Abstract
DNA microarrays were used to examine the transcriptional response of Pseudomonas aeruginosa to anaerobiosis and nitrate. In response to anaerobic growth, 691 transcripts were differentially expressed. Comparisons of P. aeruginosa grown aerobically in the presence or the absence of nitrate showed differential expression of greater than 900 transcripts.
Pseudomonas aeruginosa is capable of anaerobic growth by anaerobic respiration with nitrate, nitrite, or nitrous oxide as the terminal electron acceptor (8, 9) or by generating ATP from arginine catabolism (16, 26). Biofilms display hypoxic gradients, and biofilm formation is enhanced under oxygen limitation (7, 28, 31). P. aeruginosa grows as a biofilm in the anoxic environment of the lower airway mucus plugs in cystic fibrosis patients (7, 28).
Except with genes involved in denitrification (1, 29) and several other genes (12, 14, 15, 20), little is known about anaerobic gene expression in P. aeruginosa. The present study used microarrays to identify genes differentially expressed by P. aeruginosa in response to anaerobiosis and nitrate.
Differential gene expression in response to anaerobic growth.
Growth curves for P. aeruginosa PAO1 cultured aerobically or anaerobically were generated to establish the RNA sampling points (27) (supplemental Fig. A at http://www.urmc.rochester.edu/smd/mbi/bhi/). Total RNA from three independent P. aeruginosa PAO1 aerobic or anaerobic cultures was isolated and processed as previously described (27). RNA integrity was assessed by reverse transcriptase PCR (RT-PCR) using primers specific for pilA, and purity was confirmed by PCR. Processing of RNA, microarray data generation, analysis, and validation by quantitative RT-PCR were performed as previously described (27) (supplemental Table C at http://www.urmc.rochester.edu/smd/mbi/bhi/). All RT-PCR data were normalized using PA4232, as the expression does not change under the conditions examined (our data and reference 27).
The expression of total transcripts (72% to 80%) was comparable to that seen in other P. aeruginosa microarray studies (19, 27). A total of 691 transcriptional changes, representing approximately 12% of the genome, resulted in statistically significantly different levels of expression in response to anaerobic growth, with 245 transcripts up-regulated and 446 transcripts down-regulated (supplemental Table A at http://www.urmc.rochester.edu/smd/mbi/bhi/). Those transcripts (n = 153) demonstrating a threefold or higher change are listed in Table 1 and grouped into functional categories (supplemental Fig. B at http://www.urmc.rochester.edu/smd/mbi/bhi/).
TABLE 1.
Differentially expressed transcripts between cultures grown aerobically with nitrate and cultures grown anaerobically with nitratea
| Transcript | Gene | Fold changeb | Growth phasec | Protein descriptiond |
|---|---|---|---|---|
| PA0024 | hemF | 4.5 | ES | Coproporphyrinogen III oxidase, aerobic |
| PA0025 | aroE | 3.5 | ES | Shikimate dehydrogenase |
| PA0049 | −5.1 | ES | Hypothetical protein | |
| PA0149 | 4.1 | ML | Probable sigma-70 factor, ECF subfamily | |
| PA0179 | −4.3 | ML | Probable two-component response regulator | |
| PA0200 | −5.5 | ML | Hypothetical protein | |
| PA0430 | metF | 3.4 | ES | 5,10-Methylenetetrahydrofolate reductase |
| PA0433 | 6.5 | ML | Hypothetical protein | |
| PA0447 | gcdH | −5.2 | ES | Glutaryl-CoA dehydrogenase |
| PA0471 | 9.7 | ML | Probable transmembrane sensor | |
| PA0472 | 4.1 | ML | Probable sigma-70 factor, ECF subfamily | |
| PA0586 | −4.6 | ML | Hypothetical protein | |
| PA0587 | −4.9 | ML | Hypothetical protein | |
| PA0747 | −3.8 | ML | Probable aldehyde dehydrogenase | |
| PA0792 | prpD | −3.1 | ML | Propionate catabolic protein PrpD |
| PA0802 | 5.3 | ML | Hypothetical protein | |
| PA0836 | −3.1 | ML | Probable acetate kinase | |
| PA0918 | −3.6 | ML | Cytochrome b561 | |
| PA0930 | 3.9 | ML | Two-component sensor | |
| PA0997 | pqsB | −4.3/−18 | B | Beta keto acyl-acyl carrier protein synthase |
| PA0998 | pqsC | −11.1/−43.7 | B | Beta keto acyl-acyl carrier protein synthase |
| PA0999 | pqsD | −21 | ES | 3-Oxoacyl (acyl carrier protein) synthase III |
| PA1000 | pqsE | −10.8 | ML | Quinolone signal reponse signal |
| PA1002 | phnB | −9.6 | ES | Anthranilate synthase component II |
| PA1045 | 3 | ML | Hypothetical protein | |
| PA1052 | −3.5 | ML | Hypothetical protein | |
| PA1076 | −3.8 | ML | Hypothetical protein | |
| PA1140 | −3.2 | ML | Hypothetical protein | |
| PA1173 | napB | −4.1 | ML | Cytochrome c-type protein NapB precursor |
| PA1174 | napA | −3.2 | ML | Periplasmic nitrate reductase protein NapA |
| PA1175 | napD | −3.8 | ML | NapD protein of periplasmic nitrate reductase |
| PA1176 | napF | −3.8 | ML | Ferredoxin protein NapF |
| PA1179 | phoP | −3.4 | ES | Two-component response regulator PhoP |
| PA1180 | phoQ | −4.8 | ES | Two-component sensor PhoQ |
| PA1195 | −6.1 | ML | Hypothetical protein | |
| PA1282 | −6.1 | ES | Probable MFS transporter | |
| PA1323 | −3.8 | ML | Hypothetical protein | |
| PA1324 | −3 | ML | Hypothetical protein | |
| PA1331 | 3.3 | ML | Hypothetical protein | |
