Modelled microgravity cultivation modulatesN-acylhomoserine lactone production inRhodospirillum rubrum S1H independently of celldensity

Felice Mastroleo,1 Rob Van Houdt,1 Steve Atkinson,2 Max Mergeay,1

Larissa Hendrickx,1 Ruddy Wattiez3 and Natalie Leys1

Correspondence

Felice Mastroleo

Felice.Mastroleo@sckcen.be

Received 23 January 2013

Accepted 10 September 2013

1Unit for Microbiology, Belgian Nuclear Research Centre (SCK$CEN), Mol, Belgium

2School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK

3Research Institute of Biosciences, Proteomic and Microbiology Laboratory, Universite de Mons,Mons, Belgium

The photosynthetic alphaproteobacterium Rhodospirillum rubrum S1H is part of the Micro-

Ecological Life Support System Alternative (MELiSSA) project that is aiming to develop a closed

life support system for oxygen, water and food production to support human life in space in

forthcoming long-term space exploration missions. In the present study, R. rubrum S1H was

cultured in a rotating wall vessel (RWV), simulating partial microgravity conditions on Earth. The

bacterium showed a significant response to cultivation in simulated microgravity at the

transcriptomic, proteomic and metabolic levels. In simulated microgravity conditions three

N-acyl-L-homoserine lactones (C10-HSL, C12-HSL and 3-OH-C14-HSL) were detected in

concentrations that were twice those detected under normal gravity, while no differences in cell

density was detected. In addition, R. rubrum cultivated in modelled microgravity showed higher

pigmentation than the normal gravity control, without change in culture oxygenation. When

compared to randomized microgravity cultivation using a random positioning machine, significant

overlap for the top differentially expressed genes and proteins was observed. Cultivation in this

new artificial environment of simulated microgravity showed new properties of this well-known

bacterium, including its first, to our knowledge, complete quorum-sensing-related N-

acylhomoserine lactone profile.

INTRODUCTION

The Micro-Ecological Life Support System Alternative(MELiSSA) is a closed regenerative life support system forfuture space flights under development by the EuropeanSpace Agency. It consists of interconnected processes (i.e.bioreactors, higher plant compartments, filtration units,etc.) targeting the production of oxygen, water and food bythe recycling of organic and mineral waste (Mergeay et al.,1988). Within the MELiSSA loop, the purple non-sulfur

alphaproteobacterium Rhodospirillum rubrum S1H occu-pies a key position, processing the carbon and nitrogencompounds coming from the upstream raw waste digester(Hendrickx et al., 2006) and mainly preventing volatilefatty acids from endangering the functioning of thedownstream nitrifying compartment (Oguz et al., 2006).Among the challenges of the project, the functionalstability of the bioreactors under space flight conditionsis of paramount importance for the efficiency of the lifesupport system and therefore for the crew safety.

Culture conditions in the microgravity environment ofspace flight are characterized by a lack of sedimentationand by fluid quiescence (Hammond et al., 2000). Cellsexperience low fluid shear, and transport is limited todiffusion because convection currents are essentially absentin microgravity (Klaus et al., 1998) when no artificialmixing is applied.

Actual space flight experiments are severely constrained bylimited availability of electrical power, volume and weight

Abbreviations: AHL, acylhomoserine lactone; DO, dissolved oxygen;HSL, homoserine lactone; ISS, International Space Station; LC, liquidchromatography; LSMMG, low-shear modelled microgravity; MELiSSA,Micro-Ecological Life Support System Alternative; RPM, randompositioning machine; RWV, rotating wall vessel; QS, quorum sensing.

All microarray data have been deposited at the Gene ExpressionOmnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numberGSE19942.

Three supplementary figures and five supplementary tables are availablewith the online version of this paper.

Microbiology (2013), 159, 2456–2466 DOI 10.1099/mic.0.066415-0

2456 066415 G 2013 SGM Printed in Great Britain

for up- and download, stowage space and crew time, and ageneral lack of sophisticated analytical equipment andexpertise aboard the spacecraft. As such, former space flightand associated ground experiments involving R. rubrumS1H were only conducted on solid agar medium(Mastroleo et al., 2009b). During the MESSAGE 2 spaceflight experiment, R. rubrum S1H was cultivated on richagar medium onboard the International Space Station(ISS) and ground simulation included rich agar mediumrandom positioning machine (RPM) cultivation. For theBASE-A space flight experiment, R. rubrum S1H wascultivated on minimal agar medium onboard the ISS andground simulation included minimal agar medium RPMcultivation. While giving us a first glimpse of how thebacterium would react to space flight conditions, theculture conditions remained far from the actual liquidculture condition foreseen in the MELiSSA loop. Since in-flight research has many limitations, this has driven thedevelopment of Earth-based systems to simulate micro-gravity (Manti, 2006; Marco et al., 2007).

The most commonly used microgravity simulator is therotating wall vessel (RWV) culture apparatus developed bythe National Aeronautics and Space Administration(Hammond & Hammond, 2001). RWV cultivation doesnot alter the gravity force (microgravity) as on the ISS,rather it mimics the low turbulence of a space environmentand has been referred to as low-shear modelled micro-gravity (LSMMG) (Nickerson et al., 2004; Wilson et al.,2007, 2008). Previous reports on bacterial cultivation in aRWV mainly focused on pathogenic bacteria (Crabbe et al.,2008; Nickerson et al., 2000) since the astronauts’ immunesystems appeared to be impaired in space flight conditionsincluding change in gravity (Sonnenfeld, 2005).

Considering cultivation process properties, the RWV wasshown to maintain low-shear-stress conditions, meaningbelow 0.01 Pa (Anderson et al., 2007; Nauman et al., 2007).Because this is approximately 50 times lower shear stressthan in a typical stirred vessel (Tsao et al., 1994), thisobviously affects mixing conditions inside the culturevessels compared to classical culture. For that reason, theformation of a microenvironment around the cells thatcould be responsible for the induction of species-specificphenotypes in bacteria cultivated in the RWV washypothesized (Vukanti et al., 2008).

Our lab previously suggested the induction of rhl quorumsensing (QS)-system-related genes in Pseudomonas aerugi-nosa PAO1 when cultivated in LSMMG (Crabbe et al.,2008) and a potential functionality of the QS system inR. rubrum S1H was observed once (Pycke, 2009), butremained to be characterized in detail. In silico analysis ofthe R. rubrum genome showed components of the N-acylhomoserine lactone (AHL)-type QS system, where theLuxI-type autoinducer synthase catalyses the synthesis ofthe signalling molecule and the LuxR-type regulatorinteracts with the AHLs and controls the target genes(Case et al., 2008; Reading & Sperandio, 2006).

Here, we studied R. rubrum S1H at the transcriptomic,proteomic and metabolic levels under simulated gravity inliquid conditions using the RWV technology.

METHODS

Inoculum preparation. R. rubrum S1H (ATCC 25903) was grown tostationary phase in Sistrom medium A containing 2 g sodiumsuccinate l21 (Sistrom, 1960) at 30 uC in dark aerobic conditions onan orbital shaker at 150 r.p.m. using a cell culture flask with a ventedcap (Greiner Bio-One) to ensure adequate aeration. Cells from fourindependent cultures were harvested by centrifugation and resus-pended in 0.85 % NaCl to a final OD680 y 0.6 to constitute inoculumsuspension.

