APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 1221–1230
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 4
Transcriptional and Proteomic Responses of Pseudomonas aeruginosa
PAO1 to Spaceflight Conditions Involve Hfq Regulation and
Reveal a Role for Oxygen?
Aure ´lie Crabbe ´,1Michael J. Schurr,2Pieter Monsieurs,3Lisa Morici,7Jill Schurr,9
James W. Wilson,4C. Mark Ott,5George Tsaprailis,8Duane L. Pierson,5
Heidi Stefanyshyn-Piper,6and Cheryl A. Nickerson1*
The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona1; School of
Medicine, University of Colorado, Aurora, Colorado2; Belgian Nuclear Research Center (SCK-CEN), Mol, Belgium3;
Department of Biology, Villanova University, Villanova, Pennsylvania4; Habitability and Environmental Factors
Division, NASA-Johnson Space Center, Houston, Texas5; Astronaut Office, NASA-Johnson Space Center,
Houston, Texas6; Tulane University Health Sciences Center, New Orleans, Louisiana7; Center for Toxicology,
University of Arizona, Tucson, Arizona8; and Affymetrix Inc., Santa Clara, California9
Received 2 July 2010/Accepted 20 November 2010
Assessing bacterial behavior in microgravity is important for risk assessment and prevention of infectious
diseases during spaceflight missions. Furthermore, this research field allows the unveiling of novel connections
between low-fluid-shear regions encountered by pathogens during their natural infection process and bacterial
virulence. This study is the first to characterize the spaceflight-induced global transcriptional and proteomic
responses of Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat. P.
aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins,
with Hfq as a global transcriptional regulator. Since Hfq was also differentially regulated in spaceflight-grown
Salmonella enterica serovar Typhimurium, Hfq represents the first spaceflight-induced regulator acting across
bacterial species. The major P. aeruginosa virulence-related genes induced in spaceflight were the lecA and lecB
lectin genes and the gene for rhamnosyltransferase (rhlA), which is involved in rhamnolipid production. The
transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data for this
organism grown in microgravity analogue conditions using the rotating wall vessel (RWV) bioreactor. Inter-
esting similarities were observed, including, among others, similarities with regard to Hfq regulation and
oxygen metabolism. While RWV-grown P. aeruginosa mainly induced genes involved in microaerophilic me-
tabolism, P. aeruginosa cultured in spaceflight presumably adopted an anaerobic mode of growth, in which
denitrification was most prominent. Whether the observed changes in pathogenesis-related gene expression in
response to spaceflight culture could lead to an alteration of virulence in P. aeruginosa remains to be
determined and will be important for infectious disease risk assessment and prevention, both during space-
flight missions and for the general public.
The microgravity environment associated with spaceflight is
unique and has a profound effect on both host and pathogen
cells, with potential implications for infectious disease. From
the host point of view, astronauts experience a compromised
immune response under spaceflight conditions, as reflected in
cellular alterations of both the innate and adaptive immune
systems (23, 26, 40). Spaceflight has been shown to alter the
response of monocytes, isolated from astronauts preflight and
in flight, to Gram-negative toxins (27). Further, simulation of
aspects of this microgravity-associated decreased immune re-
sponse, using the hind limb unloaded mouse model, showed an
enhanced susceptibility of these animals to bacterial infection
(3, 6). From the pathogen’s perspective, bacterial obligate and
opportunistic pathogens have been found to exhibit enhanced
stress resistance phenotypes following growth under both true
spaceflight and microgravity analogue conditions (13, 30, 33,
46–49). In response to the spaceflight environment, global
transcriptional and proteomic changes were observed for the
enteric pathogen Salmonella enterica serovar Typhimurium
grown in the complex medium Lennox L broth base (LB),
which were associated with an increased virulence in a murine
model of infection (46). Moreover, the small RNA binding
protein Hfq was identified as a major transcriptional regulator
of S. Typhimurium responses to the spaceflight environment.
A subsequent study demonstrated that cultivation of S. Typhi-
murium in M9 minimal medium abolished the spaceflight-
induced virulence (47). While M9 spaceflight cultures of S.
Typhimurium exhibited virulence characteristics dramatically
different from those in LB, microarray and proteomic analyses
revealed a role for Hfq in both the M9 and LB spaceflight
stimulons (47). Complementary experiments using the rotating
wall vessel (RWV) bioreactor, in which cells are cultured in a
microgravity analogue low-fluid-shear environment (i.e., low-
shear modeled microgravity [LSMMG]), identified that a low
phosphate concentration in LB could be the origin of the
spaceflight-induced virulence of S. Typhimurium (47). Inter-
estingly, LSMMG and spaceflight induced similar increased
* Corresponding author. Mailing address: The Biodesign Institute,
Center for Infectious Diseases and Vaccinology, Arizona State Uni-
versity, 1001 S. McAllister Avenue, Tempe, AZ 85287. Phone: (480)
727-7520. Fax: (480) 727-8943. E-mail: firstname.lastname@example.org.
