Bacteriophage and Phenotypic Variation in Pseudomonas aeruginosa Biofilm Development

Abstract

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using antiPf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.

Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms

“…To answer this question, it is necessary to understand that most phages that target bacteria in natural environments are thought to exist in biofilm ecosystems. The reciprocal effects of biofilm structure and the activity of the prophages released have previously been demonstrated for different bacterial species (25,44,57). However, the reaction of biofilms to ubiquitous viral particles in the environment is poorly documented (52).…”

“…Furthermore, prophages can cause DNA inversions and phenotypic variations and can mediate cell death, which is an important differentiation mechanism within microcolonies (40,56,57).…”

Accurate localization and excision of genomic islands in four strains of Pseudomonas aeruginosa and Pseudomonas fluorescens

“…The genomic location of six GIs in P. aeruginosa was identical to those in our previous report [18], but their mobility was not determined. A number of studies have also described the regions of GIs in these four strains [14,[19][20][21][22][23]. These data have been presented in comparison with the 11 GIs of the present study ( Table 4).…”

“…Webb et al [14] extracted the replicative-form (RF) DNA of Pf4 (PAO1GI-1) from Pf4-infected cells, and used primers Pf4F and Pf4R to confirm recirculation of prophage Pf4 by the repeat sequence (3′-TGGAGCGGGCGAAGGGAATCGAACCCT-5′). This result was similar to our study, however, we selected a more precise crossing-combining approach of primers [11], and also determined that PAO1GI-1 (prophage Pf4) not only exists in the chromosome, but also can be excised from the chromosome to form a circular intermediate.…”

“…P. aeruginosa PAO1 was grown at 37°C and P. fluorescens Pf-5 was grown at 28°C in Luria-Bertani (LB) medium. Solid medium contained 1.5% agar [14].…”