RhlR represses biofilm formation via KinB.

(A) Colony biofilm phenotypes of WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth. kinB::Tn refers to a mutant identified in a genetic screen harboring a transposon insertion in kinB. pkinB, prhlR, and palgB refer to kinB, rhlR, and algB, respectively, under the Plac promoter on pUCP18. Scale bar for all images is 2 mm. (B) Relative expression levels of rpoD and pelA measured by qRT-PCR in WT and ΔkinB mutant colony biofilms grown as in (A). The pelABCDEFG operon encodes proteins required for the production of the Pel polysaccharide, which is essential for the formation of pellicles, SSA biofilms, and colony biofilms [29]. (C) Phos-tag western blot analysis of the indicated strains probed for 3xFLAG-AlgB. (D) Relative algB transcript levels measured by qRT-PCR in WT PA14 and the ΔrhlR mutant grown planktonically to OD600 = 1.0. (E) Relative rhlR transcript levels measured by qRT-PCR in WT PA14 and the ΔkinB mutant grown planktonically to OD600 = 1.0. (F) Pyocyanin production (OD695) was measured in WT PA14 and the designated mutants. Production from the WT was set to 100%. (G) Relative expression of rpoD, hsiC2, hcnA, lecA, and lecB measured by qRT-PCR in WT PA14 and the designated mutants grown planktonically to OD600 = 1.0. rpoD is used as the control for comparison. For panels B, D, E, and G, data were normalized to 16S RNA levels, and the WT levels were set to 1.0. For data in panels B, D, E, F, and G, error bars represent SEM for 3 biological replicates. Data for panels B, D, E, F, and G can be found in supplemental file S1 Data. The original western blot showing the data for panel C is available in supplemental file S2 Data. AU, arbitrary unit; OD, optical density; qRT-PCR, quantitative Reverse Transcriptase-Polymerase Chain Reaction; SEM, standard error of the mean; SSA, solid-surface–associated; WT, wild type.</p

Far-red light intensity controls biofilm formation.

(A) Colony biofilm phenotypes are shown for WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth under the indicated far-red light intensities. Scale bar is 2 mm for all images. (B) Relative expression of rpoD (squares) and pelA (circles) measured by qRT-PCR in WT PA14 (black) and in the bphPSTOP mutant (green) grown as colony biofilms as in (A). Data were normalized to 16S RNA levels, and the WT levels at 0 mW/m2 far-red light were set to 1.0. Error bars represent SEM for 3 biological replicates. Data for panel B can be found in supplemental file S1 Data. AU, arbitrary unit; qRT-PCR, quantitative Reverse Transcriptase-Polymerase Chain Reaction; SEM, standard error of the mean; WT, wild type.</p

The BphP–AlgB phosphotransfer relay is conserved in diverse bacteria.

(A) Maximum likelihood based phylogenetic tree for BphP showing the 150 closest orthologs to P. aeruginosa BphP. Co-occurrences of AlgB and KinB are depicted in red and blue, respectively. BphR is shown in purple. The other colors indicate bacterial phyla. The black square indicates Arabidopsis thaliana as the root of the tree. (B) In vitro phosphorylation of AlgB orthologs from the α-Proteobacterium Rce, the β-Proteobacterium Axy, and the γ-Proteobacterium Ppu by P. aeruginosa BphP–BV that had been autophosphorylated for 30 min. The bottom panel shows that phosphotransfer from P. aeruginosa phospho-BphP-BV to P. aeruginosa NtrC does not occur. To assess the quality of the protein preparations used in panel B, see S4B Fig. The original autoradiographs with the data for panel B are available in supplemental file S2 Data. Axy, Achromobacter xylosocidans; BV, biliverdin; Ppu, P. putida; Rce, Rhodospirillum centenum.</p

Photosensing, via the BphP–AlgB phosphorelay, represses group behaviors in <i>P</i>. <i>aeruginosa</i>.

(A) Colony biofilm phenotypes are shown for WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth under the indicated light conditions. Scale bar is 2 mm for all images. (B) Autophosphorylation of the BphP–BV complex was carried out for 30 min (left lane in each pair), followed by addition of AlgB (right lane in each pair) for an additional 30 min under the indicated light conditions. (C) Relative expression of rpoD and pelA as measured by qRT-PCR in WT PA14 and the designated mutant strains grown as colony biofilms as in (A) in darkness, ambient light, and far-red light. (D) Relative expression of hsiC2, hcnA, lecA, and lecB measured by qRT-PCR in WT PA14 and the designated mutants grown as colony biofilms as in (A) and light conditions as in (B). For panels B and C, data were normalized to 16S RNA levels, and the WT levels were set to 1.0. Error bars represent SEM for 3 biological replicates. Data for panels C and D can be found in supplemental file S1 Data. The original autoradiograph with the data for panel B is available in supplemental file S2 Data. AU, arbitrary unit; BV, biliverdin; qRT-PCR, quantitative Reverse Transcriptase-Polymerase Chain Reaction; SEM, standard error of the mean; WT, wild type.</p

BphP and AlgB are a two-component HK–RR pair.