| PA1418 | −8.4 | ML | Probable sodium-solute symport protein | |
| PA1419 | −14.2 | ML | Probable transporter | |
| PA1420 | −15.5 | ML | Hypothetical protein | |
| PA1421 | gbuA | −15 | ML | Guanidinobutyrase |
| PA1429 | −3 | ML | Probable cation-transporting P-type ATPase | |
| PA1516 | −3.3 | ML | Hypothetical protein | |
| PA1517 | −4.3 | ML | Hypothetical protein | |
| PA1518 | −4.1 | ML | Hypothetical protein | |
| PA1523 | xdhB | −4.4 | ML | Xanthine dehydrogenase |
| PA1546 | hemN | −4 | ML | Oxygen-independent coproporphyrinogen III oxidase |
| PA1556 | −3.2 | ML | Probable cytochrome c oxidase subunit | |
| PA1561 | aer | −4.5 | ML | Aerotaxis receptor Aer |
| PA1673 | −5.3 | ML | Hypothetical protein | |
| PA1789 | −3.9 | ML | Hypothetical protein | |
| PA1806 | fabI | −3.3 | ML | NADH-dependent enoyl-ACP reductase |
| PA1882 | −3 | ES | Probable transporter | |
| PA1883 | −3 | ES | Probable NADH-ubiquinone plastoquinone oxidoreductase | |
| PA1939 | −3.7 | ES | Hypothetical protein | |
| PA2012 | −6.5 | ML | Probable acyl-CoA carboxylase alpha chain | |
| PA2013 | −4.8 | ML | Probable enoyl-CoA hydratase isomerase | |
| PA2014 | −4.9 | ML | Probable acyl-CoA carboxyltransferase beta chain | |
| PA2015 | −6.8 | ML | Probable acyl-CoA dehydrogenase | |
| PA2016 | −9.4 | ML | Probable transcriptional regulator | |
| PA2024 | −4.7 | ML | Probable ring-cleaving dioxygenase | |
| PA2026 | −3.2 | ES | Hypothetical protein | |
| PA2073 | −6.6 | ML | Probable transporter (membrane subunit) | |
| PA2110 | −7.4 | ES | Hypothetical protein | |
| PA2112 | −4 | ML | Hypothetical protein | |
| PA2113 | −3.5/−3.9 | B | Probable porin | |
| PA2114 | −4.5/−4.4 | B | Probable MFS transporter | |
| PA2119 | −5.7 | ML | Alcohol dehydrogenase (Zn dependent) | |
| PA2128 | cupA1 | 4.2 | ES | Fimbrial subunit CupA1 |
| PA2129 | cupA2 | 9.5 | ML | Chaperone CupA2 |
| PA2130 | cupA3 | 3.6 | ML | Usher protein CupA3 |
| PA2193 | hcnA | −3.1 | ES | Hydrogen cyanide synthase HcnA |
| PA2259 | ptxS | −4.8 | ML | Transcriptional regulator PtxS |
| PA2261 | −9.4 | ML | Probable 2-ketogluconate kinase | |
| PA2264 | −3.2 | ML | Conserved hypothetical protein | |
| PA2265 | −3.4 | ML | Gluconate dehydrogenase | |
| PA2302 | −3.8 | ES | Probable nonribosomal peptide synthetase | |
| PA2303 | −3.2 | ES | Hypothetical protein | |
| PA2366 | −3.9 | ES | Hypothetical protein | |
| PA2423 | −3.3 | ES | Hypothetical protein | |
| PA2478 | 8.4 | ML | Probable thiol-disulfide interchange protein | |
| PA2552 | −5.1 | ML | Probable acyl-CoA dehydrogenase | |
| PA2553 | −7.6 | ML | Probable acyl-CoA thiolase | |
| PA2554 | −7.1 | ML | Probable short-chain dehydrogenase | |
| PA2555 | −7.9 | ML | Probable AMP-binding enzyme | |
| PA2557 | −4.3 | ML | Probable AMP-binding enzyme | |
| PA2572 | −3 | ML | Probable two-component response regulator | |
| PA2573 | −3.6 | ML | Probable chemotaxis transducer | |
| PA2753 | −9 | ML | Hypothetical protein | |
| PA2754 | −5 | ML | Hypothetical protein | |
| PA2759 | −3.1 | ML | Hypothetical protein | |
| PA2780 | −3.5 | ML | Hypothetical protein | |
| PA2790 | −3.3 | ML | Hypothetical protein | |
| PA3181 | −3.2 | ML | 2-Keto-3-deoxy-6-phosphogluconate aldolase | |
| PA3329 | −7.2 | ES | Hypothetical protein | |
| PA3330 | −4.7 | ES | Probable short chain dehydrogenase | |
| PA3333 | fabH2 | −3.8 | ES | 3-Oxoacyl (acyl carrier protein) synthase III |
| PA3334 | −3.6 | ES | Probable acyl carrier protein | |
| PA3337 | rfaD | −12.2 | ML | ADP-l-glycero-d-manno-heptose 6-epimerase |
| PA3418 | idh | −4.2 | ML | Leucine dehydrogenase |
| PA3425 | −3.2 | ES | Hypothetical protein | |
| PA3458 | −6.3 | ML | Probable transcriptional regulator | |
| PA3465 | −5.4 | ML | Hypothetical protein | |
| PA3520 | −3.9/−11.2 | B | Hypothetical protein | |
| PA3531 | bfrB | −8 | ML | Bacterioferritin |
| PA3552 | −11.5 | ES | Hypothetical protein | |
| PA3553 | −14.8 | ES | Probable glycosyl transferase | |
| PA3688 | −3 | ML | Hypothetical protein | |
| PA3724 | lasB | −5.7 | ES | Elastase LasB |
| PA3790 | oprC | 10.2 | ML | Outer membrane protein OprC |
| PA3866 | 3 | ES | Pyocin protein | |
| PA3876 | narK2 | −5.9 | ML | Nitrite extrusion protein 2 |
| PA3877 | narK1 | −7.3 | ML | Nitrite extrusion protein 1 |
| PA3914 | moeA1 | −8.8 | ES | Molybdenum cofactor biosynthetic protein A1 |
| PA3915 | moaB1 | −3.9/−7.8 | B | Molybdopterin biosynthetic protein B1 |
| PA3916 | moaE | −3.4/−5.3 | B | Molybdopterin converting factor, large subunit |
| PA3917 | moaD | −5.7 | ES | Molybdopterin converting factor, small subunit |
| PA3918 | moaC | −3.4/−6 | B | Molybdopterin biosynthetic protein C |
| PA3919 | −4.8 | ML | Hypothetical protein | |
| PA4063 | −3 | ES | Hypothetical protein | |
| PA4064 | −3.1 | ES | Probable ATP-binding component of ABC transporter | |
| PA4070 | −5.2 | ML | Probable transcriptional regulator | |
| PA4129 | −4.9 | ES | Hypothetical protein | |
| PA4134 | −6.7 | ES | Hypothetical protein | |
| PA4211 | phzBI | −3.5 | ES | Probable phenazine biosynthesis protein |
| PA4309 | pctA | −4.3 | ML | Chemotactic transducer PctA |
| PA4354 | 3.1 | ES | Hypothetical protein | |
| PA4371 | 8.8 | ML | Hypothetical protein | |
