An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence
- Columbia University, United States
Figures
Figure 1
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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. …
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 …
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 …
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. …
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. …
Figure 6
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 …
Tables
Key resource table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (UCBPP-PA14 Pseudomonas aeruginosa) | wild type (WT) | PMID: 7604262 | ||
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆phz | PMID: 16879411 | LD24 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1; ∆N1 | this study | LD1784 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN2; ∆N2 | this study | LD1614 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN3; ∆N3 | this study | LD1620 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN4; ∆N4 | this study | LD2833 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2; ∆N1∆N2 | this study | LD1888 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN4; ∆N1∆N4 | this study | LD1951 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN2∆ccoN4; ∆N2∆N4 | this study | LD1692 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN3∆ccoN4; ∆N3∆N4 | this study | LD1649 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆ccoN3; ∆N1∆N2∆N3 | this study | LD1977 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆ccoN4; ∆N1∆N2∆N4 | this study | LD1976 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆ccoN4 ∆ccoN3; ∆N1∆N2∆N4∆N3 | this study | LD2020 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆cco1cco2 | this study | LD1933 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆cox∆cyo∆cio | this study | LD2587 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆hcn | this study | LD2827 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆phz∆hcn | this study | LD2828 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN4∆hcn; ∆N4∆hcn | this study | LD2829 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆hcn; ∆N1∆N2∆hcn | this study | LD2830 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆ccoN4 ∆hcn; ∆N1∆N2∆N4∆hcn | this study | LD2831 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆cco1cco2∆hcn | this study | LD2832 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | gacA::Tn | PMID: 16477005 | LD1560 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN4::ccoN4; ∆N4::N4 | this study | LD1867 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆ccoN4:: ccoN4; ∆N1∆N2∆N4::N4 | this study | LD2576 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | MCS-gfp | this study | LD2820 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | Pcco1-gfp; cco1Pr-gfp | this study | LD2784 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | Pcco2-gfp; cco2Pr-gfp | this study | LD2786 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | PccoN4-gfp; ccoN4Pr-gfp | this study | LD2788 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | PA14-yfp | this study | LD2780 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2-yfp; ∆N1∆N2-yfp | this study | LD2013 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN4-yfp; ∆N4-yfp | this study | LD2834 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆ccoN1∆ccoN2∆cco N4-yfp; ∆N1∆N2∆N4-yfp | this study | LD2136 | |
| strain, strain background (UCBPP-PA14 P. aeruginosa) | ∆cco1cco2-yfp | this study | LD2012 | |
| strain, strain background (Escherichia coli) | UQ950 | other | From D. Lies, Caltech; LD44 | |
| strain, strain background (E. coli) | BW29427 | other | From W. Metcalf, University of Illinois; LD661 | |
| strain, strain background (E. coli) | β2155 | PMID: 8990308 | LD69 | |
| strain, strain background (E. coli) | S17-1 | doi:10.1038/nbt1183-784 | LD2901 | |
| strain, strain background (Saccharomyces cerevisiae) | InvSc1 | Invitrogen | LD676 | |
| recombinant DNA reagent | pMQ30 (plasmid) | PMID: 16820502 | For generation of deletion constructs listed above; further information can be found in the Materials and Methods section. | |
| recombinant DNA reagent | pAKN69 (plasmid) | PMID: 15186351 | For generation of strains constitutively expression eyfp; further information can be found in the Materials and Methods section. | |
| recombinant DNA reagent | pLD2722 (plasmid) | this study | For generation of gfp reporter constructs; further information can be found in the Materials and Methods section. | |
| recombinant DNA reagent | pFLP2 (plasmid) | PMID: 9661666 | For generation of gfp reporter constructs; further information can be found in the Materials and Methods section. | |
| software, algorithm | EggNOG Database | PMID: 26582926 | http://eggnogdb.embl.de/#/app/home | |
| software, algorithm | SensorTrace Profiling | Unisense | For data acquisition for redox and oxygen microprofiling; further information can be found in the Materials and Methods section. | |
