Rhamnolipid‐Dependent Spreading Growth of Pseudomonas aeruginosa on a High‐Agar Medium: Marked Enhancement under CO₂‐Rich Anaerobic Conditions

Abstract

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions.

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

“…Cultivation was carried out aerobically for 2 days at 37 °C in a sealed plastic box with wet tissue to maintain a high level of humidity (≥90%). In some special anaerobic cultivations, an ASKA anaerobic system (ASKA Diagnostics Inc., Tokyo) in a transparent plastic bag was used as described previously (Nozawa et al , 2007). Spreading growth was examined by measuring colony diameters.…”

“…In the present study, however, an ability of P. aeruginosa PAO1 to develop thin expanding colonies on 1.5% agar medium in 2 days was demonstrated. For surface‐spreading growth on hard agar medium, three independent bacterial factors have been elucidated so far: flagella for swarming (Allison & Hughes, 1991), pili for twitching motility (Semmler et al , 1999), and biosurfactants for slow‐spreading growth of Serratia marcescens (Matsuyama et al , 1989) and P. aeruginosa (Nozawa et al , 2007). Thus, the involvement of these factors in the spreading growth of P. aeruginosa PAO1 and related cellular and environmental conditions were analyzed.…”

Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

“…However, others have shown that P. aeruginosa also swarms as an expanding circle [10], [11], [18], [19], [23], [24]. Differences in these varied reports for P. aeruginosa swarming seem to be explained by a variety of factors including strain effects, media composition, and surface hardness.…”

Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

“…Numerous studies have been published in recent years that address some aspect of P. aeruginosa swarming. P. aeruginosa is generally reported to swarm agar of between 0.4 and 0.7%, and the swarming of this bacterium is greatly influenced by the production of the surfactant rhamnolipid (9,15,29,34,41). Several recent studies have addressed the importance of swarm motility to the biofilm formation and antibiotic resistance of P. aeruginosa (8,32,38,41).…”