| PA4504 | −3.5 | ML | Probable permease of ABC transporter | |
| PA4571 | −6.4 | ML | Probable cytochrome c | |
| PA4577 | −6 | ML | Hypothetical protein | |
| PA4602 | glyA3 | 3 | ES | Serine hydroxymethyltransferase |
| PA4619 | −3.1 | ML | Probable c-type cytochrome | |
| PA4739 | −3.8 | ES | Hypothetical protein | |
| PA4852 | 3.1 | ML | Hypothetical protein | |
| PA4878 | −4.3 | ES | Probable transcriptional regulator | |
| PA5027 | −5 | ML | Hypothetical protein | |
| PA5170 | arcD | −17.1 | ML | Arginine-ornithine antiporter |
| PA5171 | arcA | −6.6 | ML | Arginine deiminase |
| PA5172 | arcB | −5.7 | ML | Ornithine carbamoyltransferase, catabolic |
| PA5208 | −3.2 | ML | Conserved hypothetical protein | |
| PA5216 | 3.8 | ML | Probable permease of ABC iron transporter | |
| PA5217 | 5.2 | ML | Probable binding protein component of ABC iron transporter | |
| PA5496 | 3.2 | ES | Hypothetical protein | |
| PA5497 | 3.5 | ES | Hypothetical protein | |
| PA5504 | 3.1 | ML | Probable permease of ABC transporter | |
| PA5506 | −3.9 | ML | Hypothetical protein | |
| PA5507 | −4.1 | ML | Hypothetical protein | |
| PA5508 | −3 | ML | Probable glutamine synthetase | |
| PA5510 | −3.4 | ML | Probable transporter | |
| PA5570 | rpmH | 3.3 | ML | 50S ribosomal protein L34 |
Only transcripts identified as anaerobically regulated that demonstrated a change of equal to or greater than threefold are reported. A list of all transcripts identified as being anaerobically regulated exhibiting a statistically significant change (P ≤ 0.05) is available online ( http://www.urmc.rochester.edu/smd/mbi/bhi/). Genes are identified by transcript number, gene name, and protein description (http://www.pseudomonas.com).
The change (n-fold) was calculated by comparing PAO1 grown aerobically with nitrate present (baseline) to PAO1 grown anaerobically with nitrate present (experimental). A positive change represents an induction caused by the lack of oxygen, and a negative change represents repression caused by the lack of oxygen. When two values are given, the first is for the ML phase and the second is for the ES phase.
Growth stage(s) during which statistically significantly differential transcript expression was observed. ML, midexponential phase; ES, early stationary phase; B, both midexponential and early stationary phases.
Protein descriptions are from the Pseudomonas Genome Project website (www.pseudomonas.com). ECF, extracytoplasmic function; CoA, coenzyme A; MSF, major facilitator superfamily; ABC, ATP-binding cassette.
We found numerous genes with expression patterns consistent with anaerobic growth and previous reports, such as the repression of napBAD, napF (17, 32), hcnAC (20), flgB, flgE, flgI, flgL, fliC, fliD, fleS, fleR, fliE, fliF, fliM, flhA, and flhF (10) and increased expression of hemF (22) (supplemental Table A at http://www.urmc.rochester.edu/smd/mbi/bhi/).
Many genes involved in quorum sensing (lasR, lasA, lasB, rhlR, rhlI, rhlA, and mvfR) were repressed under oxygen limitation. Consistent with reduced mvfR expression, transcripts involved in the biosynthesis of the Pseudomonas quinolone signal (pqsB and pqsE) and anthranilate synthase components I and II (phnAB) (4) were decreased.
Genes involved in cytochrome c maturation, ccmB, ccmC, ccmE, and ccmF, were up-regulated under anaerobic conditions, which is consistent with observations of Escherichia coli (24). Additionally, transcript levels for PA5491 (a probable cytochrome) were increased, suggesting that this previously uncharacterized cytochrome may play a role in anaerobic respiration. Transcript levels for several other putative cytochromes (PA0918, PA1555, PA1556, PA2266, PA2482, PA3331, PA4571, and PA4619) were repressed, suggesting that they may not be required for anaerobic respiration.
Our data implicated many novel genes in anaerobic growth. There were 284 transcripts classified as genes encoding hypothetical proteins differentially expressed under anaerobic conditions. Several genes which play a role in virulence (PA0930) (21) or biofilm formation (PA2128, PA2129, and PA2130) (25) were induced during anaerobic growth.
Differential gene expression in response to nitrate.
In contrast to previous studies (32), no significant changes were observed (Table 1) for most of the genes involved in denitrification (nar, nir, nos, and nor), suggesting that nitrate may induce their expression. This is supported by the capacity of P. aeruginosa for aerobic denitrification (5) and aerobic Nir activity when nitrate is available (1, 13). Many denitrification genes are influenced by the presence of an N-oxide (2). To investigate this, microarray analysis using RNA from cultures grown aerobically in the presence or the absence of nitrate was performed. Nearly 18% of the genome (919 transcripts; 415 transcripts induced and 504 repressed) exhibited differential expression in response to nitrate (supplemental Table B at http://www.urmc.rochester.edu/smd/mbi/bhi/). The 266 genes demonstrating a threefold or greater change are listed in Table 2. Functional categories are shown in supplemental Fig. C at http://www.urmc.rochester.edu/smd/mbi/bhi/.
TABLE 2.