?Published ahead of print on 17 December 2010.
virulence when LB medium was used, and overlapping gene
expression profiles, including Hfq and members of the Hfq
regulon, were obtained for both growth conditions (47). The
latter indicates that molecular and phenotypic similarities be-
tween LSMMG and spaceflight conditions could be attributed
to the analogous low-fluid-shear environment.
Recently, the global transcriptional response of the oppor-
tunistic pathogen Pseudomonas aeruginosa PAO1 to LSMMG
was determined (13). As a ubiquitous organism colonizing both
environmental niches and the human body, P. aeruginosa is
found in spacecrafts and has previously caused infections in
astronauts (8, 24, 35, 43). Cultivation of P. aeruginosa in the
LSMMG environment of the RWV induced molecular path-
ways known to be of importance for virulence, compared to
control conditions. In agreement with the microarray data, an
increased production of the exopolysaccharide alginate, en-
hanced resistance to heat and oxidative stress, and a decreased
oxygen transfer rate were observed. The alternative sigma fac-
tor AlgU and Hfq were both proposed as important mediators
of the LSMMG response in P. aeruginosa. In addition, by
comparing the behavior of P. aeruginosa cultured in LSMMG
to that in a higher-fluid-shear control at body temperature,
clinically relevant traits were found to be induced, such as
biofilm formation, rhamnolipid production, and the C4-homo-
serine lactone quorum sensing system (12). Collectively, these
data indicate that low fluid shear has an impact on the behav-
ior, and possibly on the pathogenicity, of P. aeruginosa.
Importantly, low-fluid-shear zones are believed to be en-
countered by pathogens during their natural course of infec-
tion in vivo, including in the intestinal, respiratory, and uro-
genital tracts (12, 34). Therefore, in addition to the importance
of spaceflight research for the evaluation of infectious disease
risk during long-term missions, this research field has the po-
tential to provide novel insights in the role of fluid shear in
virulence and the disease process.
This study describes the global transcriptional and transla-
tional responses of P. aeruginosa PAO1 to the microgravity
environment of spaceflight. Our aim was to assess whether the
microgravity environment of spaceflight could induce virulence
traits in P. aeruginosa and if evolutionarily conserved pathways
in common with those of spaceflight-grown S. Typhimurium
were regulated in a similar fashion. Furthermore, the space-
flight (this study) and LSMMG (13) responses of P. aeruginosa
were compared and revealed interesting similarities. In addi-
tion to the role of low fluid shear in these observations, the
possible involvement of the adopted experimental setup is dis-
cussed. The present study is the first to assess the molecular
response of an important opportunistic pathogen following
growth under actual spaceflight conditions and provides im-
portant insights into the evaluation and, eventually, the pre-
vention of P. aeruginosa infections during spaceflight missions.
MATERIALS AND METHODS
Bacterial strain and growth media. A derivative of the wild-type P. aeruginosa
PAO1 (ATCC 15692), which contained a gentamicin resistance cassette in the
attB site, was used for the spaceflight experiment. The gentamicin-resistant strain
was constructed through homologous recombination as described previously
(38). P. aeruginosa PAO1 was grown in LB medium containing 25 ?g/ml genta-
micin in the spaceflight hardware (see below) to avoid growth of any contami-
nants. The bacterial inoculum (1.5 ? 108CFU/ml) in the spaceflight hardware
was suspended in 0.5 ml phosphate-buffered saline (PBS) (Invitrogen) and re-
mained viable but static (not growing) during launch and until 9 days into the
flight. After this time, growth was initiated by the addition of LB as described
below. Cells were fixed in flight using the RNA and protein fixative RNA Later
II (Ambion). At 2.5 h after landing of the space shuttle at the Kennedy Space
Center (KSC), samples were recovered and subsequently used for whole-genome
transcriptional microarray and proteomic analyses. In each case, the flight cul-
ture samples were compared with synchronous culture samples grown under
identical conditions on the ground at KSC using coordinated activation and
termination times (by means of real-time communications with the shuttle
astronauts) in an insulated room that maintained temperature and humidity
levels identical to those on the shuttle (orbital environment simulator).