Culture setup. All cultures were grown aerobically in the dark for10 days at 21 uC, which are typical time frame and temperatureconditions of a Soyuz space flight mission (Leys et al., 2009;Mastroleo et al., 2009b). RWV vessels (Cellon) were filled to capacity(zero headspace) with approximately 58 ml Sistrom medium A(Sistrom, 1960) and 1 % inoculum (v/v) from the stock culture, whileair bubbles were removed to eliminate turbulence and ensure asustained low-shear environment as previously described (Crabbeet al., 2008). In the vessel rotating around a horizontal axis (Fig. S1a,available in Microbiology Online), the liquid moves as a single body offluid in which the gravitational vector is offset by hydrodynamic,centrifugal and Coriolis forces resulting in maintenance of cells in acontinuous suspended orbit. By placing cells along the axis of rotationand spinning them perpendicular to the gravity vector, they rotatethrough the vector. Because the cell spins at a constant rate andgravity remains constant, the gravity vector is nulled from the cell’sperspective (Hammond & Hammond, 2001). Gas exchange in theRWV vessels during growth was ensured by the gas-permeablesilicone membrane present at the back of each RWV vessel. Bacterialgrowth was allowed at a rotational speed of 25 r.p.m. The horizontalRWV position was used as a normal gravity control for the modelledmicrogravity cultivation (Fig. S1a). After cultivation, dissolvedoxygen concentration (DO) was measured by inserting the oxygenprobe (910 oxy; Knick Portamess) directly into the vessel withouthomogenization as previously described (Crabbe et al., 2008). Noexperimental data could be found in the literature concerning theoxygen transport inside the RWV vessels. Current estimation comesfrom 2D mathematical calculations performed by Kwon et al. (2008)that suggest uniform diffusion of oxygen starting from 18 r.p.m.vessel rotation speed, which causes sufficient convective flux ofoxygen. After homogenization, OD was measured at 600 nm and cellswere harvested for viable count and pigment quantification afteracetone/methanol (7 : 2, v/v) extraction (Favier-Teodorescu, 2004)and for transcriptomic, proteomic and AHLs analysis. RWV setup hasintrinsically some technical constraints that do not allow samplingduring the cultivation test without compromising the low-shearcultivation state. In addition, because the RWV vessels are filled tocapacity (zero headspace), regular sampling would imply refilling theRWV vessels with fresh medium at a certain point to avoid anydecrease in pressure inside the vessels. For these reasons, only end-point measurements were performed.

The same RWV vessel type was used to cultivate R. rubrum S1H inliquid aerobic conditions using a RPM. The horizontal RWV positionwas also used as normal gravity control for the RPM cultivation (Fig.S1a). The RPM was operated as a random walk three-dimensionalclinostat (basic mode) with an angular velocity of rotation of 60u s21

as previously described (Mastroleo et al., 2009b).

Transcriptomic analysis. Cells (10 ml) were harvested after 10 daysof cultivation using the RWV technology or the RPM technology. The

Rhodospirillum rubrum in modelled microgravity

http://mic.sgmjournals.org 2457

transcriptomic study was performed using whole-genome R. rubrum

S1 DNA microarrays, in biological and technical triplicates, as

previously described (Mastroleo et al., 2009b). Genes were considered

as significantly differentially expressed when the fold change was

higher than two or lower than 0.5 with a P value lower than 0.05. All

microarray data have been deposited at the Gene Expression

Omnibus website (http://www.ncbi.nlm.nih.gov/geo/) under acces-

sion number GSE19942.

Proteomic analysis. Cells (40 ml) were harvested after 10 days of

cultivation using the RWV technology or the RPM technology.

Proteome analyses were conducted using multi-dimensional protein

identification technology (MudPIT) coupled to the isotope-coded

protein label (ICPL) technology as previously described (Mastroleo

et al., 2009b). Repeated sample injection of pooled biological

quadruplicate was performed. Proteins were considered as signifi-

cantly differentially expressed when the fold change was higher than

1.5- or lower than 0.7-fold. Thresholds were adapted when necessary

(Wang et al., 2009).

QS bioassay. R. rubrum S1H extracts for TLC analysis were

prepared from 200 ml cultures in Sistrom medium A in dark aerobic

conditions as mentioned above for the inoculum preparation. Whole

cultures were extracted twice with equal volumes of analysis-grade

dichloromethane (Merck) and the combined extracts were dried

over anhydrous magnesium sulfate, filtered and evaporated to

dryness using a Rotavapor (Buchi Labortechnik). Residues were

dissolved in 500 ml dichloromethane. Five microlitre bacterial

extracts and AHL standards, [N-butyryl-L-homoserine lactone

(C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL) and N-

dodecanoyl-L-homoserine lactone (C12-HSL)] (Sigma-Aldrich) were

applied to C18 reversed-phase TLC plates (Merck) and the

chromatograms were resolved in methanol/water (60 : 40, v/v) until

the solvent front was ~2 cm from the top of the TLC plate. The

solvent was then allowed to evaporate and the dried TLC plates were

overlaid with a culture of the indicator bacterium. Agrobacterium

tumefaciens NT1(pZLR4) was used as an AHL reporter (Shaw et al.,

1997). The A. tumefaciens indicator strain was grown at 30 uC in

modified MGM minimal medium (11 g Na2HPO4, 3 g KH2PO4,

0.5 g NaCl, 1 g glutamate, 10 g glucose, 1 mg biotin, 27.8 mg CaCl2and 246 mg MgSO4 per litre) containing 20 mg gentamicin ml21. A

10 ml overnight culture of A. tumefaciens NT1(pZRL4) was used to

inoculate 150 ml of modified MGM minimal medium containing

20 mg gentamicin ml21 and the new culture was grown to late

exponential phase. The entire 150 ml of culture was added to 150 ml

of the same medium containing 1.2 g melted agar (Select Agar;

Oxoid) and 40 mg X-Gal ml21 (Fermentas) maintained at 45 uC.

When sensing HSLs, this biosensor produces b-galactidosidase,

which is able to hydrolyse X-Gal yielding a blue signal. The culture

was mixed thoroughly and immediately spread over the surface of

the developed TLC plate placed in a 478 cm2 bioassay dish (VWR).

After the agar solidified, the coated plates were incubated at 30 uCfor 96 h.

AHL analysis. Identification and relative quantification of the N-

acyl-L-homoserine lactones produced by R. rubrum S1H were

performed using liquid chromatography (LC) coupled to hybrid

quadrupole-linear ion trap (QqQLIT) mass spectrometry in a

series of enhanced product trap experiments (EPI) triggered by

precursor ion scanning across the m/z range 150–500 and in

particular m/z 102, which is characteristic of the homoserine lactone

ring moiety. To confirm the identity of the AHLs, the EPI spectra (m/

z range 80–400) containing the fragment ion at m/z 102 were

compared to the retention time and spectral properties of a series of

corresponding synthetic AHL standards as previously described

(Ortori et al., 2007).

RESULTS

Culture analysis

R. rubrum S1H liquid cultures were subjected to simulatedmicrogravity conditions on Earth using the RWV technol-ogy, which produces low fluid shear modelled micro-gravity. After 10 days of cultivation, cell distribution wasdifferent in LSMMG and in the control vessels. In thelatter, part of the culture sedimented on the membrane sideof the vessels while cells were homogeneously distributed inthe LSMMG vessels (Fig. S1b). The use of RWV does notallow collecting time-course data for the reasons explainedin Methods. However, all cultures reached the same endpoint in relation to OD, viable count, DO and pH (approx.7.8, data not shown) while higher pigment content of thesimulated microgravity cultures compared to the controlcultures could be measured after 10 days of cultivation indark aerobic conditions (Fig. 1 and Table 1). Nauman et al.(2007) reported that the addition of a bead into the RWVdid not affect the growth curves or oxygen utilization ofbacterial cells in the vessel, suggesting that changes inmicrobial characteristics due to mass transfer are unlikely.Therefore we can assume that the cells are in the samegrowth state (stationary phase) at the end of the 10 dayexperiment.