Differentially expressed transcripts between cultures grown aerobically without nitrate and cultures grown aerobically with nitratea
| Transcript | Gene name | Fold changeb | Growth phasec | Protein descriptiond |
|---|---|---|---|---|
| PA0045 | −3.5 | ES | Hypothetical protein | |
| PA0046 | −3.4/−5.4 | B | Hypothetical protein | |
| PA0047 | −3.1/−3.8 | B | Hypothetical protein | |
| PA0129 | gabP | 4.3 | ML | Gamma-aminobutyrate permease |
| PA0130 | 4.2 | ML | Probable aldehyde dehydrogenase | |
| PA0131 | 3.9 | ML | Hypothetical protein | |
| PA0132 | 3.5 | ML | Beta-alanine-pyruvate transaminase | |
| PA0160 | −3.8 | ML | Hypothetical protein | |
| PA0164 | −4.8 | ML | Probable gamma-glutamyltranspeptidase | |
| PA0174 | 3.2 | ML | Conserved hypothetical protein | |
| PA0175 | 4.8 | ML | Probable chemotaxis protein methyltransferase | |
| PA0176 | 3.7 | ML | Probable chemotaxis transducer | |
| PA0177 | 3.8 | ML | Probable purine-binding chemotaxis protein | |
| PA0178 | 3 | ML | Probable two-component sensor | |
| PA0179 | 3.5 | ML | Probable two-component response regulator | |
| PA0200 | 8.7 | ES | Hypothetical protein | |
| PA0386 | −3.2 | ES | Probable oxidase | |
| PA0447 | gcdH | 3.4 | ES | Glutaryl-CoA dehydrogenase |
| PA0459 | 4 | ML | Probable ClpA/B protease ATP-binding subunit | |
| PA0494 | 3.2 | ML | Probable acyl-CoA carboxylase subunit | |
| PA0509 | nirN | 11.8 | ES | Probable c-type cytochrome |
| PA0510 | 17.5 | ES | Probable uroporphyrin-III C-methyltransferase | |
| PA0512 | 13.9 | ES | Conserved hypothetical protein | |
| PA0513 | 16 | ES | Probable transcriptional regulator | |
| PA0514 | nirL | 16.1 | ES | Heme d1 biosynthesis protein NirL |
| PA0516 | nirF | 16.3 | ES | Heme d1 biosynthesis protein NirF |
| PA0518 | nirM | 4.9 | ES | Cytochrome c551 precursor |
| PA0519 | nirS | 9.3 | ES | Nitrite reductase precursor |
| PA0521 | 23.6 | ES | Probable cytochrome c oxidase subunit | |
| PA0526 | 8.1 | ES | Hypothetical protein | |
| PA0586 | 4.1 | ML | Conserved hypothetical protein | |
| PA0587 | 4.5 | ML | Conserved hypothetical protein | |
| PA0614 | 3.3 | ML | Hypothetical protein | |
| PA0623 | 3.5 | ML | Probable bacteriophage protein | |
| PA0624 | 4.1 | ML | Hypothetical protein | |
| PA0627 | 4.6 | ML | Conserved hypothetical protein | |
| PA0628 | 4.5 | ML | Conserved hypothetical protein | |
| PA0632 | 4.3 | ML | Hypothetical protein | |
| PA0633 | 4 | ML | Hypothetical protein | |
| PA0639 | 3.8 | ML | Conserved hypothetical protein | |
| PA0742 | 3.6 | ML | Hypothetical protein | |
| PA0743 | 3.3 | ML | Probable 3-hydroxyisobutyrate dehydrogenase | |
| PA0744 | 3.7 | ML | Probable enoyl-CoA hydratase/isomerase | |
| PA0746 | 5.5 | ML | Probable acyl-CoA dehydrogenase | |
| PA0747 | 5.5 | ML | Probable aldehyde dehydrogenase | |
| PA0782 | putA | −5.1 | ES | Proline dehydrogenase PutA |
| PA0792 | prpD | 3.9 | ML | Propionate catabolic protein PrpD |
| PA0793 | 4 | ML | Hypothetical protein | |
| PA0795 | prpC | 4.3 | ML | Citrate synthase 2 |
| PA0887 | acsA | 3.8 | ML | Acetyl-CoA synthetase |
| PA0889 | aotQ | −3.2 | ES | Arginine/ornithine transport protein AotQ |
| PA0910 | 3.4 | ML | Hypothetical protein | |
| PA0985 | 7.6 | ML | Probable colicin-like toxin | |
| PA1070 | braG | 4.1 | ML | Branched-chain amino acid transport protein BraG |
| PA1071 | braF | 3.7 | ML | Branched-chain amino acid transport protein BraF |
| PA1072 | braE | 3.4 | ML | Branched-chain amino acid transport protein BraE |
| PA1073 | braD | 3 | ML | Branched-chain amino acid transport protein BraD |
| PA1173 | napB | 6 | ML | Cytochrome c-type protein NapB precursor |
| PA1174 | napA | 3.8 | ML | Periplasmic nitrate reductase protein NapA |
| PA1175 | napD | 3.3 | ML | NapD protein of periplasmic nitrate reductase |
| PA1249 | aprA | 6.7 | ML | Alkaline metalloproteinase precursor |
| PA1282 | 6.3 | ES | Probable MFS transporter | |
| PA1317 | cyoA | −3.7 | ML | Cytochrome o ubiquinol oxidase subunit II |
| PA1318 | cyoB | −3.1 | ML | Cytochrome o ubiquinol oxidase subunit I |
| PA1319 | cyoC | −3 | ML | Cytochrome o ubiquinol oxidase subunit III |
| PA1320 | cyoD | −4.8 | ML | Cytochrome o ubiquinol oxidase subunit IV |
| PA1337 | ansB | 4.8 | ML | Glutaminase-asparaginase |
| PA1338 | ggt | 3.4 | ML | Gamma-glutamyltranspeptidase precursor |
| PA1418 | 7.5 | ML | Probable sodium-solute symport protein | |
| PA1419 | 10.9 | ML | Probable transporter | |
| PA1420 | 6 | ML | Hypothetical protein | |
| PA1421 | gbuA | 5.6 | ML | Guanidinobutyrase |
| PA1428 | −7.4 | ES | Conserved hypothetical protein | |
| PA1559 | −3.6 | ML | Hypothetical protein | |
| PA1566 | −5.9 | ES | Conserved hypothetical protein | |
| PA1596 | htpG | −3.2 | ML | Heat shock protein HtpG |
| PA1657 | −5 | ES | Conserved hypothetical protein | |
| PA1660 | −4.8 | ES | Hypothetical protein | |
| PA1661 | −3.4 | ES | Hypothetical protein | |
| PA1662 | −3.3 | ES | Probable ClpA/B-type protease | |
| PA1663 | −3.3 | ES | Probable transcriptional regulator | |