(A) Shown is a representative isolation of a suppressor mutation of the ΔkinB smooth colony biofilm phenotype. The white arrow in the left panel indicates a region of rugose sectoring in the ΔkinB smooth colony biofilm that is diagnostic of the emergence of a suppressor mutation. The right panel shows the colony biofilm phenotype of a mutant following isolation. (B) Chromosomal arrangements of the algB (red), kinB (blue), bphO (yellow), and bphP (green) genes. Large white arrows represent open reading frames (lengths not to scale), black bent arrows indicate promoters, and black circles indicate the locations of suppressor mutations. (C) Colony biofilm phenotypes of WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth. palgB refers to algB under the Plac promoter on the pUCP18 plasmid. Scale bar is 2 mm for all images. (D) Pyocyanin production (OD695) was measured in WT PA14 and the designated mutants. pbphP refers to bphP under the Plac promoter on the pBBR-MCS5 plasmid. Error bars represent SEM for 3 biological replicates. (E) Autophosphorylation of BphP–BV and phosphotransfer to AlgB. (Left) Autophosphorylation of BphP–BV was carried out for 30 min and samples were removed at the indicated times for electrophoresis. (Right) An equimolar amount of AlgB was added to phospho-BphP-BV for 30 min and samples were removed at the indicated times for electrophoresis. (F) Dephosphorylation of AlgB-P by KinB or KinBP390S. Phosphotransfer to AlgB from phospho-BphP-BV was carried out for 30 min. ATP was removed from the reaction, and either KinB or KinBP390S was added. Samples were removed at the indicated times for electrophoresis. The top panel shows representative images of gels. The bottom graph shows percent AlgB-P levels at each time point with SEM for 3 independent replicates. Band intensities for AlgB-P when KinB was added (circles) and when KinBP390S was added (squares) were normalized to the levels at time 0. To assess the quality of protein preparations used in panels E and F, see S4B Fig. Data for the graphs in panels D and F can be found in supplemental file S1 Data. The original autoradiographs with the data for panels E and F are available in supplemental file S2 Data. BV, biliverdin; OD, optical density; SEM, standard error of the mean; WT, wild type.</p

Model for <i>P</i>. <i>aeruginosa</i> integration of quorum-sensing and photosensing information into the control of virulence and biofilm development.

The RhlR quorum-sensing receptor binds its partner AI produced by either the RhlI or PqsE autoinducer synthase (black circles) at high cell density [22]. The RhlR–AI complex represses biofilm formation and virulence gene expression by activating transcription of the algB–kinB operon encoding the KinB HK and the AlgB RR, the latter an indirect repressor of biofilm formation. KinB antagonizes AlgB by dephosphorylation. The stimulus (blue hexagon) for KinB is unknown. In the presence of this putative stimulus, the hypothesis is that KinB functions as a kinase for AlgB (dashed arrow). Photosensing stimulates the BphP HK to autophosphorylate and subsequently transfer the phosphoryl group to AlgB to activate AlgB. AlgB-P activates transcription of genes required for repression of group behaviors such as biofilm formation and virulence. A “P” in a circle denotes addition or removal of a phosphate moiety (dotted line). X denotes that the genes functioning downstream of AlgB in the process are not known. The RhlR–AI complex directly activates virulence gene expression and also represses biofilm formation by additional unknown mechanisms (dashed T-bar). AI, autoinducer; HK, histidine kinase; RR, response regulator.</p

<i>Vibrio</i> and <i>Aeromonas</i> have distinct community architectures and biogeographies within the larval zebrafish intestine.

(A) A larval zebrafish at 5 dpf; the intestine is highlighted by phenol red dye via microgavage [27]. Scale bar: 500 μm. (B) A maximum intensity projection (MIP) of Vibrio in the larval intestine. Scale bar: 100 μm. (C) The probability density of Vibrio along the intestinal axis. From (B) and (C), we see that Vibrio is predominantly localized in the anterior bulb. (D) MIP of Aeromonas in the larval intestine. Scale bar: 100 μm. (E) The probability density of Aeromonas along the intestinal axis. (D) and (E) show that Aeromonas is predominantly localized in the midgut, with a smaller population in the anterior bulb. Underlying data for C and E are provided in S1 Data.</p

Populations of <i>Vibrio</i> and <i>Aeromonas</i> exhibit different dynamics within the zebrafish intestine.