To confirm the validity of the DO measurements, another10 day experiment was performed but control vessels wereplaced upside-down (data not shown). That way, the cellssedimented on the sample ports side (instead of the gas-permeable silicon membrane side, Fig. S1b). Keeping thesame protocol for oxygen measurement, only 4 mg l21 O2

was measured in the control ‘upside-down’ and cellsappeared as pigmented as the LSMMG samples, where DOconcentration was still close to saturation [8 mg (l O2)21].Therefore, oxygen depletion leading to expected pigmentinduction (Ghosh et al., 1994; Niederman, 2013) could beartificially reproduced inside the control ‘upside-down’vessels. These data support the fact that our DOmeasurement is representative of the oxygen availabilityinside the culture vessels and that the observed effectscannot only be induced by oxygen limitation.

S1H control cultures

S1H cultures in low-shear modelled microgravity

Fig. 1. R. rubrum S1H culture pellets after 10 days of cultivationin dark aerobic conditions using the rotating wall vesseltechnology.

F. Mastroleo and others

2458 Microbiology 159

Proteomic and transcriptomic analysis

For the differential proteomic analysis of the LSMMGsamples versus the control samples, 422 proteins wereidentified including 273 proteins quantified with at leasttwo peptides (Table S1), representing, respectively, 11 %and 7 % of the total candidate protein-encoding genes witha computed false discovery rate of 1.38 % at the peptidelevel. The median value of the fold-change distribution was0.77 (Fig. S2). Therefore, the threshold of significance wasadapted by multiplying it by the correction factor (0.77)giving 1.16 and 0.54 rendering, respectively, 27 and 43 pro-teins that passed the threshold for up- and downregulation(Table 2). Interestingly, among the top 10 overexpressedproteins, seven were membrane proteins including the toptwo occupied by pufM (Rru_A2974) and puhA (Rru_

A0617) related to the photosynthetic apparatus.

In addition, the translation of 41 hypothetical proteins of R.rubrum has been shown for the first time (Table S1). Amongthese, Rru_A1096 (putative periplasmic protein), Rru_A1353(putative outer-membrane protein), Rru_A3373 and Rru_

A3662 have been shown to be significantly downregulated inLSMMG culture conditions (Table 2).

Using our R. rubrum whole-genome oligonucleotides chip,only 13 genes (out of 3824 genes retained after qualitycontrol) were identified as significantly upregulated after10 days of culturing in LSMMG compared to the normalgravity control (Table 3). No genes were found to besignificantly downregulated during LSMMG cultur-ing compared to the control conditions. The gene Rru_

A3396, which codes for an AHL synthase, was induced4.01-fold in LSMMG. The associated LuxR-type regu-lator (Rru_A3395) was not differentially expressed duringLSMMG cultivation. Interestingly, in R. rubrum the AHLsynthase gene appeared to be transcriptionally coupledto a gene of unknown function. The latter (Rru_A3397)was the third most upregulated gene (3.16-fold). The‘unclassified’ (Un) COG category ranked as the mostnumerically abundant functional category of genes whoseexpression was significantly changed with four genes up-regulated in LSMMG including Rru_A1537 and Rru_

A2850 (Table 3) that could be reannotated using theMaGe platform for genome expert annotation (Vallenetet al., 2006).

QS bioassay and AHL analysis

Because the AHL synthase gene (Rru_A3396) was the mostupregulated gene in simulated microgravity conditions, thegeneral functionality of the R. rubrum S1H QS system wastested. The production of AHLs by R. rubrum S1H culturedin regular culture flasks was first examined using a bioassaycombining TLC with the reporter strain A. tumefaciensNT1(pZRL4). The reporter strain sensed multiple AHLswith acyl chains ranging from four carbons to at least 12carbons (Fig. 2).

To achieve unequivocal structural identification and togive a quantitative dimension of the previous results, LCcoupled with MS analysis was performed on R. rubrumwhole-cell extracts from LSMMG cultures. Ten AHLs withacyl side chains ranging from C6 to C14 with or without 3-hydroxy substituents were identified (Table 4) and amongthese, C10-HSL, C12-HSL and 3-OH-C14-HSL weresignificantly more abundant in LSMMG samples, respec-tively 3.28-fold, 1.98-fold and 1.96-fold, supporting whatwas observed at the transcriptomic level (individual AHLsmeasurements are presented in Table S2).

Comparison with other experiments involvingchange in gravity conditions

The RPM is another microgravity simulator that randomlychanges the position of an accommodated (biological)experiment in three-dimensional space (Hoson et al., 1997).The RPM was shown to maintain similar low-shear-stressconditions to the RWV, meaning below 0.01 Pa (Pardo et al.,2005). At the proteomic level, the two most highly inducedproteins (out of 16) in the RPM samples were PuhA (3.43-fold) and PufM (3.34-fold), related to the photosyntheticapparatus, as already observed for the RWV experiment. Inaddition, the translation of 42 hypothetical proteins of R.rubrum has been shown for the first time (Table S4)including 25 already detected in the LSMMG sample (TableS3). Among the latter, Rru_A1096 was the only protein ofunknown function differentially expressed in both the RPMand the LSMMG samples.

At the transcriptomic level, all 13 genes upregulated in theLSMMG culture were also upregulated in the RPMexperiment but with higher fold induction in the latter

Table 1. R. rubrum S1H cell count, cell pigmentation and culture oxygen concentration after 10 days of cultivation in LSMMG versuscontrol conditions using the RWV technology

Results are the mean±standard deviation of at least three biological replicates. O2 saturation, 8.46 mg l21.

Culture condition OD600 Viable count (c.f.u. ml”1) Carotenoid

concentration (mg l”1)

Bacteriochlorophyll a

concentration (mg l”1)

O2 (mg l”1)

Control 1.00±0.04 2.166107±1.126107 0.17±0.01 0.76±0.17 7.08±0.20

LSMMG 0.93±0.03 2.376107±1.006107 0.32±0.07* 1.13±0.14* 6.97±0.20

*Significantly different from control with P,0.05.

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Table 2. Proteomic analysis of the LSMMG experiment compared to the control conditions

The genes were sorted following the fold-change values. Rru_A refers to genes located on the chromosome of R. rubrum. FC, fold change (proteins are quantified by mean±SD); No. (H/L), number

of H/L occurrences used for protein identification and quantification (see Methods); COG, clusters of orthologous groups; E, amino acid transport and metabolism; G, carbohydrate transport and

metabolism; D, cell division and chromosome partitioning; M, cell envelope biogenesis, outer membrane; H, coenzyme metabolism; C, energy production and conversion; S, function unknown; R,

general function prediction only; P, inorganic ion transport and metabolism; U, intracellular trafficking, secretion and vesicular transport; I, lipid metabolism; F, nucleotide transport and

metabolism; O, post-translational modification, protein turnover, chaperones; T, signal transduction mechanisms; K, transcription; J, translation, ribosomal structure and biogenesis; Un,

unclassified.

