Figure 1.
Interactions between the YfiBNR proteins.
A) A model of yfiBNR interaction. YfiN is a membrane-localized DGC controlled by YfiR. YfiB, the outer-membrane bound Pal-like protein, activates YfiN by sequestering YfiR. B) Co-immunoprecipitation of YfiN with flag-tagged YfiN and YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum shows YfiN (lower band) co-precipitating with YfiN-flag (upper band) or YfiR-flag. C) Membrane localization of YfiB and YfiR. Immunoblots of fractionated membrane samples with anti-YfiB (upper panel) and M2 antisera (lower 2 panels). The left two panels show membrane fractions for PA01 yfiR-M2, the right panel for ΔyfiBN yfiR-M2. IM1/2: inner membrane fractions, OM1/2: outer membrane fractions.
Figure 2.
A) Locations of activating mutations in YfiN. Arrows indicate the positions of activating mutations throughout the structure of YfiN. TM: transmembrane helix. The cartoon is drawn to scale. B) Co-immunoprecipitation of active YfiN alleles with flag-tagged YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum. N-WT shows the DyfiNR screening strain with both yfiN (WT) and yfiR-flag plasmids present. Ctrl. shows ΔyfiNR with pGm-yfiprom-N only. The point mutation present in YfiN is indicated for the remaining lanes, which show YfiN immunoprecipitated from ΔyfiNR strains containing both yfiR-flag and the mutated yfiN plasmids. C) Attachment of the active YfiN alleles is shown relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (p-yfiN p-yfiR). Controls containing the yfiN or yfiR-flag plasmid only are also shown. The point mutation present in YfiN is indicated for each bar. Light grey bars indicate mutants whose activity was compensated for by mutations at the C-terminus of YfiR. Mutants with mid grey bars were compensated for by mutations in the signal sequence, or by uncharacterized mutations, while those with dark grey bars were not compensated for during YfiR mutagenesis. The domain locations of mutants are indicated with TM, PAS etc. D) Cartoon showing the locations of activating substitutions (blue) on a homology model of the YfiN PAS domain (residues 44–154). The PAS model is based on the CitA periplasmic domain (see Materials and methods). E) Surface representation of the YfiN PAS model. The locations of activating mutants on the proposed homodimer interface are shown in light blue, those at the possible YfiR binding site are shown in dark blue. F) The locations of activating substitutions (blue) on a homology model of the YfiN HAMP domain (residues 183–236). N and C termini are marked in D) and F). The HAMP model is based on the Aer2 HAMP structure (see Materials and methods).
Table 1.
YfiR-insensitive YfiN alleles.
Figure 3.
A) Immunoblots with M2 antiserum, showing levels of compensatory YfiR-flag variants in whole cell lysates. pyfiR: ΔyfiNR pMR-yfiR-flag, ΔyfiNR: strain without vector, pyfiN/pyfiR: ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag, lanes 1–13: ΔyfiNR pGm-yfiprom-N with compensatory pMR-yfiR-flag plasmids, proposed activating mutations are highlighted in bold. Mutants in lane 1, 4–8, 10–13 harbor mutations in the signal peptide that enhance expression, see also Table 2. B) Cartoon showing the locations of activating substitutions (green) on a homology model of YfiR (comprising residues 68–190). N and C termini are marked. The YfiR model is based on multiple structures (see Materials and methods). C) Surface representation of the YfiR model. The locations of activating mutants are shown in green, hydrophobic residues forming the possible YfiN binding surface are shown in dark blue.
Table 2.
Compensatory YfiR alleles.
Figure 4.