Experimental setup adopted for spaceflight culturing. Growth of P. aeruginosa
PAO1 was initiated in flight, and cells were cultured in space and on the ground
in specialized hardware termed the fluid-processing apparatus (FPA) as de-
scribed previously (46, 47) (Fig. 1). Briefly, FPAs are glass barrels, containing a
bevel on the side, in which rubber stoppers are inserted for compartmentaliza-
tion. The bottom stopper contained a gas exchange membrane. Glass barrels and
rubber stoppers were coated with a silicone lubricant (Sigmacote; Sigma) and
autoclaved separately before assembly. The subsequent insertion of rubber stop-
pers into the FPAs resulted in the creation of three separate compartments
which contained, from top to bottom, (i) RNA Later II fixative (2.5 ml), (ii)
bacteria suspended in PBS (0.5 ml), and (iii) LB culture medium (2 ml). The last
compartment was created at the level of the bevel. Each FPA was loaded into a
lexan sheet that contained a gas-permeable membrane at the bottom, and eight
FPAs were subsequently loaded into larger containers, termed group activation
packs (GAPs). This experimental setup created a triple level of containment for
crew safety. At specific time points in flight, an astronaut manually inserted a
hand crank into the end of the GAP and turned it, which pushed down on a
pressure plate underneath, resulting in a plunging action on the rubber stoppers
of each FPA. This plunging action, which allowed for mixing of fluids between
different compartments through the bevel, was performed twice in flight. The
first plunging action, referred to as activation, served to add LB growth medium
to the cells, and the second (following a 25-h growth period) added fixative to
preserve samples for gene expression analysis. All phases of the experiment on
orbit were conducted at ambient temperature (23°C). Shuttle landing occurred at
approximately 58 h postfixation.
RNA extraction, labeling, and Affymetrix GeneChip analysis. Total cellular
RNA extraction was performed using the RNeasy minikit (Qiagen) per the
manufacturer’s instructions. Conversion to fluorescently labeled cDNA, hybrid-
ization to Affymetrix GeneChip arrays, and image acquisition were performed as
previously described (29). Raw Affymetrix data were normalized and processed
utilizing tools identical to those for the study of P. aeruginosa PAO1 under
microgravity analogue conditions (13). The Benjamini-Hochberg method was
used for multiple-testing correction (7). Only fold change ratios with P values
below 0.05 (corrected for multiple testing) were considered statistically signifi-
cant. Microarray analysis was performed on all three biological replicates.
Protein identification analysis. Proteins from spaceflight and ground cell ly-
sates were precipitated with acetone and subjected to multidimensional protein
identification technology (MudPIT) analysis using the tandem mass spectrome-
try (MS)-dual nano-liquid chromatography technique (11, 37). Tandem mass
spectra of peptides were analyzed with TurboSEQUEST version 3.1 (18) and
XTandem (14) software. Data were further processed and organized using the
Scaffold program. A probability threshold of 90% was adopted, and only proteins
present in at least two biological replicates were considered expressed. Spectra
were also assessed for good quality based on TurboSEQUEST correlation and
DeltaCorrelation scores as previously described (11).
Biostatistics. To calculate the overlap of up- and downregulated genes be-
tween P. aeruginosa and S. Typhimurium under spaceflight and simulated mi-
crogravity culture conditions, homology was determined using the BLAST soft-
ware (blastp) (1). Genes in different organisms were defined as orthologues when
they fulfilled the following criteria: (i) a cutoff on the BLAST E value of 1e?10,
(ii) a minimal alignment coverage of 80% of the shortest DNA or protein
sequence, (iii) a minimal sequence identity of 35%, and (iv) appearance as each
other’s reciprocal best BLAST hit. The statistical significance of the number of
overlapping genes between different species and conditions was determined
using the hypergeometric distribution method (20).
Microarray data accession number. The microarray data have been deposited
in the Gene Expression Omnibus database (NCBI) (http://www.ncbi.nlm.nih.gov
/geo/query/acc.cgi?token?vdmfveeoysewghy&acc?GSE22684) under accession
1222 CRABBE´ET AL.APPL. ENVIRON. MICROBIOL.
P. aeruginosa PAO1 transcriptome and proteome in re-
sponse to spaceflight. (i) General observations. Transcrip-
tional analysis of P. aeruginosa PAO1 grown and fixed under
spaceflight conditions revealed the induction of 52 genes and
the downregulation of 115 genes (2-fold threshold; P ? 0.05)
compared to those in identical synchronous ground control
samples (Table 1). The genes that were differentially regulated
under spaceflight conditions were distributed throughout the
P. aeruginosa PAO1 genome and were often adjacent, indicat-
ing organization in transcriptional units (operons). Based on
functional classification of differentially expressed genes using
the Kyoto Encyclopedia of Genes and Genomes (KEGG) (25),
several categories were significantly (P ? 0.05) overrepre-
sented in either the up- or downregulated gene group. The
functional category that was significantly more represented
under spaceflight conditions was nitrogen metabolism, while
downregulated gene categories comprised purine and pyrimi-
dine metabolism, fatty acid biosynthesis, oxidative phosphory-
lation, and ribosome synthesis.