Gene number Gene name Gene product name COG FC LSMMG No. (H/L) Mascot score MW (kDa)

Rru_A2974 pufM Photosynthetic reaction centre M subunit Un 3.00±0.10 4 225.76 34.24

Rru_A0617 puhA Photosynthetic reaction centre, H-chain Un 2.82±0.10 6 367.29 27.92

Rru_A0437 ompA OmpA family protein M 2.69±0.33 9 544.13 35.60

Rru_A3328 ompA OmpA/MotB M 2.52±0.13 5 322.98 28.87

Rru_A1095 ompA OmpA/MotB M 2.05±0.04 3 545.42 17.29

Rru_A0931 putA Aldehyde dehydrogenase C 1.88±0.06 12 591.66 55.73

Rru_A0930 eutG Iron-containing alcohol dehydrogenase C 1.72±0.18 2 180.81 39.91

Rru_A2211 ompC Porin M 1.64±0.11 8 707.05 37.49

Rru_A3548 – Magnesium-protoporphyrin IX monomethyl ester

anaerobic oxidative cyclase

C 1.52±0.11 8 215.04 61.72

Rru_A3566* – HemY-like S 1.49±0.04 2 141.93 46.63

Rru_A1310 paaJ Acetyl-CoA C-acyltransferase I 1.44±0.10 9 790.75 39.28

Rru_A1765 yajC Protein translocase subunit YajC U 1.42±0.04 3 103.72 15.60

Rru_A3794 – Cysteine synthase E 1.40±0.03 2 216.92 35.49

Rru_A1798 tolC Type I secretion outer-membrane protein, TolC M 1.39±0.06 2 146.30 59.16

Rru_A1218 pspA Phage shock protein A, PspA K 1.38±0.11 2 251.05 25.26

Rru_A0595 tktA Transketolase G 1.35±0.16 6 540.13 70.01

Rru_A3785 pnp Polynucleotide phosphorylase/polyadenylase J 1.33±0.07 7 316.05 76.40

Rru_A0355 guaA Bifunctional GMP synthase/glutamine amidotransferase

protein

F 1.32±0.05 2 68.03 57.34

Rru_A1309 fadB 3-Hydroxyacyl-CoA dehydrogenase I 1.30±0.09 12 1031.11 83.07

Rru_A1312 – AMP-dependent synthase and ligase I 1.28±0.05 3 279.15 61.88

Rru_A1313 fadL Aromatic hydrocarbon degradation protein I 1.24±0.06 14 748.31 45.34

Rru_A2694 rpoC DNA-directed RNA polymerase K 1.20±0.07 15 641.63 155.85

Rru_A2193 aarF Abc1 protein R 1.19±0.17 2 185.61 51.24

Rru_A1203 sdhD Succinate dehydrogenase subunit D C 1.19±0.05 4 184.34 13.59

Rru_A2956 ppsA Pyruvate phosphate dikinase G 1.18±0.03 5 268.94 97.15

Rru_A2691 fusA Translation elongation factor 2 (EF-2/EF-G) J 1.16±0.08 7 513.97 76.45

Rru_A1316 caiC AMP-dependent synthase and ligase I 1.16±0.07 3 412.42 58.79

Rru_A0247 rbsB Periplasmic binding protein/LacI transcriptional regulator G 0.54±0.08 5 166.25 35.83

Rru_A0217 citE Citrate lyase G 0.54±0.05 4 514.04 35.09

Rru_A2152 rplM 50S ribosomal protein L13 J 0.54±0.05 8 256.75 17.10

Rru_A2171 – Extracellular ligand-binding receptor E 0.54±0.05 5 422.88 38.63

Rru_A1214 aceF 2-Oxoglutarate dehydrogenase E2 component C 0.54±0.03 6 522.26 45.30

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Table 2. cont.

Gene number Gene name Gene product name COG FC LSMMG No. (H/L) Mascot score MW (kDa)

Rru_A2663 rplQ 50S ribosomal protein L17P J 0.54±0.02 7 419.37 15.33

Rru_A1418 ahpC Alkyl hydroperoxide reductase/thiol specific antioxidant/

Mal allergen

O 0.54±0.01 3 288.22 20.69

Rru_A3400 sbp Thiosulfate-binding protein P 0.54±0.01 3 210.14 38.52

Rru_A2677 rplX 50S ribosomal protein L24P J 0.53±0.04 8 368.91 11.29

Rru_A0210 rpmB 50S ribosomal protein L28 J 0.53±0.01 3 343.40 11.06

Rru_A0662 infA Translation initiation factor 1 J 0.53±0.01 3 99.05 10.73

Rru_A2683 rplV 50S ribosomal protein L22 J 0.52±0.04 12 427.70 14.14

Rru_A1829 – MucR family transcriptional regulator K 0.52±0.03 3 186.36 15.71

Rru_A2575D – Conserved protein of unknown function S 0.52±0.03 5 116.49 14.27

Rru_A3373D – Conserved protein of unknown function – 0.52±0.03 6 149.49 18.70

Rru_A2689 rpsJ 30S ribosomal protein S10 J 0.52±0.00 2 81.74 11.69

Rru_A1185 rpsP 30S ribosomal protein S16 J 0.51±0.08 9 323.97 13.90

Rru_A1595 hlpA Outer-membrane chaperone Skp (OmpH) M 0.51±0.02 5 263.24 21.72

Rru_A2041 bioA Aminotransferase H 0.51±0.02 3 240.83 49.34

Rru_A2681 rplP 50S ribosomal protein L16 J 0.50±0.09 5 232.86 15.43

Rru_A1232 prc C-terminal processing peptidase S41A M 0.50±0.08 6 206.48 47.87

Rru_A0332 gst Glutathione S-transferase-like protein O 0.50±0.04 4 359.65 25.43

Rru_A2356 ddpA Extracellular solute-binding protein E 0.50±0.02 20 1511.78 59.24

Rru_A3662D – Conserved protein of unknown function Un 0.50±0.02 2 100.71 12.62

Rru_A3506 livK Putative branched-chain amino acid transport system

substrate-binding protein

E 0.50±0.00 2 130.80 44.51

Rru_A1096D – Putative Tol-Pal system protein YbgF S 0.49±0.04 4 149.08 29.28

Rru_A2087 hisJ Extracellular solute-binding protein E 0.49±0.04 4 235.92 32.55

Rru_A3802 rpsT SSU ribosomal protein S20P J 0.49±0.04 5 523.38 9.57

Rru_A3205 rpmG 50S ribosomal protein L33P J 0.49±0.03 5 404.82 6.49

Rru_A2964 maoC MaoC-like dehydratase I 0.47±0.08 4 195.34 15.40

Rru_A1353D – Conserved hypothetical protein (outer membrane) S 0.47±0.07 5 129.49 18.19

Rru_A3744 – Signal transduction protein T 0.47±0.05 4 291.53 16.39

Rru_A1760 sodA Superoxide dismutase P 0.46±0.05 4 192.10 24.93

Rru_A0701 rpsD 30S ribosomal protein S4 J 0.45±0.03 5 124.98 23.62

Rru_A3782 rbfA Ribosome-binding factor A J 0.44±0.03 2 70.89 17.84

Rru_A3728 livK Extracellular ligand-binding receptor E 0.41±0.05 5 173.97 40.22

Rru_A1043 rpsU 30S ribosomal protein S21 J 0.41±0.02 5 108.05 9.42

Rru_A1589 frr Ribosome recycling factor J 0.39±0.06 5 149.48 21.14

Rru_A1665 rpmF 50S ribosomal protein L32 J 0.39±0.05 3 91.77 7.03

Rru_A2723 – Rubrerythrin S 0.39±0.05 2 121.47 19.25

Rru_A1362 – Xylose isomerase-like TIM barrel G 0.39±0.02 3 163.94 33.69

Rru_A1223 atpH F0F1 ATP synthase subunit delta C 0.35±0.14 6 412.35 19.56

Rru_A3283 – Phasin S 0.35±0.01 2 69.99 17.53

*Rru_A3566 gene product has two predicted transmembrane domains (TMHMM website).