| PA1664 | −4 | ES | Hypothetical protein | |
| PA1665 | −4.4 | ES | Hypothetical protein | |
| PA1666 | −3 | ES | Hypothetical protein | |
| PA1667 | −4.8 | ES | Hypothetical protein | |
| PA1668 | −3.2 | ES | Hypothetical protein | |
| PA1669 | −4.7 | ES | Hypothetical protein | |
| PA1761 | 3.2 | ML | Hypothetical protein | |
| PA1762 | 3.7 | ML | Hypothetical protein | |
| PA1790 | −4.8 | ES | Hypothetical protein | |
| PA1847 | 4.3 | ES | Conserved hypothetical protein | |
| PA1855 | 12.7 | ML | Hypothetical protein | |
| PA1856 | 29/4.1 | B | Probable cytochrome oxidase subunit | |
| PA1883 | 3 | ES | Probable NADH-ubiquinone/plastoquinone oxidoreductase | |
| PA1888 | 3.9 | ML | Hypothetical protein | |
| PA1902 | phzD2 | −13.5 | ES | Phenazine biosynthesis protein PhzD |
| PA1903 | phzE2 | −12.3 | ES | Phenazine biosynthesis protein PhzE |
| PA1904 | phzF2 | −12.9 | ES | Probable phenazine biosynthesis protein |
| PA1971 | braZ | −3.3 | ES | Branched chain amino acid transporter BraZ |
| PA1985 | pqqA | 3.6 | ML | Pyrroloquinoline quinone biosynthesis protein A |
| PA1986 | pqqB | 3 | ML | Pyrroloquinoline quinone biosynthesis protein B |
| PA1987 | pqqC | 4 | ML | Pyrroloquinoline quinone biosynthesis protein C |
| PA1988 | pqqD | 5 | ML | Pyrroloquinoline quinone biosynthesis protein D |
| PA1989 | pqqE | 3.3 | ML | Pyrroloquinoline quinone biosynthesis protein E |
| PA1990 | 5.6 | ML | Probable peptidase | |
| PA1999 | 8.2 | ML | Probable CoA transferase, subunit A | |
| PA2003 | bdhA | 3.1 | ML | 3-Hydroxybutyrate dehydrogenase |
| PA2011 | 9.1 | ML | Hydroxymethylglutaryl-CoA lyase | |
| PA2012 | 9.1 | ML | Probable acyl-CoA carboxylase alpha chain | |
| PA2013 | 5.2 | ML | Probable enoyl-CoA hydratase/isomerase | |
| PA2014 | 4.4 | ML | Probable acyl-CoA carboxyltransferase beta chain | |
| PA2024 | 3.7 | ML | Probable ring-cleaving dioxygenase | |
| PA2052 | cynS | 3.2 | ML | Cyanate lyase |
| PA2066 | −4.4 | ES | Hypothetical protein | |
| PA2067 | −3.9 | ES | Probable hydrolase | |
| PA2068 | −7.5 | ES | Probable MFS transporter | |
| PA2069 | −7.7 | ES | Probable carbamoyl transferase | |
| PA2110 | 7.9 | ES | Hypothetical protein | |
| PA2112 | 4.1 | ML | Conserved hypothetical protein | |
| PA2114 | 3.1 | ES | Probable MFS transporter | |
| PA2194 | hcnB | −3.3 | ES | Hydrogen cyanide synthase HcnB |
| PA2250 | lpdV | 3.7 | ES | Lipoamide dehydrogenase-Val |
| PA2266 | −3 | ES | Probable cytochrome c precursor | |
| PA2300 | chiC | −6.1 | ES | Chitinase |
| PA2306 | −3.1 | ES | Conserved hypothetical protein | |
| PA2327 | −4 | ES | Probable permease of ABC transporter | |
| PA2329 | −4.1 | ML | Probable ATP-binding component of ABC transporter | |
| PA2330 | −5.2/−3.6 | B | Hypothetical protein | |
| PA2331 | −6.7/−3.2 | B | Hypothetical protein | |
| PA2358 | −3.7 | ML | Hypothetical protein | |
| PA2370 | 4.8 | ML | Hypothetical protein | |
| PA2404 | −3.5 | ES | Hypothetical protein | |
| PA2405 | −6.2/−5 | B | Hypothetical protein | |
| PA2406 | −3 | ES | Hypothetical protein | |
| PA2408 | −3.2 | ML | Probable ATP-binding component of ABC transporter | |
| PA2442 | gcvT2 | 3.2 | ES | Glycine cleavage system protein T2 |
| PA2443 | sdaA | 6 | ML | l-Serine dehydratase |
| PA2444 | glyA2 | 6.8 | ML | Serine hydroxymethyltransferase |
| PA2445 | gcvP2 | 3.1 | ML | Glycine cleavage system protein P2 |
| PA2446 | gcvH2 | 3.1 | ML | Glycine cleavage system protein H2 |
| PA2460 | −3.3 | ES | Hypothetical protein | |
| PA2483 | 3 | ES | Conserved hypothetical protein | |
| PA2552 | 8.9 | ML | Probable acyl-CoA dehydrogenase | |
| PA2553 | 10.9 | ML | Probable acyl-CoA thiolase | |
| PA2554 | 6.7 | ML | Probable short-chain dehydrogenase | |
| PA2555 | 3.9 | ML | Probable AMP-binding enzyme | |
| PA2570 | palL | −5.1 | ES | PA-I galactophilic lectin |
| PA2573 | 3.1 | ML | Probable chemotaxis transducer | |
| PA2593 | −5.2 | ES | Hypothetical protein | |
| PA2629 | purB | −3.6 | ES | Adenylosuccinate lyase |
| PA2663 | 41.4 | ES | Hypothetical protein | |
| PA2691 | 3 | ES | Conserved hypothetical protein | |
| PA2763 | −6.5 | ES | Hypothetical protein | |
| PA2798 | −3.2 | ES | Probable two-component response regulator | |
| PA2968 | fabD | −3.2 | ES | Malonyl-CoA-(acyl carrier protein) transacylase |
| PA2969 | plsX | −3.3 | ES | Fatty acid biosynthesis protein PlsX |
| PA3019 | −3.2 | ES | Probable ATP-binding component of ABC transporter | |
| PA3188 | 3.4 | ML | Probable permease of ABC sugar transporter | |
| PA3221 | csaA | 8.1 | ES | CsaA protein |
| PA3222 | 7 | ES | Hypothetical protein | |
| PA3232 | 3.2 | ML | Probable nuclease | |
| PA3234 | 5 | ML | Probable sodium-solute symporter | |
| PA3235 | 3.5 | ML | Conserved hypothetical protein | |
| PA3294 | −4 | ES | Hypothetical protein | |
| PA3327 | −3.9 | ES | Probable nonribosomal peptide synthetase | |
| PA3328 | −7.9 | ES | Probable FAD-dependent monooxygenase | |
| PA3329 | −9.6 | ES | Hypothetical protein | |
| PA3330 | −10.9 | ES | Probable short-chain dehydrogenase | |
