INRS-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7
Correspondence
Eric Déziel
deziel{at}molbio.mgh.harvard.edu
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ABSTRACT |
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Present address: Department of Surgery, Molecular Surgery Laboratory, Massachusetts General Hospital, Boston, MA 02114, USA.
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INTRODUCTION |
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Swarming cells need to overcome the strong surface tension of water surrounding the colony to efficiently colonize the surface (Matsuyama & Nakagawa, 1996). This surface conditioning is often achieved by the production of surface-active compounds, which act as wetting agents (Lindum et al., 1998
; Matsuyama et al., 1992
, 1995
; Mendelson & Salhi, 1996
; Toguchi et al., 2000
). Spreading growth of Serratia marcescens depends on the production of various extracellular lipopeptides named serrawetins (Matsuyama et al., 1986
, 1992
, 1995
). Synthesis of serrawettin W2, a cyclic lipodepsipentapeptide required for swarming motility of Serratia liquefaciens, is regulated in a cell-density-dependent manner by an acyl-homoserine lactone-based quorum sensing system (Lindum et al., 1998
). Swarming of Bacillus subtilis relies on the production of surfactin (Mendelson & Salhi, 1996
), a cyclic lipopeptide biosurfactant whose synthesis is also controlled by quorum sensing via two pheromone signal peptides, ComX and CSF (Lazazzera et al., 1997
; Solomon et al., 1996
).
P. aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid (rhamnolipids) (Hauser & Karnovsky, 1957; Jarvis & Johnson, 1949
). Although their exact physiological function is still unclear, these amphiphilic molecules are usually considered biosurfactants, acting as solubilizing agents promoting the uptake of hydrophobic substrates, especially n-alkanes (Beal & Betts, 2000
; Itoh et al., 1971
). Moreover, rhamnolipids are virulence factors found in high concentrations in sputa of P. aeruginosa-colonized cystic fibrosis patients (Kownatzki et al., 1987
). Rhamnolipids interfere with the normal tracheal ciliary function (Read et al., 1992
), inhibit the phagocytic response of macrophages (McClure & Schiller, 1996
) and act as heat-stable extracellular haemolysins (Johnson & Boese-Marrazzo, 1980
). In liquid cultures, they are produced as a complex mixture of congeners containing one or two 3-hydroxy fatty acids of various length, linked to a mono- or dirhamnose moiety (Déziel et al., 1999
, 2000
). In general, the two more abundant rhamnolipids are L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate and L-rhamnosyl-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Lang & Wullbrandt, 1999
; Maier & Soberón-Chávez, 2000
) (Fig. 1
; m, n=6). According to the biosynthetic pathway proposed by Burger et al. (1963)
, rhamnolipid synthesis proceeds by two sequential glycosyl transfer reactions, each catalysed by a different rhamnosyltransferase. The first rhamnosyltransferase, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA; Fig. 1
), is encoded by the rhlAB operon (Ochsner et al., 1994a
, b
, 1995
). Both genes, co-expressed from the same promoter, are essential for rhamnolipid synthesis but, whereas rhlB is known to encode the catalytic subunit of the rhamnosyltransferase, the function of rhlA is still unresolved. RhlA is probably an inner-membrane-bound protein (Rahim et al., 2001
), presumably involved in the synthesis or transport of rhamnosyltransferase precursor substrates or in the stabilization of the RhlB protein (Ochsner et al., 1994a
). Environmental factors, especially nutritional conditions, influence rhamnolipid production (Guerra-Santos et al., 1986
). Furthermore, cell-to-cell signalling regulates the expression of the rhlAB operon (Ochsner et al., 1994b
; Ochsner & Reiser, 1995
; Pearson et al., 1997
; Pesci et al., 1997
). This quorum sensing system is composed of rhlI, the N-butyrylhomoserine lactone autoinducer synthase gene, and rhlR, which encodes the transcriptional activator (Ochsner et al., 1994b
; Ochsner & Reiser, 1995
). The second rhamnosyltransferase, encoded by rhlC, has been characterized and its expression shown to be co-ordinately regulated with rhlAB by the same quorum sensing system (Rahim et al., 2001
). Köhler et al. (2000)
reported that cell-to-cell signalling, and both flagella and type IV pili, are required for swarming motility of P. aeruginosa. They observed that an rhlA mutant was unable to swarm and therefore concluded that rhamnolipid production is required for swarming motility of P. aeruginosa.
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METHODS |
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Expression of the rhlAB operon was investigated by monitoring -galactosidase activity of the wild-type strain PG201 containing an rhlA'-lacZ translational fusion (plasmid pECP60) (Pesci et al., 1997
). pECP60 was introduced into strain PG201 by electroporation (Smith & Iglewski, 1989
) and PG201(pECP60) transformants were selected with carbenicillin (200 µg ml-1). X-Gal (40 µg ml-1) was added to culture media when required.
