1. Microbiology and Infectious Disease
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An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

Research Article
Cite this article as: eLife 2017;6:e30205 doi: 10.7554/eLife.30205
6 figures, 5 tables and 1 additional file

Figures

The respiratory chain and arrangement of cco genes and protein products in P. aeruginosa, and the phylogenetic distribution of orphan ccoN genes.

(A) Branched electron transport chain in P. aeruginosa, containing five canonical terminal oxidases. (B) Organization of cco genes in the P. aeruginosa genome. The cartoon of the Cco complex is based on the Cco structure from P. stutzeri (PDB: 3mk7) (Buschmann et al., 2010). (C) Left: graphical representation of the portion of genomes in each bacterial phylum that contain ccoO and N homologs. The clades Chrysiogenetes, Gemmatimonadetes, and Zetaproteobacteria were omitted because they each contain only one species with ccoO and N homologs. The height of each rectangle indicates the total number of genomes included in the analysis. The width of each shaded rectangle represents the portion of genomes that contain ccoN homologs. Middle: genomes that contain more ccoN than ccoO homologs (indicating the presence of orphan ccoN genes) are listed. Right: numbers of ccoO and ccoN homologs in each genome. Blue highlights genomes containing more than one orphan ccoN homolog.

https://doi.org/10.7554/eLife.30205.003
Figure 2 with 4 supplements
CcoN4-containing heterocomplexes make biofilm-specific contributions to morphogenesis and respiration.

(A) Top: Five-day-old colony biofilms of PA14 WT and cco mutant strains. Biofilm morphologies are representative of more than 10 biological replicates. Images were generated using a digital microscope. Scale bar is 1 cm. Bottom: 3D surface images of the biofilms shown in the top panel. Images were generated using a wide-area 3D measurement system. Height scale bar: bottom (blue) to top (red) is 0–0.7 mm for WT, ∆N1N2, and ∆N4; 0–1.5 mm for ∆N1N2N4 and ∆cco1cco2. (B) TTC reduction by WT and cco mutant colonies after 1 day of growth. Upon reduction, TTC undergoes an irreversible color change from colorless to red. Bars represent the average, and error bars represent the standard deviation, of individually-plotted biological replicates (n = 5). p-Values were calculated using unpaired, two-tailed t tests comparing each mutant to WT (****p≤0.0001). (C) Mean growth of PA14 WT and cco mutant strains in MOPS defined medium with 20 mM succinate. Error bars represent the standard deviation of biological triplicates.

https://doi.org/10.7554/eLife.30205.004
Figure 2—figure supplement 1
Effects of individual and combined cco gene deletions on colony biofilm morphogenesis.

(A) Morphologies of WT, ∆phz, and cco single, combinatorial, and ccoN4 complementation strains after 3 and 5 days of incubation. Images shown are representative of at least 5 biological replicates and were generated using a digital microscope. Scale bar is 1 cm. (B) Development of WT, ∆N4 and N subunit double mutants containing ∆N4. Images shown are representative of at least 3 biological replicates and were generated using a digital microscope. Scale bar is 1 cm. (C) Development of WT and the triple mutant ∆coxcyocio in which only the cbb3-type terminal oxidases are present. Images were generated using a flatbed scanner and are representative of at least 3 biological replicates. Scale bar is 1 cm.

https://doi.org/10.7554/eLife.30205.005
Figure 2—figure supplement 2
PA14 WT, ∆phz, and cco mutant growth phenotypes are unaffected by endogenous cyanide production.

(A) Colony development over 4 days for ∆phz, ∆hcnABC, and cco combinatorial mutants. Images were generated using a flatbed scanner and are representative of at least 3 biological replicates. Scale bar is 1 cm. (B) Growth of ∆phz, ∆hcnABC, and cco combinatorial mutants in MOPS defined medium with 20 mM succinate. Error bars represent the standard deviation of biological triplicates and are not shown in cases where they would be obscured by the point marker.

https://doi.org/10.7554/eLife.30205.006
Figure 2—figure supplement 3
Pseudomonads with CcoN homologs.