By means of MudPIT analysis, 40 proteins were identified in
ground and spaceflight samples (present in at least two repli-
cates), among which 28 were differentially expressed (Table 2).
Seven of these 28 proteins were also differentially regulated at
the transcriptional level.
(ii) Hfq and the Hfq regulon. The gene encoding the RNA
binding protein Hfq and genes under the control of Hfq were
differentially expressed under spaceflight conditions. More
specifically, 13.4% of the genes from the previously described
Hfq regulon (42) were induced (17 of 38 genes) or downregu-
lated (21 of 38 genes) in response to spaceflight, accounting for
23% of the P. aeruginosa spaceflight stimulon. The overlap
between the spaceflight data set and the Hfq regulon was
significant (P ? 0.05), indicating that this transcriptional reg-
ulator, at least in part, mediated the spaceflight response of P.
aeruginosa. While the downregulation of Hfq under spaceflight
conditions presumably resulted in the downregulation of genes
under positive control of Hfq (such as sigX, adk, and fabA) and
the upregulation of genes under negative control of Hfq (such
as bkdA2, bdhA, and glcC), other genes showed a direction of
fold change opposite to what would be expected based upon
the described Hfq regulon. Examples include the upregulation
of nirS, chiC, and rhlA, which have been documented to be
under positive control of Hfq under conventional culture con-
ditions (42). This finding indicates that other (post)transcrip-
tional or posttranslational regulators (or regulatory networks)
may have played a role in the differential expression of these
genes in the microgravity environment of spaceflight. Addi-
tionally, three proteins whose mRNA expression levels are
controlled by Hfq (i.e., PA0070, PA0456, and PA1555) were
found to be differentially expressed at the proteomic level.
(iii) Anaerobic metabolism. The majority of genes that were
upregulated under spaceflight conditions (60%) were associ-
ated with growth under anaerobic conditions (Table 1) (19).
Furthermore, 13% of the genes that were downregulated in
spaceflight are known to be downregulated during anaerobic
FIG. 1. Diagram of a fluid-processing apparatus (FPA) used as hardware for the spaceflight experiment. In the preactivation setting of the FPA
(setting 1), the bacterial inoculum (suspended in PBS) is separated from the culture medium and fixative agent (RNA Later II) through rubber
stoppers. The FPAs are brought on board the shuttle in their preactivation setting until activation in low-Earth orbit. Upon activation in flight
(setting 2), the plunger is pushed downwards in order to bring the bacteria in contact with the medium, allowing for bacterial growth; the plunger
is pushed until the middle stopper is located at the top part of the bevel. After 25 h of bacterial growth (setting 3), the plunger is pushed again
in order to bring the middle stopper below the top part of the bevel, which brings the fixative in contact with the bacterial culture.
VOL. 77, 2011RESPONSE OF PSEUDOMONAS AERUGINOSA TO SPACEFLIGHT 1223
TABLE 1. P. aeruginosa PAO1 genes differentially expressed under spaceflight conditions compared to identical ground controls
Acetyl coenzyme A carboxylase
Nitrite reductase precursor
Nitric oxide reductase subunit C
Nitric oxide reductase subunit B
Probable dinitrification protein NorD
30S ribosomal protein S21
Organic solvent tolerance protein OstA precursor
Ribonucleotide diphosphate reductase alpha subunit
C4-dicarboxylate transport protein
Probable cytochrome c
Probable cytochrome oxidase subunit (cbb3-type)
Succinate dehydrogenase (C subunit)
Succinate dehydrogenase (D subunit)
Succinate dehydrogenase catalytic subunit
3-Hydroxydecanoyl-acyl carrier protein dehydratase
ECF sigma factor SigX
Molybdate binding periplasmic protein precursor ModA
Probable ring-cleaving dioxygenase
2-Oxoisovalerate dehydrogenase (alpha subunit)
2-Oxoisovalerate dehydrogenase (beta subunit)
PA-I galactophilic lectin
Probable chemotaxis transducer
Translation initiation factor IF-1
ATP binding protease component ClpA
NADH dehydrogenase I chain C
Translation initiation factor IF-3
Probable chemotaxis transducer
Elongation factor P
Acyl carrier protein
50S ribosomal protein L32
30S ribosomal protein S1
Fucose binding lectin PA-IIL
Regulatory protein NosR
Nitrous oxide reductase precursor
Probable dihydrolipoamide acetyltransferase
Probable pyruvate dehydrogenase E1 component
Probable pyruvate dehydrogenase E1 component
Continued on following page
1224CRABBE´ET AL.APPL. ENVIRON. MICROBIOL.