<p>(A) An optical section of the intestinal bulb from a larval zebrafish mono-associated with <i>Vibrio</i> (the cyan box in the diagram below outlines the region imaged). The population consists of discrete, highly motile individuals (inset: single <i>Vibrio</i> cells). (B) A montage of images taken from the time-series in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s009" target="_blank">S3 Movie</a> shows that the highly motile and planktonic <i>Vibrio</i> cells maintain their overall distribution despite repeated intestinal contractions. Time between frames: 1 second. (C) An optical section of the intestinal midgut from a larval zebrafish mono-associated with <i>Aeromonas</i> (the magenta box in the diagram below outlines the region imaged). Cells are largely non-motile and densely aggregated. (D) A montage of images taken from the time-series in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s013" target="_blank">S7 Movie</a> shows an aggregate of <i>Aeromonas</i> in the midgut that is spatially dynamic, entering and exiting the field of view multiple times. Time between frames: 1 second. (A–D) Scale bars: 50 μm.</p

<i>Aeromonas</i> and <i>Vibrio</i> exhibit a competitive interaction within the larval zebrafish intestine.

<p>(A) Graphical overview of succession schemes used to characterize <i>Aeromonas</i>–<i>Vibrio</i> interactions. <i>Aeromonas</i> is allowed to colonize GF larvae at 4 dpf followed by addition of <i>Vibrio</i> to the water column at 5 or 6 dpf for 24 or 48 hr prior to enumeration of abundances by dissection and serial plating techniques. (B, left) <i>Aeromonas</i> abundances after different mono-association durations and (B, right) <i>Aeromonas</i> and <i>Vibrio</i> abundances after different <i>Vibrio</i> challenge periods. Statistical significance of <i>Aeromonas</i> abundances after <i>Vibrio</i> challenge compared to respective mono-association reference populations (i.e., 5–6 versus 4–6; 6–7 versus 4–7; 5–7 versus 4–7) was determined by an unpaired <i>t</i> test. (C) Time course analysis of <i>Aeromonas</i> and <i>Vibrio</i> abundances determined by dissection and plating at 3 hr intervals over a 12 hr period starting at 6 dpf. Additionally plotted are an <i>Aeromonas</i> mono-association reference population and 24 hr <i>Aeromonas</i> and <i>Vibrio</i> populations previously plotted in 1B (4–6 and 6–7, respectively). Statistical significance of <i>Aeromonas</i> abundances to the mono-association reference population (ref.) was determined by an unpaired <i>t</i> test. CFU = colony-forming units; *** = <i>p</i> < 0.0001; ns = not significant; <i>n</i> > 19/condition. Gray and black dashed lines in panels B and C denote limits of quantification and detection, respectively. Underlying data for B and C are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s001" target="_blank">S1 Data</a>.</p

<i>Aeromonas</i> experiences sharp drops in population size that are intensified during <i>Vibrio</i> challenge.

<p>(A) MIPs of <i>Aeromonas</i> (magenta) and <i>Vibrio</i> (cyan) in a larval zebrafish intestine. Scale bar: 200 μm. The fish was initially colonized at 4 dpf with <i>Aeromonas</i>, challenged 24 hr later by inoculation with <i>Vibrio</i>, and then imaged every 20 min for 14 hr. The times indicated denote hours post-challenge. In all images, the region shown spans about 80% of the intestine, with the anterior on the left. Image contrast in both color channels is enhanced for clarity. Yellow dotted line roughly indicates the lumenal boundary. As time progresses, the anterior growth of <i>Vibrio</i> as well as abrupt changes in the <i>Aeromonas</i> distribution (arrows) are evident. (B,C) Total bacterial abundance, derived from image data, for <i>Aeromonas</i> and <i>Vibrio</i> in two representative fish inoculated and challenged as in panel A, as a function of time following the <i>Vibrio</i> inoculation. Sharp drops of over an order of magnitude in the <i>Aeromonas</i> population, but not the <i>Vibrio</i> population, are evident. (D,E) Total abundance for <i>Aeromonas</i> in mono-associations as a function of time post-inoculation, in two representative fish. Sudden declines are also observed, though in general the populations recover to approximately pre-collapse levels. (F) The ratio, <i>f</i>, of the abundance immediately after to that before population drops, for <i>Aeromonas</i> challenged by <i>Vibrio</i>; this ratio spans many orders of magnitude (horizontal axis). At the same time points, the <i>Vibrio</i> populations are essentially unchanged, with ratios of populations afterward to before being close to one (vertical axis). (G) Characteristics of <i>Aeromonas</i> population collapses. Circles and bars indicate the mean and standard deviation, respectively, of <i>f</i> and <i>p</i>_c, the magnitude and rate of collapse occurrence, for both mono-associations and <i>Aeromonas</i> challenged by <i>Vibrio</i>. The dashed line at <i>f</i> = 0.1 indicates the threshold for identification of collapses. Underlying data for B–G are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s001" target="_blank">S1 Data</a>.</p