We examined genomes available in the Pseudomonas Genome Database (Winsor et al., 2016) for CcoN homologs by performing a protein BLAST search on CcoN1 from P. aeruginosa PA14. All hits from full genomes, excluding other P. aeruginosa strains, were aligned using ClustalW and a tree was built using the geneious tree builder (Geneious 10 (Kearse et al., 2012)). We also included draft genomes that contained genes involved in phenazine biosynthesis (highlighted in purple). The tree revealed four clusters, each being more closely related to one of the four N subunits from PA14, which allowed us to annotate the N subunits accordingly. We next probed all genomes with N subunits for the presence of genes involved in cyanide synthesis (hcnABC) and phenazine biosynthesis (phzABCDEFG). We did not find a clear correlation between the presence of CcoN4 and Hcn proteins (Hirai et al., 2016). We note that with the exception of two P. fluorescens strains, those containing phzABCDEFG operons also contained ccoN4.

https://doi.org/10.7554/eLife.30205.007
Figure 2—figure supplement 4
Comparison of the PA14 CcoN subunit sequences and analysis of the predicted structure of CcoN4.

(A) Amino acid alignment (ClustalW) of the four CcoN subunits encoded by the PA14 genome. Residues conserved among all four N subunits are highlighted in black; residues conserved among any three of the four N subunits in gray; residues shared exclusively between CcoN1 and CcoN4 in yellow; and residues unique to CcoN4 in purple. (B) Predicted structure of CcoN4 from P. aeruginosa PA14, obtained by threading the PA14 sequence through the reported structure for the CcoN subunit of P. stutzeri (PDB: 5DJQ; Buschmann et al., 2010) using SWISS-MODELL (Biasini et al., 2014). Surface-exposed residues that are shared exclusively between CcoN1 and CcoN4 are shown in yellow, while residues that are unique to CcoN4 are shown in magenta. Ribbon structures of the CcoO and CcoP subunits from P. stutzeri are shown in red and green, respectively. Structures were generated using PyMol (Schrödinger, LLC, 2015).

https://doi.org/10.7554/eLife.30205.008
Figure 3 with 1 supplement
CcoN4 confers a competitive advantage in biofilms, particularly when O2 becomes limiting.

(A) Relative fitness of various YFP-labeled cco mutants when co-cultured with WT in mixed-strain biofilms for 3 days. Error bars represent the standard deviation of biological triplicates. p-Values were calculated using unpaired, two-tailed t tests (**p≤0.01; ***p≤0.001; ****p≤0.0001). (B) Time course showing relative fitness, over a period of 3 days, of various cco mutants when co-cultured with WT in mixed-strain biofilms. Results are shown for experiments in which the WT was co-cultured with various ‘labeled’ strains, that is, those that were engineered to constitutively express YFP. (See Figure 3—figure supplement 1 for results from experiments in which the labeled WT was co-cultured with unlabeled mutants.) Error bars represent the standard deviation of biological triplicates. (C) Change in thickness over 3 days of development for colony biofilms of WT and ∆phz as assessed by thin sectioning and DIC microscopy. After the onset of wrinkling, thickness was determined for the base (i.e. the ‘valley’ between wrinkles). Error bars represent the standard deviation of biological triplicates. (D) O2 profiles of colonies at selected timepoints within the first 3 days of biofilm development. Gray point markers indicate measurements made in the agar directly below the colony. Error bars denote standard deviation of biological triplicates.

https://doi.org/10.7554/eLife.30205.009
Figure 3—figure supplement 1
CcoN4 is necessary for optimal fitness in biofilms, particularly when O2 becomes limiting.

(A) Relative fitness of YFP-labeled WT when co-cultured with various cco mutant strains in mixed-strain biofilms for 3 days. Error bars represent the standard deviation of biological triplicates. p-Values were calculated using unpaired, two-tailed t tests (***p≤0.001; ****p≤0.0001). (B) Time course showing relative fitness, over a period of 3 days, of YFP-labeled WT when co-cultured with various cco mutant strains in mixed-strain biofilms. Error bars represent the standard deviation of biological triplicates. (C) DIC image of a 3-day-old WT biofilm, which is representative of at least 10 biological replicates.

https://doi.org/10.7554/eLife.30205.010
Figure 4 with 1 supplement
cco genes are differentially expressed over biofilm depth.