Autoinducer synthesis protein RhlI
Rhamnosyltransferase chain A
(3R)-Hydroxymyristoyl acyl carrier protein dehydratase
UDP-3-O-(3-hydroxymyristoyl) glucosamine N-acyltransferase
Elongation factor Ts
30S ribosomal protein S2
Probable flavin mononucleotide oxidoreductase
50S ribosomal protein L19
16S rRNA-processing protein
30S ribosomal protein S16
Nucleoside diphosphate kinase
L-Cysteine desulfurase (pyridoxal phosphate-dependent)
Probable metal-transporting P-type ATPase
Riboflavin synthase subunit beta
DNA-directed RNA polymerase alpha subunit
30S ribosomal protein S4
30S ribosomal protein S11
30S ribosomal protein S13
50S ribosomal protein L36
Preprotein translocase SecY
50S ribosomal protein L30
30S ribosomal protein S5
50S ribosomal protein L18
50S ribosomal protein L6
30S ribosomal protein S8
50S ribosomal protein L24
30S ribosomal protein S17
30S ribosomal protein S3
50S ribosomal protein L22
30S ribosomal protein S19
50S ribosomal protein L2
50S ribosomal protein L23
50S ribosomal protein L4
50S ribosomal protein L3
Elongation factor G
30S ribosomal protein S7
30S ribosomal protein S12
50S ribosomal protein L7/L12
50S ribosomal protein L10
50S ribosomal protein L11
Probable two-component response regulator
Probable cytochrome b
Probable iron-sulfur protein
30S ribosomal protein S9
50S ribosomal protein L13
Aspartyl/glutamyl-tRNA amidotransferase subunit C
30S ribosomal protein S20
50S ribosomal protein L21
Continued on following page
VOL. 77, 2011 RESPONSE OF PSEUDOMONAS AERUGINOSA TO SPACEFLIGHT1225
growth (19). Using hypergeometric distribution, the overlap
between the genes induced under anaerobic conditions and the
genes upregulated in spaceflight was significant (P ? 0.05).
Similarly, a significant overlap was found between genes down-
regulated during anaerobic growth and in spaceflight. Only a
few genes which are typically induced under microaerophilic
growth conditions (2) (i.e., PA4306, PA4352, rhlI, and PA1123)
were differentially expressed in spaceflight compared to syn-
chronous ground controls. Remarkably, genes involved in
denitrification were among those with the highest fold induc-
tions within this category. While genes encoding the nitrate
reductase were not induced significantly, the mRNAs of genes
encoding nitrite (nirMS), nitric oxide (norBC), and nitrous
oxide reductases (nosRZ) were more abundant in spaceflight-
grown P. aeruginosa PAO1.
Proteomic analysis of the P. aeruginosa cells grown in space-
flight revealed that 7 of the 28 differentially expressed proteins
play a role in anaerobic growth. The downregulation of ArcA,
an enzyme involved in the fermentation of arginine, was ob-
served, as well as the downregulation of CcoP2 (PA1555) (10),
a cytochrome with high affinity for oxygen. The latter is typi-
cally induced under microaerophilic conditions but not in the
anaerobic mode of growth of P. aeruginosa (2).
(iv) Virulence factors. The transcripts of several genes en-
coding known P. aeruginosa PAO1 virulence factors were in-
duced in spaceflight samples compared to the ground controls.
Among others, the genes encoding the lectins PA-I and PA-IIL
(lecA and lecB, respectively), the chitinase-encoding gene chiC,
and the rhamnolipid-encoding gene rhlA were significantly in-
duced. The lecA gene showed the highest fold induction in the
gene list (6.3-fold). On the other hand, downregulation of
genes encoding heat shock proteins (groES and hslU) and the
N-butanoyl-L-homoserine lactone (C4-HSL) synthase (rhlI)
was observed. As mentioned above, hfq, which is a transcrip-
tional regulator involved in the virulence of P. aeruginosa, was
downregulated during spaceflight.
(v) Other functional categories. Of 115 genes that were
downregulated under spaceflight conditions, 40 are involved in
the synthesis of ribosomes. Genes involved in the dehydroge-
nation of succinate to fumarate (i.e., sdhBCD) and ATP syn-
thesis (atpGH) were less expressed in spaceflight-grown P.
Comparative bioinformatic analysis of the P. aeruginosa
PAO1 and S. Typhimurium spaceflight stimulons. In order to
compare the gene expression profiles of P. aeruginosa (this
study) and S. Typhimurium (46) following exposure to space-
flight conditions, orthologues of P. aeruginosa genes were iden-
tified in the S. Typhimurium genome. Of 167 differentially
expressed genes in P. aeruginosa, 102 orthologues were iden-
tified in S. Typhimurium, among which 92 (of 115) belonged to
the downregulated group and 10 (of 52) belonged to the up-
regulated gene list.