| other | Agar | Teknova | For colony morphology assays; further information can be found in the Materials and Methods section. | |
| other | Lectin | Vector Laboratories | For 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| Strain | Number | Description | Source |
|---|---|---|---|
| Pseudomonas aeruginosa strains | |||
| UCBPP-PA14 | Clinical isolate UCBPP-PA14. | Rahme et al. (1995) | |
| PA14 ∆phz | LD24 | PA14 with deletions in phzA1-G1 and phzA2-G2 operons. | Dietrich et al., 2006a |
| PA14 ∆ccoN1 | LD1784 | PA14 with deletion in PA14_44370. | this study |
| PA14 ∆ccoN2 | LD1614 | PA14 with deletion in PA14_44340. | this study |
| PA14 ∆ccoN3 | LD1620 | PA14 with deletion in PA14_40510. | this study |
| PA14 ∆ccoN4 | LD2833 | PA14 with deletion in PA14_10500. | this study |
| PA14 ∆ccoN1 ∆ccoN2 | LD1888 | PA14 with deletions in PA14_44370 and PA14_44340. Made by mating pLD1610 into LD1784. | this study |
| PA14 ∆ccoN1 ∆ccoN4 | LD1951 | PA14 with deletions in PA14_44370 and PA14_10500. Made by mating pLD1264 into LD1784. | this study |
| PA14 ∆ccoN2 ∆ccoN4 | LD1692 | PA14 with deletions in PA14_44340 and PA14_10500. Made by mating pLD1264 into LD1614. | this study |
| PA14 ∆ccoN3 ∆ccoN4 | LD1649 | PA14 with deletions in PA14_40510 and PA14_10500. Made by mating pLD1264 into LD1620. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆ccoN3 | LD1977 | PA14 with deletions in PA14_443470, PA14_44340, and PA14_40510. Made by mating pLD1616 into LD1888. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4 | LD1976 | PA14 with deletions in PA14_443470, PA14_44340, and PA14_10500. Made by mating pLD1264 into LD1888. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4 ∆ccoN3 | LD2020 | PA14 with deletions in PA14_443470, PA14_44340, PA14_10500, and PA14_40510. Made by mating pLD1264 into LD1977. | this study |
| PA14 ∆cco1cco2 | LD1933 | PA14 with both cco operons (PA14_44340-PA14_44400) deleted simultaneously. | this study |
| PA14 ∆cox ∆cyo ∆cio | LD2587 | PA14 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 ∆hcn | LD2827 | PA14 with deletion in hcnABC operon (PA14_36310–36330). | this study |
| PA14 ∆phz ∆hcn | LD2828 | PA14 with deletions in phzA1-G1, phzA2-G2, and hcnABC operons. Made by mating pLD2791 into LD24. | this study |
| PA14 ∆ccoN4 ∆hcn | LD2829 | PA14 with deletions in PA14_10500 and hcnABC operon. Made by mating pLD2791 into LD2833. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆hcn | LD2830 | PA14 with deletions in PA14_44370, PA14_44340, and hcnABC operon. Made by mating pLD2791 into LD1888. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4 ∆hcn | LD2831 | PA14 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 | LD2832 | PA14 with deletions in cco1, cco2, and hcnABC operons. Made by mating pLD2791 into LD1933. | this study |
| PA14 gacA::Tn | LD1560 | MAR2xT7 transposon insertion into PA14_30650. | Liberati et al. (2006) |
| PA14 ∆ccoN4::ccoN4 | LD1867 | PA14 ∆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 | LD2576 | PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4 strain with wild-type ccoN4 complemented back into the site of deletion. Made by mating pLD1853 into LD1976. | this study |
| PA14 MCS-gfp | LD2820 | PA14 without a promoter driving gfp expression. | this study |
| PA14 Pcco-1-gfp | LD2784 | PA14 with promoter of cco1 operon driving gfp expression. | this study |
| PA14 Pcco-2-gfp | LD2786 | PA14 with promoter of cco2 operon driving gfp expression. | this study |
| PA14 PccoN4-gfp | LD2788 | PA14 with promoter of ccoN4Q4 operon driving gfp expression. | this study |
| PA14-yfp | LD2780 | WT PA14 constitutively expressing eyfp. | this study |
| PA14 ∆ccoN1 ∆ccoN2-yfp | LD2013 | PA14 ∆ccoN1 ∆ccoN2 constitutively expressing eyfp. Made by mating pAKN69 into LD1888. | this study |
| PA14 ∆ccoN4-yfp | LD2834 | PA14 ∆ccoN4 constitutively expressing eyfp. Made by mating pAKN69 into LD2833. | this study |
| PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4-yfp | LD2136 | PA14 ∆ccoN1 ∆ccoN2 ∆ccoN4 constitutively expressing eyfp. Made by mating pAKN69 into LD1976. | this study |
| PA14 ∆cco1cco2-yfp | LD2012 | PA14 ∆cco1cco2 constitutively expressing eyfp. Made by mating pAKN69 into LD1933. | this study |
| Escherichia coli strains | |||
| UQ950 | LD44 | E. 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 |
| BW29427 | LD661 | Donor strain for conjugation. thrB1004 pro thi rpsL hsdS lacZ ∆M15RP4-1360 ∆(araBAD)567 ∆dapA1314::[erm pir(wt)] | W. Metcalf, University of Illinois |
| β2155 | LD69 | Helper 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) |
| S17-1 | LD2901 | StrR, TpR, F− RP4-2-Tc::Mu aphA::Tn7 recA λpir lysogen | Simon et al. (1983) |
| Saccharomyces cerevisiae strains | |||
| InvSc1 | LD676 | MATa/MATalpha leu2/leu2 trp1-289/trp1-289 ura3−52/ura3-52 his3-∆1/his3-∆1 | Invitrogen |
Table 2
Primers used in this study.