Swarm plates were composed of 0·5 % Bacto-agar and 8 g nutrient broth l-1, both from Difco, supplemented with 5 g glucose l-1 and dried overnight at room temperature before use (Rashid & Kornberg, 2000). Cells were point-inoculated with a sterile toothpick or 2 µl of an overnight culture and the plates were incubated at 30 °C for 2448 h. For liquid swarm medium, agar was omitted.
An iron-limited mineral salts medium (MSM) designed to promote rhamnolipid production (Déziel et al., 1996, 2000
), supplemented with 2 % (w/v) carbon source, was also used. The composition was (g l-1): KH2PO4, 0·7; Na2HPO4, 0·9; NaNO3, 2·0; MgSO4.7H2O, 0·4; CaCl2.2H2O, 0·1; FeSO4.7H2O, 0·001. The final pH was 6·7.
The effects of the nitrogen source (25 µM NH4Cl or NaNO3, or 12·5 µM both) or addition of iron (0, 1, 5 or 10 µM FeSO4.7H2O) on growth and rhlA expression of PG201(pECP60) were studied on swarm agar plates containing 1·5 % agar and carbenicillin (300 µg ml-1). The plates were spot-inoculated in triplicate with 2 µl from an overnight culture and incubated for 20 h at 30 °C. The colonies were then recovered and resuspended in 1 ml PBS. -Galactosidase activity was assayed according to Miller (1972)
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Biosurfactant production and analysis.
Cultures were grown in 50 ml iron-limited MSM supplemented with 2 % (w/v) mannitol in 250 ml Erlenmeyer flasks and incubated at 30 °C with gyratory shaking at 200 r.p.m. (Déziel et al., 1999, 2000
). The cell-free supernatant was analysed after 6 days incubation at 30 °C and 200 r.p.m. Surface and wetting activities were qualitatively compared with the drop-collapsing test (Jain et al., 1991
) and surface tension was measured by the ring method with a du Nouy tensiometer (Fisher Scientific). LC/MS analyses were performed as described previously for rhamnolipids and HAAs (Déziel et al., 2000
; Lépine et al., 2002
). Culture samples were centrifuged at 16 000 g for 5 min to remove the bacteria and the supernatant was filtered through a 0·2 µm filter. An internal standard (16-hydroxyhexadecanoic acid) was added before injecting the sample into the mass spectrometer to allow quantitative measurements. All the analyses were performed in triplicate with a triple quadrupole mass spectrometer Quattro II (Micromass) equipped with a Z-spray interface using electrospray ionization in negative mode. The spectrometer was interfaced to an HP 1100 HPLC (Agilent Technologies) equipped with a 150 mmx4 mm Zorbax C8 reverse phase column (particle size 5 µm). Quantification was performed by integration of the pseudomolecular and the proper fragment ions.
Determination of biosurfactant production on swarm plates was performed as follows. Each strain was inoculated on five MSM agar plates with 2 % mannitol and after 48 h of incubation the whole content was mixed with 100 ml 1 % KHCO3 (pH 9), incubated overnight at 4 °C and centrifuged for 10 min at 1800 g to remove solids. The supernatant was then acidified to pH 4 with concentrated HCl and extracted three times with 40 ml ethyl acetate. The organic fractions were finally pooled, dried and evaporated. The residue was resuspended in an aqueous solution containing 36 % acetonitrile and 4 mM ammonium acetate.
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RESULTS AND DISCUSSION |
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We further investigated this observation with the reference strain PG201, which was originally used to isolate the rhlAB operon, and the rhlA- UO299 and rhlB- UO287 mutants, both derived from PG201 (Ochsner et al., 1994a). UO299 does not express both rhlA and rhlB, whereas rhlA is still expressed in UO287 (Ochsner et al., 1994a
). Cultures of the mutant UO299 contained no trace of HAAs or rhamnolipids, as observed with mutant 57RP (
rhlA : : Km). However, UO287, while not producing rhamnolipids, was still secreting HAAs. These results indicate that rhlA is involved in the synthesis of HAAs, the lipidic precursors of rhamnolipids and substrate of the RhlB rhamnosyltransferase. Thus, dimers of 3-hydroxyalkanoic acids are directly excreted in the extracellular milieu or coupled to rhamnose by the rhamnosyltransferase encoded by rhlB to produce rhamnolipids.
