Article Figures & Data
Figures
- FIG. 1.
Growth of P. aeruginosa in vitro in MOPS minimal medium with 20 mM glucose (▪) and in vivo in the DMC in the rat (▴). Representative growth curves are shown. Maximum doubling times were approximately 40 min in vitro and 50 min in vivo.
- FIG. 2.
Lysis of S. aureus is necessary for iron acquisition by P. aeruginosa during coculture. (A) A BHI petri plate was swabbed with a confluent lawn of S. aureus, and 5 μl of an overnight culture of wild-type P. aeruginosa PA14 and P. aeruginosa PA14-LM1 containing a Tn5 insertion in pqsA were spotted on the plate and incubated at 37°C for 24 h. A zone of clearing (indicating lysis of S. aureus) is visible around the wild-type P. aeruginosa colony. Similar lysis results were also observed in test tube coculture experiments (data not shown). Although not shown here, approximately 20% of the time P. aeruginosa PA14-LM1 produces a small zone of lysis in this assay. (B) Semiquantitative RT-PCR analysis of the low-iron-inducible gene pvdS and the constitutively expressed clpX gene (42). P. aeruginosa was grown in vitro in glucose minimal medium containing excess FeSO4 (3.5 μM) or in vivo as a monoculture or in coculture with S. aureus. P. aeruginosa PA14-LM1, which is unable to lyse S. aureus but possesses no observed growth defects under iron-limited conditions, was also grown in vivo in coculture with S. aureus.
- FIG. 3.
Growth yields of wild-type P. aeruginosa and P. aeruginosa PA14-LM1 grown in iron-limited MOPS minimal medium (see Materials and Methods) as a monoculture, with 10 μM FeSO4, in coculture with S. aureus, and with the addition of mechanically lysed S. aureus. Data are expressed as (the ratio of P. aeruginosa cell yield in the presence of the addition)/(monoculture cell yields with no addition) (numbers >1 indicate increased cell yield during growth with the indicated addition). For the two no-addition controls, the ratio was calculated by pair-wise comparisons of four individual replicates. S. aureus does not grow in this minimal medium due to amino acid auxotrophy. Less than 103 S. aureus cells were present in PA14-S. aureus coculture colonies at the time of sampling, while >109 remained in the PA14-LM1-S. aureus cocultures. Error bars represent the standard deviation for three to four experimental repeats.
Tables
- TABLE 1.
Classes of in vivo-induced genes
Functional classa No. activatedb No. repressedb Adaptation, protection 10 2 Amino acid biosynthesis and metabolism 17 6 Biosynthesis of cofactors 7 3 Carbon compound catabolism 3 2 Cell wall-LPS 1 0 Central intermediary metabolism 4 1 Chaperones and heat shock protein 2 0 Energy metabolism 25 6 Membrane proteins 2 0 Motility and attachment 1 0 Nucleotide biosynthesis and metabolism 1 0 Protein-secretion-export apparatus 4 0 Putative enzymes 15 2 Related to phage, transposon, plasmid 0 11 Secreted factors 2 0 Transcriptional regulators 29 2 Translation, posttranslational modification, degradation 1 0 Transport of small molecules 32 18 Two-component regulatory systems 3 0 Hypothetical proteins 65 19 Unknown (conserved hypothetical) 14 6 ↵a Functional classes are from the P. aeruginosa genome website (www.pseudomonas.com).
↵b Genes that were activated or repressed in the DMC at least fivefold compared to cells grown in vitro in MOPS medium.
- TABLE 2.