https://doi.org/10.7554/eLife.30205.017| Primer number | Sequence | used to make plasmid number |
|---|---|---|
| LD717 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatCAGGACAAGCAGTGGGAAC | pLD1852 |
| LD718 | aggtgttgtaggccatcagcTGGCGGACCACCTTATAGTT | |
| LD958 | aactataaggtggtccgccaCGGTGGTTTCTTCCTCACC | |
| LD959 | ggaattgtgagcggataacaatttcacacaggaaacagctGGTCCAGCCTTTTTCCTTGT | |
| LD725 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatCCCCTCAGAGAAGTCAGTCG | pLD1610 |
| LD726 | aggtgttgtaggccatcaggGGCGGACCACCTTGTAGTTA | |
| LD727 | taactacaaggtggtccgccCCTGATGGCCTACAACACCT | |
| LD728 | ggaattgtgagcggataacaatttcacacaggaaacagctCAGCGGGTTGTCATACTCCT | |
| LD741 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatTCGAGGGCTTCGAGAAGAT | pLD1616 |
| LD742 | aggtgttgtaggccatcagcCAGGGTCATCAGGGTGAACT | |
| LD743 | agttcaccctgatgaccctgGCTGATGGCCTACAACACCT | |
| LD744 | ggaattgtgagcggataacaatttcacacaggaaacagctCGGGTGATGTCGACGTATTC | |
| LD438 | ggaattgtgagcggataacaatttcacacaggaaacagctCCGTTGATTTCCTTCTGCAT | pLD1264 (LD438 - LD441) pLD1853 (LD438 and LD441) |
| LD439 | ctacaaggtggttcgccagtCGCTGACCTACTCCTTCGTC | |
| LD440 | gacgaaggagtaggtcagcgACTGGCGAACCACCTTGTAG | |
| LD441 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatCATCGACCTGGAAGTGCTC | |
| LD725 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatCCCCTCAGAGAAGTCAGTCG | pLD1929 |
| LD1063 | gttgcccaggtgttcctgtGGCGGACCACCTTGTAGTTA | |
| LD949 | ggaattgtgagcggataacaatttcacacaggaaacagctTGTAGTCGAGGGACTTCTTGC | |
| LD1064 | taactacaaggtggtccgccACAGGAACACCTGGGCAAC | |
| LD2168 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatATGTAGGGATCGAGCGACAG | pLD2791 |
| LD2169 | acacgatatccagcccctctTGGACATCGCGCCGTTCCTC | |
| LD2170 | gaggaacggcgcgatgtccaAGAGGGGCTGGATATCGTGT | |
| LD2171 | ggaattgtgagcggataacaatttcacacaggaaacagctAAGAGGTCATAATCGGCGGT | |
| LD2120 | gattcgacatcactagtACGCCCAGCTCCAACAAA | pLD2777 |
| LD2121 | gattcgatgccctcgaGCTAGGGGTTCCACGGTTAAT | |
| LD2122 | gattcgactgcactagtCATCGACTTGCCGCCCAG | pLD2778 |
| LD2123 | g attcg atg ccctcgaGCTATGGGCTTCCATC CAC | |
| LD2124 | gattcgactgcactagtGGCTACTTCCTCTGGCTGG | pLD2779 |
| LD2125 | gattcgactgcctcgagCTGTACAGTCCCGAAAGAAATGAAC | |
| LD1118 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatTCTTCAGGTTCTCGCGGTAG | pLD1966 |
| LD1119 | aagtgccagtaccaactggcGCAGATCCAGAAGATGGTCA | |
| LD1120 | tgaccatcttctggatctgcGCCAGTTGGTACTGGCACTT | |
| LD1121 | ggaattgtgagcggataacaatttcacacaggaaacagctATCGCGAGACTCATGGTTTT | |
| LD1134 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatCGCTGCTTGTCGATCTGTT | pLD1967 |
| LD1135 | gcgacatgaccctgttcaacCTGACCGGCTACTGGACC | |
| LD1136 | ggtccagtagccggtcagGTTGAACAGGGTCATGTCGC | |
| LD1137 | ggaattgtgagcggataacaatttcacacaggaaacagctCCTCGGCGACCATGAATAC | |
| LD1126 | ccaggcaaattctgttttatcagaccgcttctgcgttctgatTTCAGGTTCTTCGGGTTCTC | pLD2044 |
| LD1187 | aacagcgcgccgaccagcatCTCTTCGTTCGTTTTCAGCC | |
| LD1188 | ggctgaaaacgaacgaagagATGCTGGTCGGCGCGCTGTT | |
| LD1189 | ggaattgtgagcggataacaatttcacacaggaaacagctGCGTTGATGAAGCGGATAAC |
Table 3
Plasmids used in this study.