A) The effect of activating yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to PA01 (PA01 ctrl.). ‘YfiB WT’ indicates pME6032-yfiB. The point mutants in YfiB are indicated for the remaining lanes. Those mutations thought to contribute to YfiB activation are marked in bold. The immunoblot shows the levels of YfiB protein present in each strain. B) Left: Cartoon showing the locations of activating substitutions (red) on a homology model of YfiB (comprising residues 27–168). The YfiB model is based on the Omp/Pal structure (see Materials and methods for details). The N terminus and peptidoglycan binding site are marked. Right: Surface representation of the YfiB model. The locations of activating mutants are shown in red, hydrophobic residues forming the possible YfiR binding surface are shown in dark blue. C) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiR-flag containing pME6032-yfiB. ‘PG-’ indicates the same background strain containing pME-yfiB-PG- (YfiB containing the D102A and G105A substitutions), while ‘F48S’ contains the hyperactive yfiB F48S plasmid. D) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). ‘PG-’ mutants ± F48S or L43P mutations as indicated. In ‘LA-’ mutants, the lipid anchor is missing, the signal peptide has been replaced with that from YfiR. ‘ΔBNR LA-’ indicates the yfiB LA- mutant in the ΔyfiBNR strain background. The immunoblot shows the levels of YfiB protein present in each strain. E) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.
Figure 5.
Effect of YfiB linker mutants.
A) Sequence of the N-terminal YfiB ‘linker’, between the outer membrane (lipid anchor on the highlighted cysteine) and the Pal-like domain. The first activating mutant position (Figure 4) is shown in red. ‘short-linker’ indicates a deletion of 5 amino acids, leaving the lipobox intact. ‘long-linker’ indicates an insertion/duplication of 9 amino acids (dashed line). B) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiNR containing pME6032-yfiB. ‘short’/‘long’ indicate the strains containing pME6032-yfiB linker mutants. C) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). The immunoblot shows the levels of YfiB protein present in each strain. D) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.
Figure 6.
Reducing effects on yfiBNR activity.
A) Colony morphologies of yfiBNR/dsbA single and double mutants on LB Congo-red agar. Indication of the following abbreviations: ΔBNR – ΔyfiBNR, ΔB – ΔyfiBNR Tn7::yfiNR, ΔR – ΔyfiR, pR-flag – pMR-yfiR-flag, pdsbA – pME6032ara-dsbA, pME – pME6032ara. B) Immunoblots of PA01 and ΔdsbA with M2 and anti-YfiN antisera, showing levels of YfiR-flag and YfiN in whole cell lysates. C) The effect of increasing concentrations of DTT on attachment (bars), of the ΔBNR and ΔB mutants is shown relative to wild-type PA01. Curves represent relative optical density with standard errors.
Figure 7.
A) Colony morphologies of ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag strains with the point mutants indicated present in either YfiR or YfiN. ‘WT’: both plasmids contain wild-type yfiN/yfiR alleles. All colony morphologies are on LB Congo-red agar. B) Colony morphologies of Clin110 and Clin163 strains. pR indicates that the strain contains pME-araC-yfiR, the asterisk indicates induction. C) Colony morphologies of ΔyfiBNR strains complemented with the yfiBNR operon inserted into the att-Tn7 site. Mutations in the complementing copy of yfiN are indicated in each case. ‘WT’: wild type yfiN. D) Attachment relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (pNpR) of clinical yfiN/yfiR mutants. ‘pR’ and ‘pN’ contain pMR-yfiR-flag or pGm-yfiprom-N only. E) Attachment of Clin110 ± pME-araC-yfiR and Clin163 relative to PA01.
Figure 8.
In silico analysis of the YfiBNR system.
A) Lineage tree illustrating the distribution of the yfiBNR genes along bacterial lineages. Individual branches indicate separate genera. Genera marked in large, bold type contain species with complete yfiBNR operons. Those marked with an asterisk (*) contain species with yfiB and yfiN only, conserved and in synteny. The remaining genera contain species with two yfi genes conserved and in synteny (usually yfiN and yfiR) and a third yfi homolog elsewhere in the genome. Bacterial classes are indicated with coloured shading. Alpha, Beta etc. refer to the respective proteobacterial class. B-D) Weblogo representations of YfiBNR residue conservation. The height of the letter in each case indicates the degree of conservation at that position. Hydrophobic residues are coloured blue, hydrophilic residues red. Asterisks (*) indicate the sites of activating mutations, while blue underlining indicates those residues suggested to contribute to a hydrophobic protein binding site. B) YfiB residues 33–72. C) YfiN PAS-domain residues 48–87, YfiN HAMP-domain residues 198–237. D) YfiR residues 122–191.