Probable chemotaxis transducer
50S ribosomal protein L25
Ribosome binding factor A
Biotin carboxyl carrier protein (BCCP)
30S ribosomal protein S6
RNA binding protein Hfq
50S ribosomal protein L31
ATP-dependent protease ATP-binding subunit
Regulatory protein TypA
Export protein SecB
Lipopeptide LppL precursor
50S ribosomal protein L28
Glycolate oxidase subunit GlcD
ATP synthase subunit C
ATP synthase subunit D
50S ribosomal protein L34
aGenes that are part of the Hfq regulon or involved in anaerobic/microaerophilic metabolism are indicated with “?” (n ? 3; P ? 0.05; fold change, ?2.
1226CRABBE´ET AL.APPL. ENVIRON. MICROBIOL.
A significant overlap was found for the downregulated genes
of the two bacteria (P ? 0.05) (Table 3). More specifically, 15
genes showed a common lower transcription in the spaceflight
samples and in the synchronous ground controls, among which
9 encoded ribosomal subunits. Interestingly, hfq and bfrB (en-
coding bacterioferritin) were part of the overlapping genes and
were identified as key role players in both the spaceflight- and
LSMMG-induced responses of S. Typhimurium (46, 49). De-
spite the observation that the overlap between spaceflight-
grown P. aeruginosa and S. Typhimurium was significant, it is
rather limited. Indeed, only 16% of the S. Typhimurium or-
thologues in P. aeruginosa were found to be commonly down-
regulated between the two bacteria. No overlap could be iden-
tified for the upregulated genes of P. aeruginosa and S.
Typhimurium under spaceflight conditions. This is presumably
because, in part, of the low presence of P. aeruginosa ortho-
logues (for the upregulated genes) in the S. Typhimurium
genome and because fewer genes were upregulated in re-
sponse to spaceflight for both of these organisms.
Comparative bioinformatic analysis of the P. aeruginosa
spaceflight and LSMMG stimulons. A small, but significant
(P ? 0.05), overlap of genes commonly upregulated in space-
flight- and LSMMG-grown P. aeruginosa was identified. These
genes encode the hypothetical protein PA0534, a protein in-
volved in microaerophilic/anaerobic metabolism (PA0200), the
ATP binding protease component ClpA, and a hypothetical
protein belonging to the Hfq regulon (PA2753). On the other
hand, 35 genes that were found to be upregulated in LSMMG
were downregulated under spaceflight conditions. The major-
ity of these genes (27 of 35) could be categorized as being
involved in the synthesis of ribosomes. Additionally, genes
encoding citric acid cycle proteins (sdhB, PA2634), bacterio-
ferritin (bfrB), a translational elongation factor (fusA1), a heat
shock protein (HslU), the sigma factor RpoA, a hypothetical
protein (PA0856), and a protein involved in glycolysis
(PA3001) were downregulated in response to spaceflight cul-
ture but upregulated in LSMMG.
In our previous study (13), LSMMG was found to induce
several genes encoding hypothetical proteins in P. aeruginosa
PAO1. Among these, only one hypothetical protein (i.e.,
PA2737) had not been reported as being differentially regu-
lated under any studied condition and was proposed as poten-
tially specific to the low-fluid-shear conditions of LSMMG.
TABLE 2. Proteome of P. aeruginosa PAO1 grown under
spaceflight versus ground conditions
Protein no. NameFunction Expressiona
aBlack cells indicate the proteins expressed in flight samples and not in ground
samples, gray stands for proteins expressed in ground samples and not in flight
samples, and white cells are for proteins expressed in both ground and space-
flight samples. X, proteins that were also found differentially expressed at the
transcriptomic level; H, proteins under the control of Hfq.
TABLE 3. Overlap of genes differentially regulated in both
spaceflight-grown P. aeruginosa and S. Typhimurium
compared to identical ground controls
Acyl carrier protein
30S ribosomal protein S2
Riboflavin synthase subunit
50S ribosomal protein L6
30S ribosomal protein S19
50S ribosomal protein L23
50S ribosomal protein L4
30S ribosomal protein S12
50S ribosomal protein L13
30S ribosomal protein S6
RNA binding protein Hfq
Export protein SecB
VOL. 77, 2011RESPONSE OF PSEUDOMONAS AERUGINOSA TO SPACEFLIGHT 1227
Interestingly, PA2737 was also significantly upregulated in
spaceflight, albeit below the 2-fold threshold (1.7-fold).
Assessing the behavior and virulence potential of obligate
and opportunistic pathogens aboard spacecraft and the Inter-
national Space Station (ISS) is of central importance to eval-
uate the risk for infectious disease in the context of long-term
manned missions. Furthermore, since bacteria encounter mi-
crogravity analogue low-fluid-shear forces in the host during
their natural course of infection, bacterial spaceflight research
can provide novel insights into the in vivo infection process.