| PA3331 | −8.4 | ES | Cytochrome P450 | |
| PA3333 | fabH2 | −9.1 | ES | 3-Oxoacyl (acyl carrier protein) synthase III |
| PA3334 | −8.4 | ES | Probable acyl carrier protein | |
| PA3335 | −11.5 | ES | Hypothetical protein | |
| PA3336 | −12.5 | ES | Probable MFS transporter | |
| PA3391 | nosR | 35.5 | ES | Regulatory protein NosR |
| PA3393 | nosD | 31.3 | ES | NosD protein |
| PA3394 | nosF | 33.4 | ES | NosF protein |
| PA3395 | nosY | 36 | ES | NosY protein |
| PA3396 | nosL | 21.6 | ES | NosL protein |
| PA3416 | 3.9 | ML | Probable pyruvate dehydrogenase E1 component, beta chain | |
| PA3418 | ldh | 3.3 | ML | Leucine dehydrogenase |
| PA3430 | −3 | ML | Probable aldolase | |
| PA3431 | −8.8 | ML | Conserved hypothetical protein | |
| PA3432 | −10.1 | ML | Hypothetical protein | |
| PA3478 | rhlB | −6.5 | ES | Rhamnosyltransferase chain B |
| PA3479 | rhlA | −6.5 | ES | Rhamnosyltransferase chain A |
| PA3486 | −12 | ES | Conserved hypothetical protein | |
| PA3487 | pldA | −3.2 | ES | Phospholipase D |
| PA3530 | 4.1 | ES | Conserved hypothetical protein | |
| PA3552 | 6.5 | ES | Conserved hypothetical protein | |
| PA3569 | mmsB | 3.9 | ML | 3-Hydroxyisobutyrate dehydrogenase |
| PA3570 | mmsA | 4.8 | ML | Methylmalonate-semialdehyde dehydrogenase |
| PA3605 | −5.3 | ES | Hypothetical protein | |
| PA3655 | tsf | −3.1 | ML | Elongation factor Ts |
| PA3700 | lysS | −3.6 | ES | Lysyl-tRNA synthetase |
| PA3723 | 4.2 | ML | Probable FMN oxidoreductase | |
| PA3741 | −4.7 | ES | Hypothetical protein | |
| PA3742 | rplS | −3.7 | ML | 50S ribosomal protein L19 |
| PA3811 | hscB | 3.2 | ES | Heat shock protein HscB |
| PA3813 | iscU | 3.6 | ES | Probable iron-binding protein IscU |
| PA3872 | narI | 37 | ES | Respiratory nitrate reductase gamma chain |
| PA3876 | narK2 | 3.4 | ML | Nitrite extrusion protein 2 |
| PA3880 | 7.6 | ES | Conserved hypothetical protein | |
| PA3881 | 3.1 | ES | Hypothetical protein | |
| PA3901 | fecA | −5.7 | ML | Fe(III) dicitrate transport protein FecA |
| PA3905 | −4.1 | ES | Hypothetical protein | |
| PA3907 | −3.8 | ES | Hypothetical protein | |
| PA3912 | 3.3 | ML | Conserved hypothetical protein | |
| PA3913 | 3.6/14.8 | B | Probable protease | |
| PA3914 | moeAI | 7.3/−62.7 | B | Molybdenum cofactor biosynthetic protein A1 |
| PA3915 | moaBI | 6.3/−15.6 | B | Molybdopterin biosynthetic protein B1 |
| PA3916 | moaE | 5.8 | ES | Molybdopterin-converting factor, large subunit |
| PA3917 | moaD | 6.7 | ES | Molybdopterin-converting factor, small subunit |
| PA3918 | moaC | 6.3 | ES | Molybdopterin biosynthetic protein C |
| PA3979 | −3.1 | ES | Hypothetical protein | |
| PA4055 | ribC | −4.1 | ES | Riboflavin synthase alpha chain |
| PA4063 | 4.1 | ES | Hypothetical protein | |
| PA4142 | −4.1 | ES | Probable secretion protein | |
| PA4170 | 5.8 | ES | Hypothetical protein | |
| PA4210 | phzAI | −11.9 | ES | Probable phenazine biosynthesis protein |
| PA4211 | phzBI | −14.8 | ES | Probable phenazine biosynthesis protein |
| PA4217 | phzS | −13.1 | ES | Flavin-containing monooxygenase |
| PA4235 | bfrA | 3.4 | ES | Bacterioferritin |
| PA4292 | −4.5 | ES | Probable phosphate transporter | |
| PA4432 | rpsI | −4 | ES | 30S ribosomal protein S9 |
| PA4479 | mreD | −3.1 | ES | Rod shape-determining protein MreD |
| PA4497 | 3.8 | ML | Probable binding protein component of ABC transporter | |
| PA4512 | lpxO1 | −3.1 | ES | Lipopolysaccharide biosynthetic protein |
| PA4610 | 4.2 | ES | Hypothetical protein | |
| PA4620 | −3 | ES | Hypothetical protein | |
| PA4665 | prfA | −3.5 | ES | Peptide chain release factor 1 |
| PA4670 | prs | −3.9 | ES | Ribose-phosphate pyrophosphokinase |
| PA4672 | −5 | ES | Peptidyl-tRNA hydrolase | |
| PA4743 | rbfA | −3.2 | ES | Ribosome-binding factor A |
| PA4762 | grpE | −3.1 | ML | Heat shock protein GrpE |
| PA4853 | fis | −3.4 | ES | DNA-binding protein Fis |
| PA4916 | −9.8 | ML | Hypothetical protein | |
| PA4917 | −6.7 | ML | Hypothetical protein | |
| PA4918 | −7.2 | ML | Hypothetical protein | |
| PA4919 | pncB1 | −6.2 | ML | Nicotinate phosphoribosyltransferase |
| PA4920 | nadE | −4.4 | ML | NH3-dependent NAD synthetase |
| PA4921 | −3.9 | ML | Hypothetical protein | |
| PA5023 | 4.3 | ES | Conserved hypothetical protein | |
| PA5048 | −4.2 | ES | Probable nuclease | |
| PA5117 | typA | −5 | ES | Regulatory protein TypA |
| PA5118 | thiI | −3.5 | ES | Thiazole biosynthesis protein Thil |
| PA5139 | −5 | ES | Hypothetical protein | |
| PA5167 | 3 | ES | Probable C4-dicarboxylate-binding protein | |
| PA5168 | 4.3 | ES | Probable dicarboxylate transporter | |
| PA5169 | 3.6 | ES | Probable C4-dicarboxylate transporter | |
| PA5275 | 3.8 | ES | Conserved hypothetical protein | |
| PA5296 | rep | −3.1 | ES | ATP-dependent DNA helicase Rep |
| PA5304 | dadA | −4.3 | ES | d-Amino acid dehydrogenase, small subunit |
| PA5372 | betA | 4.2 | ML | Choline dehydrogenase |
| PA5415 | glyAI | 4.3 | ML | Serine hydroxymethyltransferase |
| PA5436 | −3.4 | ES | Probable biotin carboxylase subunit of a transcarboxylase | |