P. aeruginosa genes differentially regulated during in vivo growth
ORFa Gene Homology or functiona Fold regulationb 0149 Probable sigma-70 factor 7 0171 Hypothetical protein −8 0200 Hypothetical protein 7 0280 cysA Sulfate transport protein −11 0281 cysW Sulfate transport protein −17 0282 cysT Sulfate transport protein −11 0283 sbp Sulfate-binding protein precursor −15 0284 Hypothetical protein −17 0440* Probable oxidoreductase 7 0466 Hypothetical protein −7 0509* nirN Probable c-type cytochrome 7 0510 Probable uroporphyrin III c-methyltransferase 13 0511* nirJ Heme d1 biosynthesis protein 7 0512 Conserved hypothetical protein 8 0513 Probable transcriptional regulator 8 0514* nirL Heme d1 biosynthesis protein 11 0515 Probable transcriptional regulator 9 0516* nirF Heme d1 biosynthesis protein 8 0517* nirC Probable c-type cytochrome precursor 11 0518* nirM Cytochrome c551 precursor 12 0519* nirS Nitrate reductase precursor 15 0521 Probable cytochrome c oxidase subunit 6 0523* norC Nitric oxide reductase subunit C 32 0524* norB Nitric oxide reductase subunit B 68 0525* Probable denitrification protein 15 0587 Conserved hypothetical protein 5 0613 Hypothetical protein −5 0614 Hypothetical protein −5 0617 Probable bacteriophage protein −10 0621 Conserved hypothetical protein −5 0622 Probable bacteriophage protein −8 0627 Conserved hypothetical protein −5 0629 Conserved hypothetical protein −7 0631 Hypothetical protein −6 0632 Hypothetical protein −7 0633 Hypothetical protein −8 0635 Hypothetical protein −11 0636 Hypothetical protein −8 0639 Conserved hypothetical protein −9 0641 Probable bacteriophage protein −19 0674 Hypothetical protein 25 0675 Probable sigma-70 factor 5 0713 Hypothetical protein 26 0714 Hypothetical protein 24 0781 Hypothetical protein 18 0782† putA Proline dehydrogenase 8 0865† hpd 4-Hydroxyphenylpyruvate dioxygenase 5 0872† phhA Phenylalanine-4-hydroxylase 31 0910 Hypothetical protein −18 0911 Hypothetical protein −6 0921 Hypothetical protein −6 0984 Colicin immunity protein 7 1123 Hypothetical protein 5 1178 oprH Outer membrane protein H1 precursor −17 1179 phoP Two-component response regulator −5 1195 Hypothetical protein 6 1196 Probable transcriptional regulator 5 1382 Probable type II secretion system protein 12 1414 Hypothetical protein 5 1516 Hypothetical protein 5 1537 Probable short-chain dehydrogenase 6 1540 Conserved hypothetical protein 7 1541 Probable drug efflux transporter 8 1552 Probable cytochrome c −12 1553 Probable cytochrome c oxidase subunit −13 1554 Probable cytochrome oxidase subunit −5 1673 Hypothetical protein 5 1742 Probable amidotransferase 7 1746 Hypothetical protein 9 1837 Hypothetical protein −13 1838 cysI Sulfite reductase −10 1871 lasA LasA protease precursor 6 1887 Hypothetical protein 11 1888 Hypothetical protein 10 1892 Hypothetical protein −9 1894 Hypothetical protein −9 1895 Hypothetical protein −10 1896 Hypothetical protein −9 1897 Hypothetical protein −35 1911 Probable transmembrane sensor 6 1912 Probable sigma-70 factor 7 1922 Probable TonB-dependent receptor 11 1924 Hypothetical protein 9 1925 Hypothetical protein 8 1999† Probable CoA transferase, subunit A 35 2000† Probable CoA transferase, subunit