Indeed, spaceflight increased the virulence of S. Typhimurium,
while global gene expression profiling revealed a general
downregulation of key virulence genes in this pathogen (46,
47). The present study demonstrated for the first time that the
opportunistic pathogen P. aeruginosa responded to culture in
the microgravity environment of spaceflight through differen-
tial regulation of 167 genes and 28 proteins. A significant part
of the spaceflight stimulon was under the control of the RNA
binding protein Hfq. Hfq is important for the virulence and
stress resistance of several (opportunistic) pathogens, includ-
ing P. aeruginosa PAO1 (17, 39, 41), by modulating the func-
tion and stability of small regulatory RNAs (sRNAs) and in-
terfering with their interactions with mRNAs (reviewed in
references 31 and 44). Interestingly, Hfq was also found to be
an important regulator in the responses of (i) P. aeruginosa to
microgravity analogue low-fluid-shear conditions (LSMMG,
using the RWV bioreactor) and (ii) S. Typhimurium to actual
spaceflight and LSMMG conditions (13, 46, 49). Hence, Hfq is
the first transcriptional regulator ever shown to be commonly
involved in the spaceflight and LSMMG responses of two bac-
Among the P. aeruginosa genes with the highest fold induc-
tions under spaceflight conditions were the genes encoding the
lectins LecA and LecB. Lectins bind galactosides, play a role in
the bacterial adhesion process to eukaryotic cells, and are thus
important virulence factors in P. aeruginosa (21, 22). P. aerugi-
nosa lectins have cytotoxic effects in human peripheral lym-
phocytes and respiratory epithelial cells in vitro and increase
alveolar barrier permeability in vivo (4, 9). Lectin production in
P. aeruginosa is regulated through the N-butanoyl-L-homo-
serine lactone (C4-HSL) quorum-sensing system (50), which
has been previously reviewed (45). However, the downregula-
tion of rhlI, the gene encoding the C4-HSL synthase, under
spaceflight conditions was unexpected. Nevertheless, rhlA,
which is dependent on C4-HSL quorum-sensing regulation and
encodes the rhamnosyltransferase I involved in rhamnolipid
surfactant biosynthesis, was induced during spaceflight culture.
Rhamnolipids are glycolipidic surface-active molecules that
have cytotoxic and immunomodulatory effects in eukaryotic
cells (5, 15, 32, 36). Interestingly, rhamnolipids and rhlA tran-
scripts were also found in P. aeruginosa in larger amounts
under low-fluid-shear compared to higher-fluid-shear growth
conditions, using the RWV bioreactor (12). These data indi-
cate that rhamnolipid production could be induced upon sens-
ing of low fluid shear.
Gene expression profiles of P. aeruginosa grown under
spaceflight conditions also revealed the differential regulation
of a significant fraction of genes involved in growth under
oxygen-limiting conditions. Spaceflight induced mainly genes
involved in anaerobic metabolism, which was reinforced by a
lower expression in spaceflight samples of CcoP2, a cyto-
chrome with high affinity for oxygen that is typically induced
under microaerophilic conditions (2, 10). At the time of mea-
surement, the most prominent way to cope with the apparent
oxygen shortage under spaceflight conditions seemed to occur
through denitrification and not through fermentation. Indeed,
under oxygen-limiting conditions, P. aeruginosa switches to an-
aerobic respiration in the presence of the alternative electron
acceptor nitrate or nitrite (16). The downregulation of ArcA, a
protein involved in arginine fermentation, accentuates that
fermentation was presumably not activated in spaceflight-
When comparing the gene expression profiles of P. aerugi-
nosa grown in spaceflight and P. aeruginosa grown in LSMMG,
a limited but significant overlap was found. Besides the role of
Hfq and its regulon in the response of P. aeruginosa PAO1 to
both spaceflight and LSMMG (see above), a significant frac-
tion of genes involved in both microaerophilic and anaerobic
metabolism were commonly induced. In contrast to P. aerugi-
nosa grown under spaceflight conditions, LSMMG-grown P.