| PA5446 | −7.2 | ML | Hypothetical protein | |
| PA5448 | wbpY | 3.2 | ML | Glycosyltransferase WbpY |
| PA5460 | −3.2 | ML | Hypothetical protein | |
| PA5496 | 3.1 | ES | Hypothetical protein | |
| PA5553 | atpC | −3 | ML | ATP synthase epsilon chain |
| PA5554 | atpD | −3.1 | ML | ATP synthase beta chain |
| PA5556 | atpA | −3.4 | ML | ATP synthase alpha chain |
| PA5557 | atpH | −3 | ML | ATP synthase delta chain |
| ig326671 | 2.9/3.5 | B | Intergenic region between PA0458 and PA0459, 326671-327284, plus strand | |
| ig4326394 | 3.2 | ES | Intergenic region between PA3969 and PA3970, 4326394-4327696, plus strand | |
| ig4713098 | −5.3 | ES | Intergenic region between PA4280 and PA4281, 4713098-4713795, plus strand |
Only transcripts identified as nitrate regulated that demonstrated a change of equal to or greater than threefold are reported. A list of all transcripts identified as being nitrate regulated exhibiting a statistically significant change (P ≤ 0.05) is available online ( http://www.urmc.rochester.edu/smd/mbi/bhi/). Genes are identified by transcript number, gene name, and protein description (http://www.pseudomonas.com).
The change (n-fold) was calculated by comparing PAO1 grown aerobically lacking nitrate (baseline) to PAO1 grown aerobically with nitrate (experimental). A positive change represents induction caused by the presence of nitrate, and a negative change represents repression caused by the presence of nitrate. When two values are given, the first is for the ML phase and the second is for the ES phase.
Growth stage(s) during which statistically significantly differential transcript expression was observed. ML, midexponential phase; ES, early stationary phase; B, both midexponential and early stationary phases.
Protein descriptions are from the Pseudomonas Genome Project website (www.pseudomonas.com). CoA, coenzyme A; FAD, flavin adenine dinucleotide; MSF, major facilitator superfamily; ABC, ATP-binding cassette; FMN, flavin mononucleotide.
The transcription of narI was up-regulated by the presence of nitrate, while narG, narH, and narJ were not found to be statistically differentially expressed. napB, napA, napD, nosRDFYL, and nirS exhibited increased expression in the presence of nitrate. Other genes encoding proteins either implicated (PA0513, PA0514, PA0516, PA0518, and PA0521) or known to be involved (nirL, nirM, nirN, and nirF) in the processing of respiratory system components were up-regulated in response to nitrate. Our results indicate that nitrate alone is sufficient to induce the expression of many enzymes involved in denitrification regardless of the presence or absence of oxygen and explain the apparent lack of differential expression of some of these genes in our anaerobic experiments.
The expression of a number of genes involved in the production of virulence factors of P. aeruginosa were influenced by the addition of nitrate. For example, while mexAB and rhlAB expression were repressed by nitrate, mexF was up-regulated. Transcription of a recently described chemotaxis cluster (PA0174-0179) found to be required for optimal chemotaxis (6) and aerotaxis (11) was induced by the nitrate. The R-type pyocins (PA0614 to PA0646) (18) were induced by nitrate. We also observed differential expression of 306 transcripts which currently do not have defined functions. Importantly, a couple of transcripts (PA0459 and PA5167) have been previously found to be required for lung infection (21).
Consistent with previous reports for Escherichia coli (23), Shewanella oneidensis, and Bacillus subtilis (3, 30), we found that P. aeruginosa significantly changes its transcriptional profile in the absence of oxygen or in the presence of nitrate. It should be noted that our results are biased towards using nitrate as the terminal electron acceptor, and using other nitric oxides or arginine may affect other genes.
Our results provide a global view of oxygen-regulated gene expression in P. aeruginosa and illustrate the complex regulation of anaerobic metabolism in this organism. Changes in genes encoding virulence factors and quorum-sensing components implicate altered pathogenic pathways during anaerobic growth. Our identification of a substantial number of genes encoding proteins of unknown function should contribute to further annotation of the genome and provide impetus for further research on the role of these genes in P. aeruginosa physiology and metabolism.
Acknowledgments
We thank A. Brooks, K. Miller, L. Ascroft, and K. Wahowski at the Microarray Core Facility in The Functional Genomics Center at the University of Rochester for technical support and assistance with the quantitative RT-PCR and Cystic Fibrosis Foundation Therapeutics, Inc., for subsidizing the P. aeruginosa Affymetrix GeneChip arrays.
This work was supported by grants IGLEWS00V0 and IGLEWS03FG0 to B.H.I., L.P., and C.G.H. from Cystic Fibrosis Foundation Therapeutics and grant R37AI33713 to B.H.I from the NIH. M.J.F is supported by an NIH fellowship (F32AI056825).