Left: Representative images of thin sections prepared from WT biofilms grown for 3 days. Each biofilm is expressing a translational GFP reporter under the control of the cco1, cco2, or ccoN4Q4 promoter. Reporter fluorescence is shown in green and overlain on respective DIC images. Right: Fluorescence values corresponding to images on the left. Fluorescence values for a strain containing the gfp gene without a promoter (the empty MCS control) have been subtracted from each respective plot. O2 concentration over depth (open circles) from 3-day-old WT biofilms is also shown. Error bars represent the standard deviation of biological triplicates and are not shown in cases where they would be obscured by the point markers. y-axis in the right panel provides a scale bar for the left panel. Reporter fluorescence images and values are representative of 4 biological replicates.

https://doi.org/10.7554/eLife.30205.011
Figure 4—figure supplement 1
Expression of cco reporters in shaken liquid cultures.

(A) Fluorescence of translational reporter strains, engineered to express GFP under the control of the cco1, cco2, or ccoN4Q4 promoter during growth in 1% tryptone. Fluorescence values for a strain containing the gfp gene without a promoter (the MCS control) were treated as background and subtracted from each growth curve. (B) Liquid-culture growth of translational reporter strains in 1% tryptone. Error bars in (A) and (B) represent the standard deviation of biological triplicates and are not drawn in cases where they would be obscured by point markers.

https://doi.org/10.7554/eLife.30205.012
Figure 5 with 1 supplement
Characterization of chemical gradients and matrix distribution in PA14 WT and mutant colony biofilms.

(A) Left: Change in O2 concentration (blue) and redox potential (orange) with depth for WT and ∆phz biofilms grown for two days. WT biofilms are ~150 µm thick while ∆phz biofilms are ~80 µm thick. For O2 profiles, error bars represent the standard deviation of biological triplicates. For redox profiles, data are representative of at least 5 biological replicates. Right: model depicting the distribution of O2 and reduced vs. oxidized phenazines in biofilms. (B) Top: Change in redox potential with depth for WT and various mutant biofilms grown for 2 days. Data are representative of at least 5 biological replicates. Bottom: Thickness of 3-day-old colony biofilms of the indicated strains. Bars represent the average of the plotted data points (each point representing a biological replicate, n ≥ 4), and error bars represent the standard deviation. p-Values were calculated using unpaired, two-tailed t tests comparing each mutant to WT (n.s., not significant; **p≤0.01; ****p≤0.0001). (C) Left: Representative thin sections of WT and cco mutant biofilms, stained with lectin and imaged by fluorescence microscopy. Biofilms were grown for 2 days before sampling. Right: Relative quantification of lectin stain signal intensity. Coloration of strain names in the left panel provides a key for the plotted data, and the y-axis in the right panel provides a scale bar for the left panel. Lectin-staining images and values are representative of 4 biological replicates.

https://doi.org/10.7554/eLife.30205.013
Figure 5—figure supplement 1
Use of a redox microelectrode to measure phenazine reduction in colony biofilms.

(A) Change in redox potential over depth for 2-day-old biofilms of PA14 WT, ∆phz, and ∆phz grown on 200 µM phenazine methosulfate (PMS). Data are representative of at least 3 biological replicates. To ensure that addition of PMS did not alter the baseline redox potential, a measurement was also taken of agar only. (B) Change in redox potential with depth for WT, ∆phz, and ∆coxcyocio biofilms grown for 2 days. Data are representative of at least 2 biological replicates. (C) Levels of phenazines extracted from the agar medium underneath the colony and separated by HPLC, adjusted for biomass, for PA14 WT and various cco mutant biofilms grown for 2 days. Data represent the area under each peak in absorbance units for the phenazines indicated, and error bars represent standard deviation of at least 3 biological replicates. The phenazines pyocyanin (PYO), phenazine-1-carboxamide (PCN), and phenazine-1-carboxylic acid (PCA) were quantified. (D) Colony biofilm morphologies on day 4 of development for WT and various cco mutant biofilms grown on colony morphology plates containing 0, 10, and 40 mM potassium nitrate. Images were generated using a flatbed scanner and are representative of at least 3 biological replicates. Scale bar is 1 cm.

https://doi.org/10.7554/eLife.30205.014
CcoN4-containing isoform(s) make unique contributions to PA14 virulence.