https://doi.org/10.7554/eLife.30205.018| Plasmid | Description | Source |
|---|---|---|
| pMQ30 | 7.5 kb mobilizable vector; oriT, sacB, GmR. | Shanks et al. (2006) |
| pAKN69 | Contains mini-Tn7(Gm)PA1/04/03::eyfp fusion. | Lambertsen et al. (2004) |
| pLD2722 | GmR, TetR flanked by Flp recombinase target (FRT) sites to resolve out resistance casettes. | this study |
| pFLP2 | Site-specific excision vector with cI857-controlled FLP recombinase encoding sequence, sacB, ApR. | Hoang et al. (1998) |
| pLD1852 | ∆ccoN1 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD1610 | ∆ccoN2 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD1616 | ∆ccoN3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD1264 | ∆ccoN4 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD1929 | ∆cco1 cco2 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD2791 | ∆hcn PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. | this study |
| pLD1853 | Full genomic sequence of ccoN4 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain InvSc1. Verified by sequencing. | this study |
| pLD1966 | ∆aa3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1. | this study |
| pLD1967 | ∆bo3 PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1. | this study |
| pLD2044 | ∆cio PCR fragment introduced into pMQ30 by gap repair cloning in yeast strain IncSc1. | this study |
| pLD2777 | PCR-amplified cco1 promoter ligated into pSEK103 using SpeI and XhoI. | this study |
| pLD2778 | PCR-amplified cco2 promoter ligated into pSEK103 using SpeI and XhoI. | this study |
| pLD2779 | PCR-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 2B | Number of values (biological replicates) | mean | median | SD | SEM | Lower 95% confidence interval of mean | Upper 95% confidence interval of mean |
|---|---|---|---|---|---|---|---|
| WT | 5 | 73.22 | 72.94 | 3.387 | 1.515 | 69.02 | 77.43 |
| ∆N4 | 5 | 68.97 | 70.6 | 6.44 | 2.88 | 60.97 | 76.96 |
| ∆N1∆N2 | 5 | 52.18 | 50.46 | 5.142 | 2.3 | 45.79 | 58.56 |
| ∆N1∆N4 | 5 | 11.57 | 12.42 | 2.011 | 0.8991 | 9.074 | 14.07 |
| ∆N1∆N2∆N4 | 5 | 0.001958 | 0.001117 | 0.001696 | 0.0007586 | −0.0001481 | 0.004064 |
| ∆cco1cco2 | 5 | 0.001367 | 0.0008644 | 0.001237 | 0.0005532 | −0.0001686 | 0.002903 |
| t-test | p value | p value summary | |||||
| WT vs. ∆N4 | 0.2273 | ns | |||||
| WT vs. ∆N1∆N2 | <0.0001 | **** | |||||
| WT vs. ∆N1∆N4 | <0.0001 | **** | |||||
| WT vs. ∆N1∆N2∆N4 | <0.0001 | **** | |||||
| WT vs. ∆cco1cco2 | <0.0001 | **** | |||||
| Figure 3A | Number of values (biological replicates) | mean | median | SD | SEM | Lower 95% confidence interval of mean | Upper 95% confidence interval of mean |
| WT-YFP | 12 | 54.95 | 54.92 | 4.387 | 1.266 | 52.16 | 57.74 |
| ∆N4-YFP | 3 | 29.92 | 30.83 | 2.234 | 1.29 | 24.37 | 35.46 |