DGene that was reannotated in MaGe.

Rho

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condition, respectively 19.91 and 14.21, for the autoinducer(Rru_A3396) and for the gene of unknown functionRru_A3397. In addition, the transcriptional regulator(Rru_A3395) appeared to be significantly upregulated(1.40-fold). Besides, upregulation was also observed forpucC (Rru_A0618) (2.65-fold), a component of the pucoperon coding for two light-harvesting antenna subunits, fora membrane stress response gene, mufM (Rru_A0424) (3.69-fold), and for a mechanosensitive ion channel-related gene,mscS (Rru_A1608) (4.34-fold), during the RPM experiment.

The response of R. rubrum S1H to LSMMG and RPM liquidgrowth conditions in minimal medium used in the presentstudy showed low overlap with previously performedMESSAGE 2 and BASE-A space flight and RPM experimentson minimal and rich solid media (Mastroleo et al., 2009b)(Fig. S3). Only the liquid RPM experiment performed in thisstudy and the previous MESSAGE 2 agar RPM experi-ments showed a significant overlap (P,0.05). The sms gene(Rru_A1537) coding for a protein with unknown functionwas upregulated in RPM liquid, MESSAGE 2 flight,MESSAGE 2 agar RPM and BASE-A agar RPM experiments.Rru_A2850, a putative chemotaxis-related gene, was upre-gulated in LSMMG and RPM liquid as well as in MESSAGE2 RPM-related and BASE-A RPM-related experiments. Rru_A0637, which codes for a putative lipoprotein with threepredicted transmembrane domains, and Rru_A3369, whichcodes for a hypothetical protein, were upregulated in RPMliquid, MESSAGE 2 flight and MESSAGE 2 agar RPM ex-periments. Rru_A1608, related to a mechanosensitive ionchannel protein, was upregulated in both the RPM liquidand the BASE-A RPM experiments. For Rru_A1758, codingfor a hypothetical protein with one predicted transmembrane

domain, joint upregulation was found in LSMMG and RPMliquid and in BASE-A agar RPM experiments. Finally,Rru_A3286 coding for a putative transcriptional regulatorwas jointly upregulated in RPM liquid, MESSAGE 2 flightand MESSAGE 2 agar RPM experiments (Table S4).

DISCUSSION

To our knowledge, the present study is the first report of anintegrated culture, proteomic and transcriptomic analysis,

Table 3. Significantly differentially expressed genes in the LSMMG experiments compared to the control conditions

Rru_A refers to genes located on the chromosome while Rru_B refers to gene located on the plasmid of R. rubrum. FC, fold change (P,0.05). Genes

were sorted following fold-change values. E, amino acid transport and metabolism; M, cell envelope biogenesis, outer membrane; R, general

function prediction only; I, lipid metabolism; Q, secondary metabolite biosynthesis, transport and catabolism; J, translation, ribosomal structure

and biogenesis; Un, unclassified.

Gene number Gene name Gene product name COG FC LSMMG

Rru_A1072 rpmE 50S ribosomal protein L31 J 4.2

Rru_A3396 – LuxI-type autoinducer synthesis protein Q 4.01

Rru_A3397 – Hypothetical protein Rru_A3397 Un 3.16

Rru_A1758 – Hypothetical protein Rru_A1758 Un 2.96

Rru_A1530 – 5,10-Methylenetetrahydrofolate reductase E 2.71

Rru_A1518 – Hypothetical protein Rru_A1518 Un 2.65

Rru_A1947 – Nucleotidyltransferase-like R 2.41

Rru_A0160 – Hypothetical protein Rru_A0160 E 2.36

Rru_B0012 – DNA polymerase, beta-like region R 2.21

Rru_A3394 – Hypothetical protein Rru_A3394 Un 2.18

Rru_A2850* cheL Chemotactic signal-response protein M 2.11

Rru_A0073 – Putative ribosomal subunit interface protein J 2.11

Rru_A3689 – MaoC-like dehydratase I 2.09

Rru_A1537* sms Protein of unknown function Un 1.78

*Gene that was reannotated in MaGe.

C4C6

C12

S1H

Fig. 2. TLC of R. rubrum S1H Sistrom-succinate dark aerobicwhole culture extract. S1H, whole culture extract; C4, N-butyryl-DL-homoserine lactone; C6, N-hexanoyl-DL-homoserine lactone;C12, N-dodecanoyl-DL-homoserine lactone.

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combined with pigment quantification and AHL profiling,of a bacterium cultured in modelled microgravity.

Modelled microgravity cultivation modulates AHLproduction in R. rubrum independently of celldensity

In both modelled microgravity experiments the genecoding for the AHL synthase Rru_A3396 responsible forthe synthesis of AHLs was highly upregulated although thecorresponding protein was not detected during proteomicanalysis. However, LC-QdQLIT-MS confirmed the activa-tion of the R. rubrum QS system in LSMMG by identifiyingthree AHLs (C10-HSL, C12-HSL and 3-OH-C14-HSL)which were at least twice as abundant in samples cultivatedin simulated microgravity conditions when compared tothose in normal gravity. A further seven AHLs were alsoidentified but were not differentially produced in simulatedmicrogravity. Probable causes for the apparent inconsistentAHL pattern like variable cell numbers and pH can beruled out since there were no significant differences in OD/live count and pH between the control and the simulatedmicrogravity samples. This observed difference in AHLproduction may be due to the type and available cellularpool of acyl-acyl carrier proteins, originating from the fattyacid biosynthetic pathway and involved in AHL biosyn-thesis, which have also been shown to influence theproduction of AHL molecules (Fast & Tipton, 2012;Watson et al., 2002).

Because the transcription of the AHL synthase may beregulated by AHLs in a positive feed-back loop, it ispossible that in (modelled) microgravity, the fluidquiescence and reduced mixing could enhance theaccumulation of the AHLs in the bacterium’s surroundingsand thus promote AHL synthase and QS-related geneexpression, independently of cell concentration (Horswill

et al., 2007). According to Hense et al. (2007), cells canindeed not distinguish between the three key determinantsof autoinducer concentration, namely cell density, spatialdistribution of the cells and mass-transfer properties.Because QS pathways are involved in the regulation of anumber of key functions in many bacterial species,especially those associated with virulence (Fuqua &Greenberg, 2002; Reading & Sperandio, 2006), the possiblecell density independent induction of QS could altersignificantly the organisms’ ability to thrive in a givenenvironmental setting (Boedicker et al., 2009; Carnes et al.,2010; Connell et al., 2010). Moreover Baysse et al. (2005)reported that alterations in membrane properties of P.aeruginosa resulted in premature production of C4-HSLand C6-HSL, which suggests that the QS system was alsoactivated independently of cell density. The present studyalso put forward the differential expression of a numberof membrane proteins, which suggests that membraneperturbation caused by either chemical or mechanicalstimuli related to simulated microgravity cultivation couldalso be a trigger to QS activation in R. rubrum.