Slow-killing kinetics of WT, gacA, and various cco mutant strains in the nematode Caenorhabditis elegans. Nearly 100% of the C. elegans population exposed to WT PA14 is killed after 4 days of exposure to the bacterium, while a mutant lacking GacA, a regulator that controls expression of virulence genes in P. aeruginosa, shows decreased killing, with ~50% of worms alive 4 days post-exposure. (A) ∆N1∆N2∆N4 and ∆cco1cco2 show comparably attenuated pathogenicity relative to WT. Error bars represent the standard deviation of at least 6 biological replicates. At 2.25 days post-exposure, significantly less C. elegans were killed by ∆N1N2N4 than by WT (unpaired two-tailed t test; p=0.0022). (B) ∆N1∆N2 displays only slightly reduced pathogenicity when compared to WT. At 2.25 days post-exposure, significantly more C. elegans were killed by ∆N1N2 than by ∆N1N2N4 (unpaired two-tailed t test; p=0.003). Error bars represent the standard deviation of at least 4 biological replicates, each with a starting sample size of 30–35 worms per replicate.

https://doi.org/10.7554/eLife.30205.015

Tables

Key resource table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
strain, strain background
(UCBPP-PA14
Pseudomonas aeruginosa)
wild type (WT)PMID: 7604262
strain, strain background
(UCBPP-PA14 P. aeruginosa)
phzPMID: 16879411LD24
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1; ∆N1this studyLD1784
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN2; ∆N2this studyLD1614
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN3; ∆N3this studyLD1620
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN4; ∆N4this studyLD2833
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2; ∆N1N2this studyLD1888
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN4; ∆N1∆N4this studyLD1951
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN2ccoN4; ∆N2∆N4this studyLD1692
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN3ccoN4; ∆N3∆N4this studyLD1649
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2ccoN3;
∆N1∆N2N3
this studyLD1977
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2ccoN4;
∆N1∆N2∆N4
this studyLD1976
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2ccoN4
ccoN3; ∆N1∆N2∆N4N3
this studyLD2020
strain, strain background
(UCBPP-PA14 P. aeruginosa)
cco1cco2this studyLD1933
strain, strain background
(UCBPP-PA14 P. aeruginosa)
coxcyociothis studyLD2587
strain, strain background
(UCBPP-PA14 P. aeruginosa)
hcnthis studyLD2827
strain, strain background
(UCBPP-PA14 P. aeruginosa)
phzhcnthis studyLD2828
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN4hcn; ∆N4∆hcnthis studyLD2829
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2hcn;
∆N1∆N2∆hcn
this studyLD2830
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2ccoN4
hcn; ∆N1∆N2∆N4hcn
this studyLD2831
strain, strain background
(UCBPP-PA14 P. aeruginosa)
cco1cco2hcnthis studyLD2832
strain, strain background
(UCBPP-PA14 P. aeruginosa)
gacA::TnPMID: 16477005LD1560
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN4::ccoN4; ∆N4::N4this studyLD1867
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2ccoN4::
ccoN4; ∆N1∆N2∆N4::N4
this studyLD2576
strain, strain background
(UCBPP-PA14 P. aeruginosa)
MCS-gfpthis studyLD2820
strain, strain background
(UCBPP-PA14 P. aeruginosa)
Pcco1-gfp; cco1Pr-gfpthis studyLD2784
strain, strain background