Editor: J. T. Barbieri
REFERENCES
- 1.Arai, H., Y. Igarashi, and T. Kodama. 1995. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR. FEBS Lett. 371:73-76. [DOI] [PubMed] [Google Scholar]
- 2.Arai, H., T. Kodama, and Y. Igarashi. 1999. Effect of nitrogen oxides on expression of the nir and nor genes for denitrification in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 170:19-24. [DOI] [PubMed] [Google Scholar]
- 3.Beliaev, A. S., D. K. Thompson, T. Khare, H. Lim, C. C. Brandt, G. Li, A. E. Murray, J. F. Heidelberg, C. S. Giometti, J. Yates III, K. H. Nealson, J. M. Tiedje, and J. Zhoui. 2002. Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors. OMICS 6:39-60. [DOI] [PubMed] [Google Scholar]
- 4.Cao, H., G. Krishnan, B. Goumnerov, J. Tsongalis, R. Tompkins, and L. G. Rahme. 2001. A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism. Proc. Natl. Acad. Sci. USA 98:14613-14618. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Chen, F., Q. Xia, and L.-K. Ju. 2003. Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(P)H fluorescence. Appl. Environ. Microbiol. 69:6715-6722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ferrández, A., A. C. Hawkins, D. T. Summerfield, and C. S. Harwood. 2002. Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response. J. Bacteriol. 184:4374-4383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Govan, J. R. W., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60:539-574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Haas, D., M. Gamper, and A. Zimmermann. 1992. Anaerobic control in Pseudomonas aeruginosa, p. 177-187. In E. Galli, S. Silver, and B. Witholt (ed.), Pseudomonas: molecular biology and biotechnology. American Society for Microbiology, Washington, D.C.
- 9.Hassett, D. J. 1996. Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. J. Bacteriol. 178:7322-7325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Hassett, D. J., J. Cuppoletti, B. Trapnell, S. V. Lymar, J. J. Rowe, S. Sun Yoon, G. M. Hilliard, K. Parvatiyar, M. C. Kamani, D. J. Wozniak, S. H. Hwang, T. R. McDermott, and U. A. Ochsner. 2002. Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Adv. Drug Delivery Rev. 54:1425-1443. [DOI] [PubMed] [Google Scholar]
- 11.Hong, C. S., M. Shitashiro, A. Kuroda, T. Ikeda, N. Takiguchi, H. Ohtake, and J. Kato. 2004. Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 231:247-252. [DOI] [PubMed] [Google Scholar]
- 12.Irani, V. R., A. Darzins, and J. J. Rowe. 1997. Snr, new genetic loci common to the nitrate reduction systems of Pseudomonas aeruginosa PAO1. Curr. Microbiol. 35:9-13. [DOI] [PubMed] [Google Scholar]
- 13.Ka, J. O., J. Urbance, R. W. Ye, T. Y. Ahn, and J. M. Tiedje. 1997. Diversity of oxygen and N-oxide regulation of nitrite reductases in denitrifying bacteria. FEMS Microbiol. Lett. 156:55-60. [DOI] [PubMed] [Google Scholar]
- 14.Kerschen, E. J., V. R. Irani, D. J. Hassett, and J. J. Rowe. 2001. snr-1 gene is required for nitrate reduction in Pseudomonas aeruginosa PAO1. J. Bacteriol. 183:2125-2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Krieger, R., A. Rompf, M. Schobert, and D. Jahn. 2002. The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor. Mol. Genet. Genomics 267:409-417. [DOI] [PubMed] [Google Scholar]
- 16.Mercenier, A., J.-P. Simon, C. Vander Wauven, D. Haas, and V. Stalon. 1980. Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa. J. Bacteriol. 144:159-163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Moreno-Vivián, C., P. Cabello, M. Martínez-Luque, R. Blasco, and F. Castillo. 1999. Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J. Bacteriol. 181:6573-6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Nakayama, K., K. Takashima, H. Ishihara, T. Shinomiya, M. Kageyama, S. Kanaya, M. Ohnishi, T. Murata, H. Mori, and T. Hayashi. 2000. The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol. Microbiol. 38:213-231. [DOI] [PubMed] [Google Scholar]
- 19.Ochsner, U. A., P. J. Wilderman, A. I. Vasil, and M. L. Vasil. 2002. GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol. Microbiol. 45:1277-1287. [DOI] [PubMed] [Google Scholar]
- 20.Pessi, G., and D. Haas. 2000. Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa. J. Bacteriol. 182:6940-6949. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Potvin, E., D. E. Lehoux, I. Kukavica-Ibrulj, K. L. Richard, F. Sanschagrin, G. W. Lau, and R. C. Levesque. 2003. In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ. Microbiol. 5:1294-1308. [DOI] [PubMed] [Google Scholar]
- 22.Rompf, A., C. Hungerer, T. Hoffmann, M. Lindenmeyer, U. Romling, U. Gross, M. O. Doss, H. Arai, Y. Igarashi, and D. Jahn. 1998. Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr. Mol. Microbiol. 29:985-997. [DOI] [PubMed] [Google Scholar]
- 23.Salmon, K., S. P. Hung, K. Mekjian, P. Baldi, G. W. Hatfield, and R. P. Gunsalus. 2003. Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR. J. Biol. Chem. 278:29837-29855. [Online.] [DOI] [PubMed] [Google Scholar]
- 24.Thöny-Meyer, L. 1997. Biogenesis of respiratory cytochromes in bacteria. Microbiol. Mol. Biol. Rev. 61:337-376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Vallet, I., J. W. Olson, S. Lory, A. Lazdunski, and A. Filloux. 2001. The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. USA 98:6911-6916. [Online.] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Vander Wauven, C., A. Piérard, M. Kley-Raymann, and D. Haas. 1984. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J. Bacteriol. 160:928-934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Wagner, V. E., D. Bushnell, L. Passador, A. I. Brooks, and B. H. Iglewski. 2003. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J. Bacteriol. 185:2080-2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Worlitzsch, D., R. Tarran, M. Ulrich, U. Schwab, A. Cekici, K. C. Meyer, P. Birrer, G. Bellon, J. Berger, T. Weiss, K. Botzenhart, J. R. Yankaskas, S. Randell, R. C. Boucher, and G. Doring. 2002. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Investig. 109:317-325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ye, R. W., D. Haas, J.-O. Ka, V. Krishnapillai, A. Zimmermann, C. Baird, and J. M. Tiedje. 1995. Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J. Bacteriol. 177:3606-3609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ye, R. W., W. Tao, L. Bedzyk, T. Young, M. Chen, and L. Li. 2000. Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions. J. Bacteriol. 182:4458-4465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Yoon, S. S., R. F. Hennigan, G. M. Hilliard, U. A. Ochsner, K. Parvatiyar, M. C. Kamani, H. L. Allen, T. R. DeKievit, P. R. Gardner, U. Schwab, J. J. Rowe, B. H. Iglewski, T. R. McDermott, R. P. Mason, D. J. Wozniak, R. E. Hancock, M. R. Parsek, T. L. Noah, R. C. Boucher, and D. J. Hassett. 2002. Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev. Cell 3:593-603. [DOI] [PubMed] [Google Scholar]
- 32.Zumft, W. G. 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61:533-616. [DOI] [PMC free article] [PubMed] [Google Scholar]