Besides, while photosystem pigment-protein componentproduction is commonly linked to change in light intensityand oxygen tension in purple photosynthetic bacteria(Ghosh et al., 1994; Niederman, 2013), R. rubrum S1Hcells cultivated in modelled microgravity in the darkshowed higher pigmentation than the normal gravitycontrol, without change in cell density and cultureoxygenation. At the proteomic level, the most highlyinduced proteins in modelled microgravity were alsorelated to the membrane-associated photosynthetic appa-ratus, namely PuhA and PufM. The common activation ofpigment biosynthesis and QS systems indicates that thephotosynthetic apparatus of R. rubrum S1H is putativelycontrolled by QS. This was also recently reported for theclosely related Rhodobacter sphaeroides 2.4.1 (Hwang et al.,2008). Rhodobacter sphaeroides mutants deficient in theproduction of the native AHL (7,8-cis-N-tetradecenoyl-homoserine lactone) were less pigmented than the wild-type and showed an aggregative phenotype at high celldensities; both could be complemented by addition of thenative AHL or by a functional copy of the AHL synthase(Puskas et al., 1997). It is possible that this QS-regulatedpigmentation and dispersion could be a response to lightlimitation: that when light becomes a limiting nutrientfactor in sufficiently large cell aggregates, increased cellpigmentation to capture the few available protons coupledto the dispersion of such aggregates would be advantageousfor the photosynthetic organisms. If so, this type ofresponse is of importance for continuous culturing of R.rubrum S1H in MELiSSA conditions in which theproduction of cell aggregates adhering to the bioreactorglass wall that eventually shade completely the incominglight has been observed (F. Mastroleo and others,unpublished results). Then, QS molecules might be apotential natural dispersive agent to counteract such anundesired event. The yet unknown response mechanisms

Table 4. Identified AHLs in R. rubrum S1H cultivated inLSMMG compared to the control conditions

Results are the mean±SD of three biological replicates. FC, fold

change; NS, not significant.

Identified AHL FC in LSMMG P value

C6-HSL NS 0.937

C8-HSL NS 0.081

C10-HSL 3.282±1.187* 0.009

C12-HSL 1.984±0.245* 0.007

C14-HSL NS 0.106

3-OH-C6-HSL NS 0.350

3-OH-C8-HSL NS 0.537

3-OH-C10-HSL NS 0.375

3-OH-C12-HSL NS 0.072

3-OH-C14-HSL 1.955±0.170* ,0.001

*Significantly different from control with P,0.05.

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and regulation of the QS system of R. rubrum S1H and itsrole in the different phenotypes is currently, however, stillunder investigation.

Comparison of LSMMG and RPM results

The RPM has been extensively used to study cytoskeletonstructure and motility of human cells (Meloni et al., 2006;Walther et al., 1998) and plant gravitropism (Barjaktarovicet al., 2009; Hoson et al., 1997) and more recently the RPMhas been used to study bacterial cultivation (Beuls et al.,2009; Crabbe et al., 2010; Leroy et al., 2010; Mastroleo et al.,2009b; Mauclaire & Egli, 2010). In the present study, at theproteomic level, only a few common proteins were foundin the response of R. rubrum S1H to LSMMG and RPMcultivation, and the LSMMG appeared to induce a highernumber of significantly regulated proteins than the RPMcompared to the control conditions. On the other hand,only a few genes were significantly induced at the RNAlevel after cultivation of R. rubrum S1H in LSMMG and allof them were included in the more pronounced transcrip-tomic response to cultivation in RPM. Moreover, thesecommon genes showed much higher fold induction (atleast threefold) in the RPM. Therefore, one must becautious to conclude which of the two simulators induceda higher response in R. rubrum S1H. Nevertheless, it isapparent that culturing R. rubrum S1H under simulatedmicrogravity in liquid conditions induced more genes withhigher fold induction than culturing under simulatedmicrogravity on agar medium as previously performed(Mastroleo et al., 2009b). As mentioned above, theincreased AHL production independent of cell densitysuggests that the response of the bacterium R. rubrum S1Hto LSMMG was related to the fluid quiescence and reducedmixing conditions. The latter was also hypothesized forEscherichia coli K-12 cultivated in LSMMG (Tucker et al.,2007; Vukanti et al., 2008) and Cupriavidus metalliduransCH34 cultivated in RPM (Leroy et al., 2010). However,membrane stress response and mechanosensitive-relatedgene induction among others indicate that a directmechanical effect on the R. rubrum cell membrane cannotbe fully excluded.

Genes coding for hypothetical proteins

The multiple experiments including space flight andsimulated microgravity on plate (Mastroleo et al., 2009b)and in liquid culture also permitted us to have a betterinsight into the genome of R. rubrum. Particularly, thegenes Rru_A1537 and Rru_A2850 could be specificallyrelated to responses associated with changes in gravity sincethese genes were the only two (within the whole genome)showing a similar trend in expression in all the space-related culture conditions mentioned above. The produc-tion of Rru_A1537 protein was recently detected using ahigh-throughput proteomics approach (Mastroleo et al.,2009a). This ‘protein of unknown function’, from now onnamed Sms (for simulated microgravity and space), has no

homology to any previously reported sequence using theMaGe platform for genome expert annotation (Vallenetet al., 2006). Further studies on this unknown proteinshould involve the construction of a specific mutant to betested in space flight conditions.

Relevance for cultivation in space and theMELiSSA system

Now that we have identified in this study that R. rubrumS1H is capable of producing a number of different AHLs,and that this production is modulated and cell densityindependent under microgravity conditions, it will be ofimportance to determine the fate of these signallingmolecules within the closed MELiSSA loop. Because theR. rubrum S1H compartment directly feeds the nitrifierscompartment, AHLs present in its effluent could poten-tially affect the biofilm formation of Nitrosomonas europaeasince the latter has been shown to possess an AHL systemtoo (Burton et al., 2005). In addition, QS systems havealready been shown not only to elicit a response inside amultispecies bacterial population but also to cross theinter-kingdom barrier (Reading & Sperandio, 2006; Shineret al., 2005). Therefore, production of bacterial signallingmolecules could not only influence the MELiSSA loop byaffecting the bacterial population within and betweenbioreactors but even affect the last compartment compris-ing higher plants. However, these AHL molecules are notubiquitously stable, and the high pH (up to pH 10) fromthe cyanobacterial compartment and the elevated temper-ature (up to 55 uC) from the first compartment wouldresult in a high rate of lactonolysis (Yates et al., 2002). So,further experiments and calculations will be performed inthe frame of the MELiSSA project to assure global stabilityof the interconnected bioreactors for safety and processefficiency during long-term manned space explorationmissions.

ACKNOWLEDGEMENTS

This research was partly funded by the Belgian Nuclear ResearchCentre (SCK$CEN) through a PhD AWM grant to F. M. We alsothank the Belgian Science Policy (Belspo) and the European SpaceAgency (ESA-PRODEX, ESA-GSTP) for additional financial supportvia the MELGEN-2 project. R. W. is a Research Associate of FNRS.We are thankful to Professor Dr P. Pippia and Dr M. A. Meloni forgiving us access to their ESA RPM facility and to G. Campus and A.Janssen for providing technical support. We are grateful to ProfessorDr Paul Williams for giving access to his metabolomic analysisplatform. This paper is dedicated to the memory of Larissa Hendrickx(1973–2008), MELiSSA consortium member, MELGEN-2 projectleader, and much appreciated scientist and colleague.

REFERENCES

Anderson, B. N., Ding, A. M., Nilsson, L. M., Kusuma, K.,Tchesnokova, V., Vogel, V., Sokurenko, E. V. & Thomas, W. E.(2007). Weak rolling adhesion enhances bacterial surface coloniza-tion. J Bacteriol 189, 1794–1802.