(UCBPP-PA14 P. aeruginosa)
Pcco2-gfp; cco2Pr-gfpthis studyLD2786
strain, strain background
(UCBPP-PA14 P. aeruginosa)
PccoN4-gfp; ccoN4Pr-gfpthis studyLD2788
strain, strain background
(UCBPP-PA14 P. aeruginosa)
PA14-yfpthis studyLD2780
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2-yfp;
N1N2-yfp
this studyLD2013
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN4-yfp; ∆N4-yfpthis studyLD2834
strain, strain background
(UCBPP-PA14 P. aeruginosa)
ccoN1ccoN2cco
N4-yfp; ∆N1∆N2N4-yfp
this studyLD2136
strain, strain background
(UCBPP-PA14 P. aeruginosa)
cco1cco2-yfpthis studyLD2012
strain, strain background
(Escherichia coli)
UQ950otherFrom D. Lies, Caltech; LD44
strain, strain background
(E. coli)
BW29427otherFrom W. Metcalf, University of Illinois; LD661
strain, strain background
(E. coli)
β2155PMID: 8990308LD69
strain, strain background
(E. coli)
S17-1doi:10.1038/nbt1183-784LD2901
strain, strain background
(Saccharomyces cerevisiae)
InvSc1InvitrogenLD676
recombinant DNA reagentpMQ30 (plasmid)PMID: 16820502For generation of deletion constructs listed above;
further information can be found in the
Materials and Methods section.
recombinant DNA reagentpAKN69 (plasmid)PMID: 15186351For generation of strains constitutively
expression eyfp; further information can be
found in the Materials and Methods section.
recombinant DNA reagentpLD2722 (plasmid)this studyFor generation of gfp reporter constructs;
further information can be found in the
Materials and Methods section.
recombinant DNA reagentpFLP2 (plasmid)PMID: 9661666For generation of gfp reporter constructs;
further information can be found in the
Materials and Methods section.
software, algorithmEggNOG DatabasePMID: 26582926http://eggnogdb.embl.de/#/app/home
software, algorithmSensorTrace ProfilingUnisenseFor data acquisition for redox and oxygen
microprofiling; further information can be
found in the Materials and Methods section.
otherAgarTeknovaFor colony morphology assays;
further information can be found in the
Materials and Methods section.
otherLectinVector LaboratoriesFor visualization of matrix;
further information can be found
in the Materials and Methods section.
Table 1
Strains used in this study.
https://doi.org/10.7554/eLife.30205.016
StrainNumberDescriptionSource
Pseudomonas aeruginosa strains
UCBPP-PA14Clinical isolate UCBPP-PA14.Rahme et al. (1995)
PA14 ∆phzLD24PA14 with deletions in phzA1-G1 and phzA2-G2
operons.
Dietrich et al., 2006a
PA14 ∆ccoN1LD1784PA14 with deletion in PA14_44370.this study
PA14 ∆ccoN2LD1614PA14 with deletion in PA14_44340.this study
PA14 ∆ccoN3LD1620PA14 with deletion in PA14_40510.this study
PA14 ∆ccoN4LD2833PA14 with deletion in PA14_10500.this study
PA14 ∆ccoN1
ccoN2
LD1888PA14 with deletions in PA14_44370 and PA14_44340. Made by mating pLD1610 into LD1784.this study
PA14 ∆ccoN1
ccoN4
LD1951PA14 with deletions in PA14_44370 and PA14_10500. Made by mating pLD1264 into LD1784.this study
PA14 ∆ccoN2
ccoN4
LD1692PA14 with deletions in PA14_44340 and PA14_10500. Made by mating pLD1264 into LD1614.this study
PA14 ∆ccoN3
ccoN4
LD1649PA14 with deletions in PA14_40510 and PA14_10500. Made by mating pLD1264 into LD1620.this study
PA14 ∆ccoN1
ccoN2ccoN3
LD1977PA14 with deletions in PA14_443470, PA14_44340, and PA14_40510. Made by mating pLD1616 into LD1888.this study
PA14 ∆ccoN1
ccoN2ccoN4