B 44 2001 atoB Acetyl-CoA acetyltransferase 13 2003 bdhA 3-Hydroxybutyrate dehydrogenase 6 2004 Conserved hypothetical protein 8 2016 Probable transcriptional regulator 5 2114 Probable MFS transporter −7 2202 Probable amino acid permease −7 2204 Probable binding protein component of ABC transporter −10 2223 Hypothetical protein 6 2247† bkdA1 2-Oxoisovalerate dehydrogenase (alpha subunit) 38 2248† BkdA2 2-Oxoisovalerate dehydrogenase (beta subunit) 14 2249† bkdB Branched-chain alpha-keto acid dehydrogenase (lipoamide component) 15 2250† lpdV Lipoamide dehydrogenase (−Val) 9 2466 Probable TonB-dependent receptor 5 2534 Probable transcriptional regulator 5 2567 Hypothetical protein 5 2648 nuoM NADH dehydrogenase I chain −5 2686 pfeR Two-component response regulator 6 2687 pfeS Two-component sensor 5 2753 Hypothetical protein 7 2807 Hypothetical protein −19 2825 Probable transcriptional regulator 5 2826 Probable glutathione peroxidase 5 2929 Hypothetical protein 5 2931 Probable transcriptional regulator 19 2932 morB Morphinone reductase 71 2933 Probable MFS transporter 31 2934 Probable hydrolase 16 2945 Conserved hypothetical protein −6 3038 Probable porin −6 3118† leuB 3-Isopropylmalate dehydrogenase −5 3120† leuD 3-Isopropylmalate dehydratase small subunit −6 3121† leuC 3-Isopropylmalate dehydratase large subunit −6 3126 ibpA Heat shock protein 7 3188 Probable permease of ABC sugar transporter −382 3189 Probable permease of ABC sugar transporter −79 3195 gapA Glyceraldehyde-3-phosphate dehydrogenase −6 3249 Probable transcriptional regulator 5 3309 Conserved hypothetical protein 6 3313 Hypothetical protein −5 3391* nosR Regulatory protein 54 3392* nosZ Nitrous oxide reductase precursor 25 3393* nosD NosD protein 11 3394* nosF NosF protein 16 3395* nosY NosY protein 8 3396* nosL NosL protein 8 3404 Probable secretion protein 7 3405 hasE Metalloprotease secretion protein 32 3406 hasD Transport protein 16 3415 Probable dihydrolipoamide acetyltransferase 9 3416 Probable pyruvate dehydrogenase E1 component, beta chain 36 3417 Probable pyruvate dehydrogenase E1 component, alpha subunit 7 3418† ldh Leucine dehydrogenase 8 3441 Probable molybdopterin-binding protein −12 3442 Probable ATP-binding component of ABC transporter −21 3443 Probable permease of ABC transporter −43 3444 Conserved hypothetical protein −161 3445 Conserved hypothetical protein −62 3446 Conserved hypothetical protein −19 3450 Probable antioxidant protein −8 3516 Probable lyase −5 3581 glpF Glycerol uptake facilitator protein 9 3582 glpK Glycerol kinase 6 3584 glpD Glycerol-3-phosphate dehydrogenase 44 3598 Conserved hypothetical protein 7 3600 Conserved hypothetical protein 139 3601 Conserved hypothetical protein 83 3719 Hypothetical protein 21 3720 Hypothetical protein 30 3721 Probable transcriptional regulator 5 3837 Probable permease of ABC transporter −5 3862 Hypothetical protein 6 3870 moaA1 Molybdopterin biosynthetic protein 7 3871 Probable peptidyl-prolyl cis-trans isomerase 7 3872* narI Respiratory nitrate reductase gamma chain 9 3873* narJ Respiratory nitrate reductase delta chain 11 3874* narH Respiratory nitrate reductase beta chain 24 3875* narG Respiratory nitrate reductase alpha