F. Mastroleo and others

2464 Microbiology 159

Barjaktarovic, Z., Schutz, W., Madlung, J., Fladerer, C., Nordheim, A.& Hampp, R. (2009). Changes in the effective gravitational field

strength affect the state of phosphorylation of stress-related proteins

in callus cultures of Arabidopsis thaliana. J Exp Bot 60, 779–789.

Baysse, C., Cullinane, M., Denervaud, V., Burrowes, E., Dow, J. M.,Morrissey, J. P., Tam, L., Trevors, J. T. & O’Gara, F. (2005).Modulation of quorum sensing in Pseudomonas aeruginosa through

alteration of membrane properties. Microbiology 151, 2529–2542.

Beuls, E., Van Houdt, R., Leys, N., Dijkstra, C., Larkin, O. & Mahillon,J. (2009). Bacillus thuringiensis conjugation in simulated microgravity.

Astrobiology 9, 797–805.

Boedicker, J. Q., Vincent, M. E. & Ismagilov, R. F. (2009).Microfluidic confinement of single cells of bacteria in small volumes

initiates high-density behavior of quorum sensing and growth and

reveals its variability. Angew Chem Int Ed Engl 48, 5908–5911.

Burton, E. O., Read, H. W., Pellitteri, M. C. & Hickey, W. J. (2005).Identification of acyl-homoserine lactone signal molecules produced

by Nitrosomonas europaea strain Schmidt. Appl Environ Microbiol 71,

4906–4909.

Carnes, E. C., Lopez, D. M., Donegan, N. P., Cheung, A., Gresham, H.,Timmins, G. S. & Brinker, C. J. (2010). Confinement-induced quorum

sensing of individual Staphylococcus aureus bacteria. Nat Chem Biol 6,

41–45.

Case, R. J., Labbate, M. & Kjelleberg, S. (2008). AHL-driven

quorum-sensing circuits: their frequency and function among the

Proteobacteria. ISME J 2, 345–349.

Connell, J. L., Wessel, A. K., Parsek, M. R., Ellington, A. D., Whiteley,M. & Shear, J. B. (2010). Probing prokaryotic social behaviors with

bacterial ‘‘lobster traps’’. MBio 1, e00202-10.

Crabbe, A., De Boever, P., Van Houdt, R., Moors, H., Mergeay, M. &Cornelis, P. (2008). Use of the rotating wall vessel technology to study

the effect of shear stress on growth behaviour of Pseudomonas

aeruginosa PA01. Environ Microbiol 10, 2098–2110.

Crabbe, A., Pycke, B., Van Houdt, R., Monsieurs, P., Nickerson, C.,Leys, N. & Cornelis, P. (2010). Response of Pseudomonas aeruginosa

PAO1 to low shear modelled microgravity involves AlgU regulation.

Environ Microbiol 12, 1545–1564.

Fast, W. & Tipton, P. A. (2012). The enzymes of bacterial census and

censorship. Trends Biochem Sci 37, 7–14.

Favier-Teodorescu, L. (2004). Etude cinetique et stoechiometrique

de la croissance de Rhodospirillum rubrum en photobioreacteur. In

Laboratoire de genie chimique et biochimique, p. 365. PhD thesis,

Universite Blaise Pascal, Aubiere, France.

Fuqua, C. & Greenberg, E. P. (2002). Listening in on bacteria: acyl-

homoserine lactone signalling. Nat Rev Mol Cell Biol 3, 685–695.

Ghosh, R., Hardmeyer, A., Thoenen, I. & Bachofen, R. (1994).Optimization of the Sistrom culture medium for large-scale batch

cultivation of Rhodospirillum rubrum under semiaerobic conditions

with maximal yield of photosynthetic membranes. Appl Environ

Microbiol 60, 1698–1700.

Hammond, T. G. & Hammond, J. M. (2001). Optimized suspension

culture: the rotating-wall vessel. Am J Physiol Renal Physiol 281, F12–

F25.

Hammond, T. G., Benes, E., O’Reilly, K. C., Wolf, D. A., Linnehan,R. M., Taher, A., Kaysen, J. H., Allen, P. L. & Goodwin, T. J. (2000).Mechanical culture conditions effect gene expression: gravity-induced

changes on the space shuttle. Physiol Genomics 3, 163–173.

Hendrickx, L., De Wever, H., Hermans, V., Mastroleo, F., Morin, N.,Wilmotte, A., Janssen, P. & Mergeay, M. (2006). Microbial ecology of

the closed artificial ecosystem MELiSSA (Micro-Ecological Life

Support System Alternative): reinventing and compartmentalizing

the Earth’s food and oxygen regeneration system for long-haul spaceexploration missions. Res Microbiol 157, 77–86.

Hense, B. A., Kuttler, C., Muller, J., Rothballer, M., Hartmann, A. &Kreft, J. U. (2007). Does efficiency sensing unify diffusion andquorum sensing? Nat Rev Microbiol 5, 230–239.

Horswill, A. R., Stoodley, P., Stewart, P. S. & Parsek, M. R. (2007).The effect of the chemical, biological, and physical environment onquorum sensing in structured microbial communities. Anal BioanalChem 387, 371–380.

Hoson, T., Kamisaka, S., Masuda, Y., Yamashita, M. & Buchen, B.(1997). Evaluation of the three-dimensional clinostat as a simulator ofweightlessness. Planta 203 (Suppl.), S187–S197.

Hwang, W., Lee, K. E., Lee, J. K., Park, B. C. & Kim, K. S. (2008). Genesof Rhodobacter sphaeroides 2.4.1 regulated by innate quorum-sensingsignal, 7,8-cis-N-(tetradecenoyl) homoserine lactone. J MicrobiolBiotechnol 18, 219–227.

Klaus, D. M., Todd, P. & Schatz, A. (1998). Functional weightlessnessduring clinorotation of cell suspensions. Adv Space Res 21, 1315–1318.

Kwon, O., Devarakonda, S. B., Sankovic, J. M. & Banerjee, R. K.(2008). Oxygen transport and consumption by suspended cells inmicrogravity: a multiphase analysis. Biotechnol Bioeng 99, 99–107.

Leroy, B., Rosier, C., Erculisse, V., Leys, N., Mergeay, M. & Wattiez, R.(2010). Differential proteomic analysis using isotope-coded protein-labeling strategies: comparison, improvements and application tosimulated microgravity effect on Cupriavidus metallidurans CH34.Proteomics 10, 2281–2291.

Leys, N., Baatout, S., Rosier, C., Dams, A., s’Heeren, C., Wattiez, R. &Mergeay, M. (2009). The response of Cupriavidus metallidurans CH34to spaceflight in the international space station. Antonie vanLeeuwenhoek 96, 227–245.

Manti, L. (2006). Does reduced gravity alter cellular response toionizing radiation? Radiat Environ Biophys 45, 1–8.

Marco, R., Lavan, D. A., van Loon, J. J., Leandro, L. J., Larkin, O. J.,Dijkstra, C., Anthony, P., Villa, A., Davey, M. R. & other authors(2007). Drosophila melanogaster, a model system for comparativestudies on the responses to real and simulated microgravity. J GravitPhysiol 14, 125–126.

Mastroleo, F., Leroy, B., Van Houdt, R., s’Heeren, C., Mergeay, M.,Hendrickx, L. & Wattiez, R. (2009a). Shotgun proteome analysis ofRhodospirillum rubrum S1H: integrating data from gel-free and gel-based peptides fractionation methods. J Proteome Res 8, 2530–2541.