LD1976PA14 with deletions in PA14_443470, PA14_44340, and PA14_10500. Made by mating pLD1264 into LD1888.this study
PA14 ∆ccoN1
ccoN2ccoN4
ccoN3
LD2020PA14 with deletions in PA14_443470, PA14_44340, PA14_10500, and PA14_40510. Made by mating pLD1264 into LD1977.this study
PA14 ∆cco1cco2LD1933PA14 with both cco operons (PA14_44340-PA14_44400) deleted simultaneously.this study
PA14 ∆coxcyocioLD2587PA14 with deletions in PA14_01290–01320 (cox/aa3 operon), PA14_47150–47210 (cyo/bo3 operon), and PA14_13030–13040 (cio operon). Made by mating pLD1966, pLD1967, and pLD2044, in that order, to PA14.this study
PA14 ∆hcnLD2827PA14 with deletion in hcnABC operon (PA14_36310–36330).this study
PA14 ∆phzhcnLD2828PA14 with deletions in phzA1-G1, phzA2-G2, and hcnABC operons. Made by mating pLD2791 into LD24.this study
PA14 ∆ccoN4hcnLD2829PA14 with deletions in PA14_10500 and hcnABC operon. Made by mating pLD2791 into LD2833.this study
PA14 ∆ccoN1
ccoN2hcn
LD2830PA14 with deletions in PA14_44370, PA14_44340, and hcnABC operon. Made by mating pLD2791 into LD1888.this study
PA14 ∆ccoN1
ccoN2ccoN4hcn
LD2831PA14 with deletions in PA14_44370, PA14_44340, PA14_10500 and hcnABC operon. Made by mating pLD2791 into LD1976.this study
Pseudomonas aeruginosa strains
PA14 ∆cco1cco2
hcn
LD2832PA14 with deletions in cco1, cco2, and hcnABC operons. Made by mating pLD2791 into LD1933.this study
PA14 gacA::TnLD1560MAR2xT7 transposon insertion into PA14_30650.Liberati et al. (2006)
PA14 ∆ccoN4::ccoN4LD1867PA14 ∆ccoN4 strain with wild-type ccoN4 complemented back into the site of deletion. Made by mating pLD1853 into LD2833.this study
PA14 ∆ccoN1
ccoN2
ccoN4::ccoN4
LD2576PA14 ∆ccoN1ccoN2ccoN4 strain with wild-type ccoN4 complemented back into the site of deletion. Made by mating pLD1853 into LD1976.this study
PA14 MCS-gfpLD2820PA14 without a promoter driving gfp expression.this study
PA14 Pcco-1-gfpLD2784PA14 with promoter of cco1 operon driving gfp expression.this study
PA14 Pcco-2-gfpLD2786PA14 with promoter of cco2 operon driving gfp expression.this study
PA14 PccoN4-gfpLD2788PA14 with promoter of ccoN4Q4 operon driving gfp expression.this study
PA14-yfpLD2780WT PA14 constitutively expressing eyfp.this study
PA14 ∆ccoN1
ccoN2-yfp
LD2013PA14 ∆ccoN1ccoN2 constitutively expressing eyfp. Made by mating pAKN69 into LD1888.this study
PA14 ∆ccoN4-yfpLD2834PA14 ∆ccoN4 constitutively expressing eyfp. Made by mating pAKN69 into LD2833.this study
PA14 ∆ccoN1
ccoN2ccoN4-yfp
LD2136PA14 ∆ccoN1ccoN2ccoN4 constitutively expressing eyfp. Made by mating pAKN69 into LD1976.this study
PA14 ∆cco1cco2-yfpLD2012PA14 ∆cco1cco2 constitutively expressing eyfp. Made by mating pAKN69 into LD1933.this study
Escherichia coli strains
UQ950LD44E. coli DH5 λpir strain for cloning. F-∆(argF- lac) 169φ80 dlacZ58(∆M15) glnV44(AS) rfbD1 gyrA96(NaIR) recA1 endA1 spoT thi-1 hsdR17 deoR λpir+D. Lies, Caltech
BW29427LD661Donor strain for conjugation. thrB1004 pro thi rpsL hsdS lacZ ∆M15RP4-1360 ∆(araBAD)567
∆dapA1314::[erm pir(wt)]
W. Metcalf, University of Illinois
β2155LD69Helper strain. thrB1004 pro thi strA hsdsS lacZ∆M15 (F’lacZ∆M15 lacIq traD36 proA + proB + ) ∆dapA::erm (Ermr)pir::RP4 [::kan (Kmr) from SM10]Dehio and Meyer (1997)
S17-1LD2901StrR, TpR, F− RP4-2-Tc::Mu aphA::Tn7 recA λpir lysogenSimon et al. (1983)
Saccharomyces cerevisiae strains
