Table 1.
Mutants from Colony Morphology Screens.
Figure 1.
Analysis of surface behaviors to determine efficacy of Varpa_4680 (wbuB) and Varpa_5900 (pilY1) complementation.
Biofilm levels after 24 h incubation and swarm diameters at 48 h for wt and mutant strains expressing either wbuB (A, B) or pilY1 (C, D) in trans compared to vector controls. 86 = Varpa_4680::Tn5, 223 = Varpa_5900::Tn5. Error was computed as +/−SEM. All p-values were calculated using the student's unpaired T-test. For all panels a = p<0.01 compared to wt+vec, b = p<0.01 compared to 86+vec (A,B) or 223+vec (C,D).
Figure 2.
Swarm edges at 24 h on 0.5% FW plates+25 µg/ml Km were photographed using a 10× phase contrast lens (scale bar = 10 microns).
A) wt+vec, B) wt+wbuB, C) mut86+vec, D) mut86+wbuB.
Figure 3.
Swarms of V. paradoxus EPS imaged using a stereomicroscope.
(A) wt+vec, (B) wt+pilY1, (C) mut223+vec, (D) mut223+pilY1.
Figure 4.
Expression of wbuB (grey bars) and pilY1(black bars) assessed directly by qPCR.
Planktonic cells, biofilm cells, and plate cultures from 0.5% agarose solidified YE plates or FW swarming plates were harvested at 48 h of growth and compared to aerated liquid culture in log phase (19 h) or stationary phase (26 h). RNA levels were determined in comparison to a luciferase spike added to each sample, and fold expression relative to log phase was assessed using the Pfaffl method.
Figure 5.
Analysis of surface behaviors to examine complementation of Varpa_4664 (shkS).
Biofilm levels after 24 h incubation (A) and swarm diameters (B) at 48 h for wt and mut99 expressing either shkS or shkR in trans compared to vector controls. Error was computed as +/−SEM. P-values were calculated using the student's unpaired T-test. For both panels, a = p<0.01 compared to wt+vec, b = p<0.01 compared to mut99+vec.
Figure 6.
Phylogenetic and nucleotide composition suggests Varpa_4680 entered the V. paradoxus EPS genome by horizontal transfer.
A) Phylogenetic tree based on WbuB amino acid sequences created using ClustalW multiple sequence alignment and subsequent Bayesian inference of phylogeny(Mr. Bayes). B) Phylogenetic tree using the 16s rDNA sequences from the same set of organisms as in (A), using Bayesian inference of phylogeny based on the HKY85 nucleotide substitution model. For both A) and B) Vp EPS = Variovorax paradoxus EPS, Pn = Polaromonas naphthlenivorans CJ2, Bp = Bordatella pertussis Tohama 1, Pao1 = Pseudomonas aeruginosa PAO1, Cv = Chromobacterium violaceum ATCC 12472, Sd = Sulfurimonas denitrificans DSM 1251 C) G/C analysis of 35 kb region of the V. paradoxus EPS genome including wbuB. Total region spans orfs Varpa_4661-4691. Low G/C region from 18.4 kb to 27.1 kb spans orfs Varpa_4679-4684.
Table 2.
Strains, Vectors, Primers.
Figure 7.
Conservation of putative active sites in three pilY1 homologs in V. paradoxus EPS.
ClustalW alignment of PilY1 around (A) the N-terminal conserved site previously described in [29] and (B) the C-terminal Ca2+-binding site [27]. Varpa_5900 and its ortholog Vapar_5179 in V. paradoxus S110 are outlined in red, and the conserved functional sites in each region are outlined in black. Ne = Nitrosomonas europaea ATCC 19718 NE_1747,Thio = Thioalkalivibrio sulfidophilus HL-EbGr7 Tgr7_3225, Pm ymp = Pseudomonas mendocina ymp Pmen_0958, PAO1 = Pseudomonas aeruginosa PAO1 PA4554, PAK = Pseudomonas aeruginosa PAK PilY1.