| ∆N1∆N2-YFP | 3 | 30.49 | 31.91 | 3.527 | 2.036 | 21.73 | 39.25 |
| ∆N1∆N2∆N4-YFP | 3 | 4.408 | 4.296 | 3.23 | 1.865 | −3.617 | 12.43 |
| ∆cco1cco2-YFP | 3 | 7.097 | 5.306 | 4.093 | 2.363 | −3.072 | 17.27 |
| t-test | p value | p value summary | |||||
| WT-YFP vs. ∆N4- YFP | <0.0001 | **** | |||||
| WT-YFP vs. ∆N1∆N2-YFP | <0.0001 | **** | |||||
| ∆N1∆N2-YFP vs. ∆N1∆N2∆N4-YFP | 0.0007 | *** | |||||
| ∆N1∆N2-YFP vs. ∆cco1cco2-YFP | 0.0017 | ** | |||||
| Figure 3—figure supplement 1A | Number of values (biological replicates) | mean | median | SD | SEM | Lower 95% confidence interval of mean | Upper 95% confidence interval of mean |
| WT) | 12 | 45.05 | 45.08 | 4.387 | 1.266 | 42.26 | 47.84 |
| ∆N4 | 3 | 28.22 | 31.31 | 7.442 | 4.297 | 9.731 | 46.71 |
| ∆N1∆N2 | 3 | 27.81 | 28.57 | 2.514 | 1.451 | 21.56 | 34.05 |
| ∆N1∆N2∆N4 | 3 | 7.002 | 6.973 | 0.7508 | 0.4335 | 5.137 | 8.867 |
| ∆cco1cco2 | 3 | 5.38 | 4.183 | 2.146 | 1.239 | 0.05034 | 10.71 |
| t-test | p value | p value summary | |||||
| WT vs. ∆N4 | 0.0002 | *** | |||||
| WT vs. ∆N1∆N2 | <0.0001 | **** | |||||
| ∆N1∆N2 vs. ∆N1∆N2∆N4 | 0.0002 | *** | |||||
| ∆N1∆N2 vs. ∆cco1cco2 | 0.0003 | *** | |||||
| Figure 5 | Number of values (biological replicates) | mean | median | SD | SEM | Lower 95% confidence interval of mean | Upper 95% confidence interval of mean |
| WT | 8 | 150.3 | 151.2 | 10.31 | 3.644 | 141.7 | 158.9 |
| ∆N1∆N2 | 4 | 139.3 | 137.6 | 12.33 | 6.166 | 119.6 | 158.9 |
| ∆N4 | 7 | 131.9 | 127.8 | 8.915 | 3.369 | 123.7 | 140.2 |
| ∆N1∆N4 | 4 | 99.96 | 99.34 | 2.726 | 1.363 | 95.62 | 104.3 |
| ∆cco1cco2 | 4 | 95.19 | 95.56 | 1.559 | 0.7793 | 92.71 | 97.67 |
| ∆N1∆N2∆N4 | 4 | 102.8 | 99.79 | 8.664 | 4.332 | 88.98 | 116.6 |
| ∆phz | 7 | 84.98 | 84.23 | 10.93 | 4.131 | 74.87 | 95.09 |
| t-test | p value | p value summary | |||||
| WT vs. ∆N1∆N2 | 0.1302 | ns | |||||
| WT vs. ∆N4 | 0.0028 | ** | |||||
| WT vs. ∆N1∆N4 | <0.0001 | **** | |||||
| WT vs. ∆cco1cco2 | <0.0001 | **** | |||||
| WT vs. ∆N1∆N2∆N4 | <0.0001 | **** | |||||
| WT vs. ∆phz | <0.0001 | **** | |||||
| Figure 6 | Number of values (biological replicates) | mean | median | SD | SEM | Lower 95% confidence interval of mean | Upper 95% confidence interval of mean |
| WT | 9 | 27.44 | 39 | 18.48 | 6.16 | 13.24 | 41.65 |
| gacA::Tn | 9 | 92.56 | 93 | 8.546 | 2.849 | 85.99 | 99.12 |
| ∆N1∆N2 | 4 | 19 | 21.5 | 14.07 | 7.036 | −3.39 | 41.39 |
| ∆N1∆N2∆N4 | 6 | 64.17 | 68 | 18 | 7.35 | 45.27 | 83.06 |
| ∆cco1cco2 | 9 | 70.56 | 76 | 22.69 | 7.565 | 53.11 | 88 |
| t-test | p value | p value summary | |||||
| WT vs. ∆N1∆N2∆N4 | 0.0022 | ** | |||||
| ∆N1∆N2 vs. ∆N1∆N2∆N4 | 0.0030 | ** | |||||
| WT vs. ∆N1∆N2 | 0.4362 | ns |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.30205.020