InvSc1LD676MATa/MATalpha leu2/leu2 trp1-289/trp1-289 ura3−52/ura3-52 his3-∆1/his3-∆1Invitrogen
Table 2
Primers used in this study.
https://doi.org/10.7554/eLife.30205.017
Primer numberSequenceused to make plasmid number
LD717ccaggcaaattctgttttatcagaccgcttctgcgttctgatCAGGACAAGCAGTGGGAACpLD1852
LD718aggtgttgtaggccatcagcTGGCGGACCACCTTATAGTT
LD958aactataaggtggtccgccaCGGTGGTTTCTTCCTCACC
LD959ggaattgtgagcggataacaatttcacacaggaaacagctGGTCCAGCCTTTTTCCTTGT
LD725ccaggcaaattctgttttatcagaccgcttctgcgttctgatCCCCTCAGAGAAGTCAGTCGpLD1610
LD726aggtgttgtaggccatcaggGGCGGACCACCTTGTAGTTA
LD727taactacaaggtggtccgccCCTGATGGCCTACAACACCT
LD728ggaattgtgagcggataacaatttcacacaggaaacagctCAGCGGGTTGTCATACTCCT
LD741ccaggcaaattctgttttatcagaccgcttctgcgttctgatTCGAGGGCTTCGAGAAGATpLD1616
LD742aggtgttgtaggccatcagcCAGGGTCATCAGGGTGAACT
LD743agttcaccctgatgaccctgGCTGATGGCCTACAACACCT
LD744ggaattgtgagcggataacaatttcacacaggaaacagctCGGGTGATGTCGACGTATTC
LD438ggaattgtgagcggataacaatttcacacaggaaacagctCCGTTGATTTCCTTCTGCATpLD1264 (LD438 - LD441)
pLD1853 (LD438 and LD441)
LD439ctacaaggtggttcgccagtCGCTGACCTACTCCTTCGTC
LD440gacgaaggagtaggtcagcgACTGGCGAACCACCTTGTAG
LD441ccaggcaaattctgttttatcagaccgcttctgcgttctgatCATCGACCTGGAAGTGCTC
LD725ccaggcaaattctgttttatcagaccgcttctgcgttctgatCCCCTCAGAGAAGTCAGTCGpLD1929
LD1063gttgcccaggtgttcctgtGGCGGACCACCTTGTAGTTA
LD949ggaattgtgagcggataacaatttcacacaggaaacagctTGTAGTCGAGGGACTTCTTGC
LD1064taactacaaggtggtccgccACAGGAACACCTGGGCAAC
LD2168ccaggcaaattctgttttatcagaccgcttctgcgttctgatATGTAGGGATCGAGCGACAGpLD2791
LD2169acacgatatccagcccctctTGGACATCGCGCCGTTCCTC
LD2170gaggaacggcgcgatgtccaAGAGGGGCTGGATATCGTGT
LD2171ggaattgtgagcggataacaatttcacacaggaaacagctAAGAGGTCATAATCGGCGGT
LD2120gattcgacatcactagtACGCCCAGCTCCAACAAApLD2777
LD2121gattcgatgccctcgaGCTAGGGGTTCCACGGTTAAT
LD2122gattcgactgcactagtCATCGACTTGCCGCCCAGpLD2778
LD2123g attcg atg ccctcgaGCTATGGGCTTCCATC CAC
LD2124gattcgactgcactagtGGCTACTTCCTCTGGCTGGpLD2779
LD2125gattcgactgcctcgagCTGTACAGTCCCGAAAGAAATGAAC
LD1118ccaggcaaattctgttttatcagaccgcttctgcgttctgatTCTTCAGGTTCTCGCGGTAGpLD1966
LD1119aagtgccagtaccaactggcGCAGATCCAGAAGATGGTCA
LD1120tgaccatcttctggatctgcGCCAGTTGGTACTGGCACTT
LD1121ggaattgtgagcggataacaatttcacacaggaaacagctATCGCGAGACTCATGGTTTT
LD1134ccaggcaaattctgttttatcagaccgcttctgcgttctgatCGCTGCTTGTCGATCTGTTpLD1967
LD1135gcgacatgaccctgttcaacCTGACCGGCTACTGGACC
LD1136ggtccagtagccggtcagGTTGAACAGGGTCATGTCGC
LD1137ggaattgtgagcggataacaatttcacacaggaaacagctCCTCGGCGACCATGAATAC
LD1126ccaggcaaattctgttttatcagaccgcttctgcgttctgatTTCAGGTTCTTCGGGTTCTCpLD2044
LD1187aacagcgcgccgaccagcatCTCTTCGTTCGTTTTCAGCC
LD1188ggctgaaaacgaacgaagagATGCTGGTCGGCGCGCTGTT
LD1189ggaattgtgagcggataacaatttcacacaggaaacagctGCGTTGATGAAGCGGATAAC
Table 3
Plasmids used in this study.
https://doi.org/10.7554/eLife.30205.018
PlasmidDescriptionSource
pMQ307.5 kb mobilizable vector; oriT, sacB, GmR.Shanks et al. (2006)
pAKN69Contains mini-Tn7(Gm)PA1/04/03::eyfp fusion.Lambertsen et al. (2004)
pLD2722GmR, TetR flanked by Flp recombinase target (FRT) sites to resolve out resistance casettes.this study
pFLP2Site-specific excision vector with cI857-controlled FLP recombinase encoding sequence, sacB, ApR.Hoang et al. (1998)
pLD1852ccoN1 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD1610ccoN2 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD1616ccoN3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD1264ccoN4 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD1929cco1 cco2 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD2791hcn PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1.this study
