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Journal of Bacteriology, December 2005, p. 8185-8190, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.8185-8190.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Pseudomonas aeruginosa Proteome during Anaerobic Growth{ddagger}

Manhong Wu,1,{dagger} Tina Guina,2,{dagger} Mitchell Brittnacher,1 Hai Nguyen,1 Jimmy Eng,3 and Samuel I. Miller1*

Departments of Medicine, Microbiology, and Genome Sciences,1 Department of Pediatrics, Division of Infectious Diseases,University of Washington,2 Fred Hutchinson Cancer Research Center, Seattle, Washington3

Received 7 September 2005/ Accepted 16 September 2005


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ABSTRACT
 
Isotope-coded affinity tag analysis and two-dimensional gel electrophoresis followed by tandem mass spectrometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metabolism. These results form the basis for future analyses of alterations in bacterial protein content during growth in various environments, including the cystic fibrosis airway.


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TEXT
 
Pseudomonas aeruginosa is a ubiquitous environmental gram-negative bacterium found in soil and water. It is also an opportunistic pathogen that causes infections in individuals with innate immune defects, including cystic fibrosis (CF) patients (8). P. aeruginosa encounters low-oxygen environments in soil and water. Evidence indicates that in humans with CF, bacteria may, at least in part, be in a low-oxygen environment within mucopurulent masses or biofilms within the respiratory tracts (19). P. aeruginosa is able to grow anaerobically in the presence of terminal electron acceptors, such as nitrate (NO3–), nitrite (NO2–), and nitrous oxide (N2O), or when L-arginine is a substrate for growth (21). The CF airway mucus is sufficiently rich in NO3– and NO2– to support the anaerobic growth of P. aeruginosa (7, 19). In this study, a comparison of the P. aeruginosa proteome during growth in the presence and absence of oxygen was performed.

P. aeruginosa strain PAO1 obtained from Steve Lory (Harvard Medical School, Boston, MA) was grown in 125-ml flasks in Luria broth (LB) supplemented with 1% KNO3 with shaking at 200 rpm at 37°C for aerobic growth. Anaerobic growth was completed as previously described (9) in 80 ml of medium in 100-ml Wheaton serum bottles (Fisher Scientific) with rubber stoppers. The medium was deprived of oxygen by being subjected to bubbling with N2 gas for 1 h. For both aerobic and anaerobic conditions, bacteria were harvested at the late logarithmic phase of growth, at which point the cell density (optical density at 600 nm) of the anaerobic culture was 44% of the density of the aerobic culture. There was no significant difference between the pHs of the harvested cultures (pH 7.6 for the anaerobic culture and pH 7.4 for the aerobic culture). Equal amounts of denatured and reduced whole-cell protein (2.0 mg from each growth state) were labeled with either light (12C) or heavy (13C) cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA), processed, and analyzed as previously described (3). The reported data are the averages of at least two independent experiments.

Six hundred ten P. aeruginosa proteins were identified and quantified using ICAT (for a complete list of proteins, see Table S1 in the supplemental material). Among 151 proteins whose abundances changed during anaerobic growth, 76 were higher in abundance (Table 1) and 75 were lower in abundance (Table 2). As expected, 13 proteins that participate in anaerobic growth and denitrification (including products of nir, nos, and nar genes) were expressed at higher levels during anaerobic growth (Table 1). These results suggest that the observed changes in protein content include those resulting specifically from growth at different oxygen levels.


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  		TABLE 1. P. aeruginosa proteins with increased abundance during anaerobic growth


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  		TABLE 2. P. aeruginosa proteins with decreased abundance during anaerobic growth

The changes in the detected proteome could also reflect differences in all density-dependent regulation in addition to effects of oxygen tension, given the lower relative cell density of the harvested anaerobic culture. Indeed, 29 proteins detected in lower abundance in anaerobically grown cells are encoded by genes previously shown to be quorum sensing induced (5, 16, 17). These include the hydrogen cyanide synthase subunits HcnB and HcnC; the Pseudomonas quinolone signal biosynthetic enzymes PqsB, PqsC, and PqsD; and PhnB (Table 2). Consistent with our results, hcn and pqs genes were also found to be transcriptionally repressed during anaerobic growth by a recent DNA microarray analysis using aerobic and anaerobic cultures harvested at the same cell density (1) (Table 2).

To identify secreted P. aeruginosa proteins with altered levels during anaerobic growth, culture supernatant proteins were concentrated (11) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). Four Coomassie-stained protein bands, corresponding to differentially expressed proteins, were identified and analyzed as in a previous study (4) (Fig. 1). The abundances of three secreted proteins appeared to decrease during anaerobic growth: the CbpD chitin-binding protein, LasB elastase, and a protein of unknown function encoded by PA0572. Previous proteomic studies found that all three of these proteins are quorum sensing induced (11). One protein appeared to be increased in abundance during anaerobic growth and was identified as either the flagellar filament protein FliC or the flagellar capping protein FliD (due to the overlap in characteristics of these two proteins).



