strains exhibiting bioremediation potential can be similarly equipped to facilitate their invasion into and colonization of resident biofilms in a variety of important environmental settings.

Supplemental Keywords:

Biofilm, Pseudomonas aeruginosa, pyocin, bacteriocin, dynamics, invasion, , Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, RFA, Environmental Microbiology, Monitoring/Modeling, DNase activity, bacteria monitoring, scanning confocal laser microscopy, S-type pyocins

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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