pLD1853Full genomic sequence of ccoN4 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. Verified by sequencing.this study
pLD1966aa3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1.this study
pLD1967bo3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1.this study
pLD2044cio PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1.this study
pLD2777PCR-amplified cco1 promoter ligated into pSEK103 using SpeI and XhoI.this study
pLD2778PCR-amplified cco2 promoter ligated into pSEK103 using SpeI and XhoI.this study
pLD2779PCR-amplified ccoN4 promoter ligated into pSEK103 using SpeI and XhoI.this study
Table 4
Statistical analysis.
https://doi.org/10.7554/eLife.30205.019
Figure 2BNumber of values (biological replicates)meanmedianSDSEMLower 95% confidence interval of meanUpper 95% confidence interval of mean
WT573.2272.943.3871.51569.0277.43
N4568.9770.66.442.8860.9776.96
N1N2552.1850.465.1422.345.7958.56
N1N4511.5712.422.0110.89919.07414.07
N1N2N450.0019580.0011170.0016960.0007586−0.00014810.004064
cco1cco250.0013670.00086440.0012370.0005532−0.00016860.002903
t-testp valuep value summary
WT vs. ∆N40.2273ns
WT vs. ∆N1N2<0.0001****
WT vs. ∆N1N4<0.0001****
WT vs. ∆N1N2N4<0.0001****
WT vs. ∆cco1cco2<0.0001****
Figure 3ANumber of values (biological replicates)meanmedianSDSEMLower 95% confidence interval of meanUpper 95% confidence interval of mean
WT-YFP1254.9554.924.3871.26652.1657.74
N4-YFP329.9230.832.2341.2924.3735.46
N1N2-YFP330.4931.913.5272.03621.7339.25
N1N2N4-YFP34.4084.2963.231.865−3.61712.43
cco1cco2-YFP37.0975.3064.0932.363−3.07217.27
t-testp valuep value summary
WT-YFP vs. ∆N4- YFP<0.0001****
WT-YFP vs.
N1N2-YFP
<0.0001****
N1N2-YFP vs.
N1N2N4-YFP
0.0007***
N1N2-YFP vs.
cco1cco2-YFP
0.0017**
Figure 3—figure supplement 1ANumber of values (biological replicates)meanmedianSDSEMLower 95% confidence interval of meanUpper 95% confidence interval of mean
WT)1245.0545.084.3871.26642.2647.84
N4328.2231.317.4424.2979.73146.71
N1N2327.8128.572.5141.45121.5634.05
N1N2N437.0026.9730.75080.43355.1378.867
cco1cco235.384.1832.1461.2390.0503410.71
t-testp valuep value summary
WT vs. ∆N40.0002***
WT vs. ∆N1N2<0.0001****
N1N2 vs.
N1N2N4
0.0002***
N1N2 vs.
cco1cco2
0.0003***
Figure 5Number of values (biological replicates)meanmedianSDSEMLower 95% confidence interval of meanUpper 95% confidence interval of mean
WT8150.3151.210.313.644141.7158.9
N1N24139.3137.612.336.166119.6158.9
N47131.9127.88.9153.369123.7140.2
N1N4499.9699.342.7261.36395.62104.3
cco1cco2495.1995.561.5590.779392.7197.67
N1N2N44102.899.798.6644.33288.98116.6
phz784.9884.2310.934.13174.8795.09
t-testp valuep value summary
WT vs. ∆N1N20.1302ns
WT vs. ∆N40.0028**
WT vs. ∆N1N4<0.0001****
WT vs. ∆cco1cco2<0.0001****
WT vs. ∆N1N2N4<0.0001****
WT vs. ∆phz<0.0001****
Figure 6Number of values (biological replicates)meanmedianSDSEMLower 95% confidence interval of meanUpper 95% confidence interval of mean
WT927.443918.486.1613.2441.65
gacA::Tn992.56938.5462.84985.9999.12
N1N241921.514.077.036−3.3941.39
N1N2N4664.1768187.3545.2783.06
cco1cco2970.567622.697.56553.1188
t-testp valuep value summary
WT vs. ∆N1N2N40.0022**
N1N2 vs.
N1N2N4
0.0030**
WT vs. ∆N1N20.4362ns

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