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  		FIG. 1. P. aeruginosa secreted proteins expressed during anaerobic growth. P. aeruginosa culture supernatant proteins were separated by 12% SDS-PAGE and detected by staining with Coomassie. Proteins that changed in abundance during anaerobic growth (relative to aerobic growth) are labeled. –O2, anaerobic growth; +O2, aerobic growth.

Most P. aeruginosa outer membrane proteins do not contain cysteine residues and hence cannot be analyzed by ICAT (4). Therefore, two-dimensional (2D) PAGE was used as a complementary method (4). Several outer membrane proteins (Fig. 2) were excised from the 2D gel and identified (4). OprE appeared to increase in abundance during anaerobic growth, while OprF and OprH appeared to decrease in abundance (Fig. 2). All three proteins migrated as multiple species during isoelectric focusing (Fig. 2). Decreased abundance of OprF during anaerobic growth was confirmed by immunoblotting of outer membrane proteins (data not shown), using a polyclonal anti-OprF antiserum (a gift from Robert Hancock, University of British Columbia at Vancouver, Canada).



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  		FIG. 2. P. aeruginosa outer membrane proteins expressed during anaerobic growth. Outer membrane proteins were separated by 12% 2D PAGE and detected by staining with Coomassie. Proteins were separated in the first dimension by isoelectric focusing (IEF) at pI ranges of 4 to 7 (A) and 6 to 11 (B). Proteins that changed in abundance during anaerobic growth (relative to aerobic growth) are labeled with arrows. –O2, anaerobic growth; +O2, aerobic growth.

Among the P. aeruginosa proteins that showed increased abundance during anaerobic growth (Table 1; Fig. 1), several contribute to functions involved in the formation and development of biofilms. These proteins include the catabolite repression control protein Crc and the twitching motility proteins PilU, PilG, and ChpA (12, 13, 18). Consistent with an increased level of Crc in anaerobically grown cells (Table 1), known targets of Crc repression were decreased in abundance (Table 2), including the hmgA and bkd gene products (6, 10). ChpA and PilG are components of a complex regulatory system controlling twitching motility (18). Taken together, these results suggest that expression or function of cell surface appendages that affect biofilm formation is altered during anaerobic growth. Such changes may contribute to the increased biofilm formation observed for P. aeruginosa growing anaerobically (20).

In addition to the changes in outer membrane proteins observed during anaerobic growth, ICAT analysis showed that several enzymes involved in the biosynthesis of P. aeruginosa lipopolysaccharide (LPS) were expressed at higher levels during anaerobic growth (Table 1). These included a homologue of beta-hydroxylase LpxO2, which hydroxylates lipid A fatty acids (14); LPS core heptosyltransferases WaaC and WaaF (2, 15); and WbpG, which is encoded by a gene cluster that participates in the synthesis of a long B-band O antigen. These results suggest that LPS content could be altered as a consequence of anaerobiosis.

In summary, the P. aeruginosa proteome changes significantly during anaerobic growth. We identified 617 proteins in total: 610 by ICAT analysis, 4 by SDS-PAGE analysis, and 3 by 2D PAGE analysis. Of the 617 identified proteins, the abundances of 158 varied between anaerobically grown and aerobically grown cells. Because P. aeruginosa reached a lower cell density under our anaerobic growth conditions than under aerobic growth conditions, density-dependent changes in protein expression may have contributed to the proteome that we detected during anaerobic growth. Nevertheless, bacterial cell density is likely to be similarly limited in many environmental niches where multiple nutrients (including oxygen) are scarce. Therefore, the changes in protein levels that we have detected contribute to an understanding of how the proteome and metabolic state of bacteria vary in response to different environments. Direct analysis of bacterial protein content is a robust technology to observe the adaptation of bacteria to specific environmental niches, including the CF airway.


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ACKNOWLEDGMENTS
 
We thank David D'Argenio and Robert Ernst for helpful discussions, Xiaojun Li and Biaoyang Lin for help with the data analysis, and Susan Farmer and Robert Hancock for providing the anti-OprF antibodies.

This work was supported by NIH grant DK 064954 to S. I. Miller and by Cystic Fibrosis Foundation grant R565-CR02 to T. Guina.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departments of Medicine, Microbiology, and Genome Sciences, University of Washington, HSB K140, Box 357710, Seattle, WA 98195. Phone: (206) 616-5107. Fax: (206) 616-5109. E-mail: millersi{at}u.washington.edu. Back

{ddagger} Supplemental material for this article may be found at http://jb.asm.org/. Back

{dagger} These authors contributed equally to this work. Back


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Journal of Bacteriology, December 2005, p. 8185-8190, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.8185-8190.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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