aeruginosa induced genes involved in arginine and pyruvate
fermentation, while denitrification did not appear to play a role
in the LSMMG response of this bacterium. The observation
that spaceflight samples were presumably more deprived of
oxygen than LSMMG-grown bacteria, compared to their re-
spective controls, could be explained by the fact that actual
spaceflight conditions are characterized by even lower fluid
shear levels than LSMMG conditions. Indeed, due to the ab-
sence of convection currents in microgravity, oxygen limitation
will be more pronounced in space than in LSMMG. Further-
more, the role of the experimental setup needs to be consid-
ered. As depicted in Fig. 2, cells grown in the bioreactors used
for growth of P. aeruginosa in LSMMG and spaceflight have
different oxygen availabilities. While the bioreactors have a
gas-permeable membrane, the membrane surface-to-volume
ratio of FPA bioreactors (used in spaceflight) is 12 times lower
than that of the RWVs (LSMMG) [based on the formula
?r2/(?r2? h) or 1/h, with r ? radius and h ? height]. Hence,
oxygen availability overall will be higher in RWVs than in the
FPA devices. It also needs to be mentioned that despite dif-
ferences in aeration and fluid shear between the spaceflight
and LSMMG studies, the RWV mimics only certain aspects of
the spaceflight environment. Indeed, enhanced irradiation and
vibration or potential direct effects of microgravity (such as
effects on the cell or cellular components instead of on the
extracellular environment) during spaceflight could lead to
differences in gene and protein expression profiles between
spaceflight and LSMMG-grown P. aeruginosa. Accordingly, the
RWV bioreactor was unable to mimic the complete repertoire
of spaceflight-induced alterations in P. aeruginosa.
Since the present study was conducted by growing P. aerugi-
nosa in a liquid environment under spaceflight conditions, our
results are relevant mainly to the assessment of bacterial vir-
ulence in fluid niches of the spacecraft. Indeed, astronauts are
in regular contact with water-containing sources that could be
contaminated with P. aeruginosa, such as drinking water,
rinseless shampoo, toothpaste, mouthwash, and water for laun-
1228 CRABBE´ET AL.APPL. ENVIRON. MICROBIOL.
dry. Similarly, water-related sites in the hospital environment
are most likely to harbor P. aeruginosa (e.g., faucets, showers,
medication, disinfectants, mouthwash, and other hygiene prod-
ucts) and are at the origin of a significant number of nosoco-
mial infections (28). Furthermore, P. aeruginosa is occasionally
part of the normal human flora of the mouth, pharynx, anterior
urethra, and lower gastrointestinal tract. In these regions of the
human body, P. aeruginosa is present in a fluid environment,
which will be affected by microgravity and will presumably
result in the exposure of P. aeruginosa to lower-fluid-shear
conditions than on Earth.
This study was the first to characterize the comprehensive
transcriptional and translational responses of an opportunistic
pathogen that is frequently found in the space habitat. We
demonstrated that spaceflight conditions activated pathways in
P. aeruginosa that have been shown previously to be involved in
the in vivo infection process. However, the regulation of sev-
eral of these pathways appears to be differentially controlled
during spaceflight compared to conventional culture. Hfq was
put forward as a main transcriptional regulator in the space-
flight response of P. aeruginosa, therefore representing the first
transcriptional regulator commonly involved in the spaceflight
responses of different bacterial species. We also identified in-
teresting similarities and differences between P. aeruginosa
grown in spaceflight and under the LSMMG conditions of the
RWV. Despite the limited overlap of identical genes between
spaceflight- and LSMMG-grown P. aeruginosa, it was observed
that different genes of the same regulon or stimulon could be
induced or downregulated in spaceflight and LSMMG. The
experimental setup was proposed as one of the putative factors
at the origin of the oxygen-related transcriptional differences
between LSMMG culture in the RWV bioreactor and space-
flight-cultured P. aeruginosa in the FPAs. These data empha-
size the importance of using identical hardware for spaceflight
experiments and ground simulations, especially when oxygen is
a limiting factor. In addition, differences in fluid shear and
other environmental conditions (such as irradiation) between
actual microgravity and LSMMG need to be considered when
comparing bacterial responses to the two test conditions. This
study represents an important step in understanding the re-
sponse of bacterial opportunistic pathogens to the unique
spaceflight environment. Furthermore, it allows assessment of
the role that low-fluid-shear regions found in the human body
play in the regulation of bacterial virulence. It remains to be
determined whether the phenotype of P. aeruginosa acquired
under spaceflight conditions will effectively lead to increased
pathogenicity, as was observed for S. Typhimurium. This will
be an important consideration and key area of future study in
order to further assess the risk for infectious disease during
We thank all supporting team members at Kennedy Space Center,
Johnson Space Center, Ames Research Center, Marshall Spaceflight
Center, and BioServe Space Technologies, the crew of STS-115, Neal
Pellis, Roy Curtiss III, and Joseph Caspermeyer. We also thank Ker-
stin Ho ¨ner zu Bentrup for training team members on use of the flight
This work was supported by NASA grant NCC2-1362 to C.A.N., the
Arizona Proteomics Consortium (supported by NIEHS grant ES06694
to the SWEHSC), NIH/NCI grant CA023074 to the AZCC, and the
BIO5 Institute of the University of Arizona.
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