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. 2022 May 25;24(8):3672–3692. doi: 10.1111/1462-2920.16008

Fig. 7.

Fig. 7

Amoeba‐adherent L. pneumophila clusters express motility and virulence genes.

A. Biofilms of L. pneumophila JR32 harbouring promoter‐reporter plasmids P flaA ‐gfp (pCM009), P 6SRNA ‐gfp (pRH049), P sidC ‐gfp (pRH035), P ralF ‐gfp (pRH032) or P csrA ‐gfp (pRH031) were grown in AYE medium for 6 days. Acanthamoeba castellanii was added to preformed biofilms and confocal microscopy images of amoebae with adherent bacterial clusters were acquired close to the bottom of microscopy dishes. The images shown are representative of at least two independent experiments. Scale bars, 30 μm. Quantification by flow cytometry of GFP‐positive L. pneumophila JR32 harbouring promoter reporters (P flaA , P 6SRNA , P sidC , P ralF , or P csrA ) using FlowJo software: (B) forward scatter area (FCS‐A) versus fluorescence intensity, or (C) counts versus fluorescence intensity [amoebae‐associated (red) and non‐associated (black)] (protocol #2, see Materials and Methods).