chain 19 3876* narK2 Nitrite extrusion protein 2 10 3877* narK1 Nitrite extrusion protein 1 6 3914 moeA1 Molybdenum cofactor biosynthetic protein 7 3915 moaB1 Molybdopterin biosynthetic protein 5 3922 Conserved hypothetical protein −6 3931 Conserved hypothetical protein −16 3933 Probable choline transporter 10 3935 tauD Taurine dioxygenase −16 3936 Probable permease of ABC taurine transporter −35 3937 Probable ATP-binding component of ABC taurine transporter −25 3938 Probable periplasmic taurine-binding protein precursor −21 4063 Hypothetical protein 15 4064 Probable ATP-binding component of ABC transporter 7 4065 Hypothetical protein 5 4156 Probable TonB-dependent receptor 10 4160 fepD Ferric enterobactin transport protein 5 4167 Probable oxidoreductase 13 4168 Probable TonB-dependent receptor 17 4181 Hypothetical protein 16 4182 Hypothetical protein 10 4197 Probable two-component sensor 10 4288 Probable transcriptional regulator 8 4333 Probable fumarase −5 4364 Hypothetical protein 113 4365 Probable transporter 54 4442† cysN ATP sulfurylase GTP-binding subunit, APS kinase −22 4443† cysD ATP sulfurylase small subunit −10 4577 Hypothetical protein 5 4588† gdhA Glutamate dehydrogenase −6 4610 Hypothetical protein 9 4621 Probable oxidoreductase 5 4657 Hypothetical protein 5 4738 Conserved hypothetical protein −6 4739 Conserved hypothetical protein −9 4834 Hypothetical protein 18 4835 Hypothetical protein 16 4836 Hypothetical protein 32 4837 Probable outer membrane protein 38 4838 Hypothetical protein 7 5024 Conserved hypothetical protein −13 5027 Hypothetical protein 5 5088 Hypothetical protein 7 5100† hutU Urocanase 7 5102 Hypothetical protein −8 5106 Conserved hypothetical protein 6 5170† arcD Arginine-ornithine antiporter 11 5171† arcA Arginine deiminase 9 5172† arcB Ornithine carbamoyltransferase, catabolic 10 5173† arcC Carbamate kinase 6 5302† dadX Catabolic alanine racemase 6 5303 Conserved hypothetical protein 9 5304† dadA d-Amino acid dehydrogenase, small subunit 9 5351 Rubredoxin −5 5372† betA Choline dehydrogenase 12 5373† betB Betaine aldehyde dehydrogenase 12 5374 betI Transcriptional regulator 11 5427 adhA Alcohol dehydrogenase 6 5446 Hypothetical protein 13 5475 Hypothetical protein 5 5481 Hypothetical protein −7 5482 Hypothetical protein −8 5499‡ np20 Transcriptional regulator 6 5500 znuC Zinc transport protein 6 5532 Hypothetical protein 8 5534 Hypothetical protein 13 5535 Conserved hypothetical protein 15 5536 Conserved hypothetical protein 52 5538 amiA N-Acetylmuramoyl-l-alanine amidase 28 5539 Hypothetical protein 8 5540 Hypothetical protein 17 5541 Probable dihydroorotase 11 ↵a From the P. aeruginosa genome website, www.pseudomonas.com. Genes involved in anaerobic metabolism (*), transport and metabolism of amino acids (†), or identified previously using IVET (‡) are listed in bold.
↵b Fold change in P. aeruginosa mRNA level when grown in vivo in the DMC compared to that in bacteria grown in glucose minimal medium in vitro. Positive numbers represent induction, and negative numbers indicate repression in the DMC. All genes listed were also >5- fold regulated when DMC-grown and succinate-grown P. aeruginosa cultures were compared.
- TABLE 3.