Mastroleo, F., Van Houdt, R., Leroy, B., Benotmane, M. A., Janssen, A.,Mergeay, M., Vanhavere, F., Hendrickx, L., Wattiez, R. & Leys, N. (2009b).Experimental design and environmental parameters affect Rhodospirillumrubrum S1H response to space flight. ISME J 3, 1402–1419.

Mauclaire, L. & Egli, M. (2010). Effect of simulated microgravity ongrowth and production of exopolymeric substances of Micrococcusluteus space and earth isolates. FEMS Immunol Med Microbiol 59,350–356.

Meloni, M. A., Galleri, G., Pippia, P. & Cogoli-Greuter, M. (2006).Cytoskeleton changes and impaired motility of monocytes atmodelled low gravity. Protoplasma 229, 243–249.

Mergeay, M., Verstraete, W., Dubertret, G., Lefort-Tran, M., Chipaux,C. & Binot, R. A. (1988). ‘MELiSSA’ – A micro-organisms-basedmodel for ‘CELSS’ development. In Proceedings of the 3rd EuropeanSymposium on Space Thermal Control & Life Support Systems,Nordwijk, The Netherlands, ESA Special Publication no. 288, pp. 65–68. Edited by T. D. Guyenne & J. Hunt. European Space Agency.

Nauman, E. A., Ott, C. M., Sander, E., Tucker, D. L., Pierson, D.,Wilson, J. W. & Nickerson, C. A. (2007). Novel quantitative biosystemfor modeling physiological fluid shear stress on cells. Appl EnvironMicrobiol 73, 699–705.

Rhodospirillum rubrum in modelled microgravity

http://mic.sgmjournals.org 2465

Nickerson, C. A., Ott, C. M., Mister, S. J., Morrow, B. J., Burns-Keliher,L. & Pierson, D. L. (2000). Microgravity as a novel environmentalsignal affecting Salmonella enterica serovar Typhimurium virulence.Infect Immun 68, 3147–3152.

Nickerson, C. A., Ott, C. M., Wilson, J. W., Ramamurthy, R. & Pierson,D. L. (2004). Microbial responses to microgravity and other low-shearenvironments. Microbiol Mol Biol Rev 68, 345–361.

Niederman, R. A. (2013). Membrane development in purplephotosynthetic bacteria in response to alterations in light intensityand oxygen tension. Photosynth Res (in press).

Oguz, M. T., Robinson, K. G., Layton, A. C. & Sayler, G. S. (2006).Volatile fatty acid impacts on nitrite oxidation and carbon dioxidefixation in activated sludge. Water Res 40, 665–674.

Ortori, C. A., Atkinson, S., Chhabra, S. R., Camara, M., Williams, P. &Barrett, D. A. (2007). Comprehensive profiling of N-acylhomoserinelactones produced by Yersinia pseudotuberculosis using liquidchromatography coupled to hybrid quadrupole-linear ion trap massspectrometry. Anal Bioanal Chem 387, 497–511.

Pardo, S. J., Patel, M. J., Sykes, M. C., Platt, M. O., Boyd, N. L.,Sorescu, G. P., Xu, M., van Loon, J. J., Wang, M. D. & Jo, H. (2005).Simulated microgravity using the Random Positioning Machineinhibits differentiation and alters gene expression profiles of 2T3preosteoblasts. Am J Physiol Cell Physiol 288, C1211–C1221.

Puskas, A., Greenberg, E. P., Kaplan, S. & Schaefer, A. L. (1997). Aquorum-sensing system in the free-living photosynthetic bacteriumRhodobacter sphaeroides. J Bacteriol 179, 7530–7537.

Pycke, B. (2009). The fate and effects of micropollutants in abiological life support system. In Faculteit Biol-ingenieurswetenschap-pen, p. 187. PhD thesis, Universiteit Gent, Belgium.

Reading, N. C. & Sperandio, V. (2006). Quorum sensing: the manylanguages of bacteria. FEMS Microbiol Lett 254, 1–11.

Shaw, P. D., Ping, G., Daly, S. L., Cha, C., Cronan, J. E., Jr, Rinehart,K. L. & Farrand, S. K. (1997). Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography.Proc Natl Acad Sci U S A 94, 6036–6041.

Shiner, E. K., Rumbaugh, K. P. & Williams, S. C. (2005). Inter-kingdom signaling: deciphering the language of acyl homoserinelactones. FEMS Microbiol Rev 29, 935–947.

Sistrom, W. R. (1960). A requirement for sodium in the growth ofRhodopseudomonas sphaeroides. J Gen Microbiol 22, 778–785.

Sonnenfeld, G. (2005). The immune system in space, includingEarth-based benefits of space-based research. Curr Pharm Biotechnol6, 343–349.

Tsao, Y.-M. D., Boyd, E., Wolf, D. A. & Spaulding, G. (1994). Fluid

dynamics within a rotating bioreactor in space and Earth environ-

ments. J Spacecr Rockets 31, 937–943.

Tucker, D. L., Ott, C. M., Huff, S., Fofanov, Y., Pierson, D. L., Willson,R. C. & Fox, G. E. (2007). Characterization of Escherichia coli MG1655

grown in a low-shear modeled microgravity environment. BMC

Microbiol 7, 15.

Vallenet, D., Labarre, L., Rouy, Z., Barbe, V., Bocs, S., Cruveiller, S.,Lajus, A., Pascal, G., Scarpelli, C. & Medigue, C. (2006). MaGe: a

microbial genome annotation system supported by synteny results.

Nucleic Acids Res 34, 53–65.

Vukanti, R., Mintz, E. & Leff, L. (2008). Changes in gene expression of

E. coli under conditions of modeled reduced gravity. Microgravity Sci

Technol 20, 41–57.

Walther, I., Pippia, P., Meloni, M. A., Turrini, F., Mannu, F. & Cogoli, A.

(1998). Simulated microgravity inhibits the genetic expression of

interleukin-2 and its receptor in mitogen-activated T lymphocytes.

FEBS Lett 436, 115–118.

Wang, Y., Mulligan, C., Denyer, G., Delom, F., Dagna-Bricarelli, F.,Tybulewicz, V. L., Fisher, E. M., Griffiths, W. J., Nizetic, D. & Groet, J.(2009). Quantitative proteomics characterization of a mouse embryonic

stem cell model of Down syndrome. Mol Cell Proteomics 8, 585–595.

Watson, W. T., Minogue, T. D., Val, D. L., von Bodman, S. B. &Churchill, M. E. (2002). Structural basis and specificity of acyl-

homoserine lactone signal production in bacterial quorum sensing.

Mol Cell 9, 685–694.

Wilson, J. W., Ott, C. M., Honer zu Bentrup, K., Ramamurthy, R.,Quick, L., Porwollik, S., Cheng, P., McClelland, M., Tsaprailis, G. &other authors (2007). Space flight alters bacterial gene expression and

virulence and reveals a role for global regulator Hfq. Proc Natl Acad

Sci U S A 104, 16299–16304.

Wilson, J. W., Ott, C. M., Quick, L., Davis, R., Honer zu Bentrup, K.,

Crabbe, A., Richter, E., Sarker, S., Barrila, J. & other authors (2008).Media ion composition controls regulatory and virulence response of

Salmonella in spaceflight. PLoS ONE 3, e3923.

Yates, E. A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S. R.,Sockett, R. E., Goldner, M., Dessaux, Y., Camara, M. & other authors(2002). N-acylhomoserine lactones undergo lactonolysis in a pH-,

temperature-, and acyl chain length-dependent manner during

growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa.

Infect Immun 70, 5635–5646.

Edited by: D. Demuth

F. Mastroleo and others

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