Expression of P. aeruginosa iron-regulated genes during monoculture and coculture growth in vivo
ORFa Gene Homology or functiona Fold regulationb In vivo vs in vitro Coculture vs monoculture 0471 Probable transmembrane sensor 7 −17 0472 Probable sigma-70 factor 8 −34 0500 bioB Biotin synthase −9 NC 0672 Hypothetical protein 44 −44 0707 toxR Transcriptional regulator 6 −4 1245 Hypothetical protein 5 −3 1300 Probable sigma-70 factor 35 −37 1301 Probable transmembrane sensor 13 −12 2033 Hypothetical protein 20 −23 2034 Hypothetical protein 20 −16 2384 Hypothetical protein 41 −46 2385 Probable acylase 34 −30 2386 pvdA l-Ornithine N5-oxygenase 85 −74 2389 Conserved hypothetical protein 9 −9 2390 Probable ATP-binding/permease fusion ABC transporter 7 −8 2391 Probable outer membrane protein 5 −7 2392 Hypothetical protein 18 −12 2393 Probable dipeptidase precursor 53 −127 2394 Probable aminotransferase 57 −60 2395 Hypothetical protein 13 −38 2396 Hypothetical protein 13 −7 2397 pvdE Pyoverdine biosynthesis protein 44 −25 2398 fpvA Ferripyoverdine receptor 30 −86 2399 pvdD Pyoverdine synthetase 40 −23 2400 Probable nonribosomal peptide synthetase 94 −28 2401 Probable nonribosomal peptide synthetase 94 −40 2402 Probable nonribosomal peptide synthetase 46 −28 2403 Hypothetical protein 58 −22 2404 Hypothetical protein 43 −39 2405 Hypothetical protein 43 −37 2406 Hypothetical protein 24 −21 2407 Probable adhesion protein 14 −18 2408 Probable ATP-binding component of ABC transporter 38 −11 2411 Probable thioesterase 84 −111 2412 Conserved hypothetical protein 197 −203 2413 Probable class III aminotransferase 51 −136 2424 Probable nonribosomal peptide synthetase 112 −53 2425 Probable thioesterase 44 −51 2426 pvdS Sigma factor 66 −117 2427 Hypothetical protein 14 −8 2451 Hypothetical protein 9 −9 2452 Hypothetical protein 73 −110 2467 Probable transmembrane sensor 5 −8 2468 Probable sigma-70 factor 6 −13 3407 hasAp Heme acquisition protein 2,180 −724 3408 hasR Heme acquisition protein 74 −74 3409 Probable transmembrane sensor 9 −7 3410 Probable sigma-70 factor 14 −18 3530 Conserved hypothetical protein 9 −39 3899 Probable sigma-70 factor 17 −26 3900 Probable transmembrane sensor 7 −11 3901 fecA Fe(III) dicitrate transport protein 9 −10 4158 fepC Ferric enterobactin transport protein 13 −13 4218 Probable transporter 40 −22 4219 Hypothetical protein 109 −66 4220 Hypothetical protein 288 −48 4221 fptA Fe(III) pyochelin receptor precursor 81 −92 4222 Probable ATP-binding component of ABC transporter 34 −13 4223 Probable ATP-binding component of ABC transporter 26 −15 4224 Hypothetical protein 58 −34 4225 pchF Pyochelin synthetase 52 −65 4226 pchE Dihydroaeruginoic acid synthetase 53 −46 4227 pchR Transcriptional regulator 21 −76 4228 pchD Pyochelin biosynthesis protein 74 −66 4229 pchC Pyochelin biosynthesis protein 99 −163 4230 pchB Salicylate biosynthesis protein 126 −118 4231 pchA Salicylate biosynthesis isochorismate synthetase 122 −89 4359 Conserved hypothetical protein 5 −10 4467 Hypothetical protein 21 −10 4468 sodM Superoxide dismutase 40 −48 4469 Hypothetical protein 48 −53 4470 fumC1 Fumarate hydratase 60 −106 4471 Hypothetical protein 100 −131 4570 Hypothetical protein 37 −25 4708 Hypothetical protein 9 −6 4709 Probable hemin degrading factor 9 −8 4710 Probable outer membrane hemin receptor 19 −30 4895 Probable transmembrane sensor 12 −13 4896 Probable sigma-70 factor 14 −25 4973 thiC Thiamin biosynthesis protein −14 NC 5312 Probable aldehyde dehydrogenase 6 NC 5313 Probable pyridoxal-dependent aminotransferase 7 NC ↵a From the P. aeruginosa genome website, www.pseudomonas.com.
↵b Regulation of P. aeruginosa iron-regulated genes (28) as determined by Affymetrix GeneChip analysis. Two conditions were compared: monoculture growth in vivo versus monoculture in vitro growth in glucose minimal medium with added FeSO4 (positive numbers represent induction in vivo); and in vivo coculture growth versus in vivo monoculture growth (positive numbers represent induction during coculture growth). NC, no change.