The RhlB rhamnosyltransferase prefers longer chain and saturated HAAs
We determined the concentrations of free HAAs congeners, along with the mono- and dirhamnolipids, in the supernatant of a PG201 culture (Table 1). The free HAA profile in strain PG201 is similar to the one previously observed for strain 57RP (Lépine et al., 2002
). Notably, the supernatant was proportionally depleted in C10-C10 HAA (23·6 %) relative to the mono- (64·4 %) and dirhamnolipids (60·8 %) and contained a large proportion of C8-C8 HAA (22·1 %), while no rhamnolipids containing C8-C8 were detected. In contrast, the total free HAA concentration in the rhlB- mutant UO287 supernatant was almost nine times higher than in the wild-type strain PG201 supernatant (Table 1
). Proportions of the various free HAAs differed between strains UO287 and PG201. Most notably, the proportion of free HAAs represented by the C10-C10 congener goes from 23·6 % for PG201 to 56·6 % for strain UO287. This value is very close to the proportion of the C10-C10 congener observed in mono- (64·4 %) and dirhamnolipids (60·8 %) in PG201. The percentage of free HAAs represented by the C8-C8 congener goes from 22·1 % for PG201, to only 0·5 % for strain UO287, while no C8-C8 congeners are observed in mono- and dirhamnolipids of PG201. The very high percentage of free HAAs represented by the C10-C12 : 1 congener (31·3 %) in PG201 becomes 8·6 % in UO287, close to the values observed for the C10-C12 : 1 congeners in mono- and dirhamnolipids of PG201. These results further strengthen our hypothesis that the RhlB rhamnosyltransferase has a preference for longer chain and saturated HAAs (Lépine et al., 2002
), leaving an HAA pool enriched in shorter chain and unsaturated congeners, as seen in the free HAAs of PG201. The residual free HAA pool of PG201 is small because the RhlB rhamnosyltranferase utilizes most of the available HAAs. The larger UO287 pool is therefore representative of the initial HAAs pool before rhamnolipid synthesis. This explains why the pool of free HAAs in UO287 cultures is very similar to the HAA profile of mono- and dirhamnolipids observed with PG201.
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HAAs are surfactants
Besides the lack of concordance between the composition of the initial HAA pool and the preference of the RhlB rhamnosyltransferase, presence of HAAs in cultures of the wild-type strain indicates that their release by the rhlB- mutant is not simply the result of overproduction. The mixture of hydroxylated fatty acids is actually excreted under normal growth conditions, suggesting that free HAAs play a specific role of their own in the bacteria. The excretion of anionic biosurfactants, such as rhamnolipids, is revealed by the formation of a dark blue halo around colonies growing on SW Blue agar plates (Siegmund & Wagner, 1991). As expected, the rhlA mutant UO299 did not produce a halo on these plates. Interestingly, the rhlB mutant UO287 produced a small halo, indicating that the cells released anionic amphiphilic compounds other than rhamnolipids.
Accordingly, significant surface activity was detected in the extracellular fluids of strains PG201 and UO287 cultivated in a liquid medium designed to promote the production of rhamnolipids (Table 2). A positive drop-collapsing test and a surface tension below 45 mN m-1 usually denote the presence of surface-active agents. The surface tension of rhlB- mutant cultures was very low and consistently lower than wild-type cultures, suggesting that HAAs display a potent surface-tension-lowering activity. Total rhamnolipids and HAAs from PG201 and UO287 cultures, respectively, were purified, diluted in water over a range of concentrations and the surface tension of these solutions was measured. Fig. 2
shows that HAAs can decrease the surface tension to a lower value than rhamnolipids, confirming that HAAs have excellent tensioactive properties. Thus, HAAs represent a new class of biosurfactants released by P. aeruginosa in addition to rhamnolipids.
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Conclusion
The finding of a second class of biosurfactants produced by P. aeruginosa as well as the complex regulation of rhlAB expression indicate that swarming motility plays a critical role in the environmental adaptation of this bacterium. The capacity for surface-colonizing migration is increasingly recognized as a widespread trait in eubacteria (Fraser & Hughes, 1999; Harshey, 1994
). The multicellular and cooperative nature of swarming motility clearly associates this phenomenon with the natural propensity of micro-organisms to form biofilms (Eberl et al., 1999
; Mireles et al., 2001
; Sharma & Anand, 2002
). Interestingly, Singh et al. (2002)
recently reported that very low available iron concentrations, resulting from sequestering, markedly stimulated surface motility, thus preventing biofilm development by P. aeruginosa. It is therefore intriguing that we also observed increased surface motility when limiting iron conditions prevailed. These results support the hypothesis that biosurfactant production and surface motility are hyper-expressed under unfavourable nutritional conditions, presumably to prevent P. aeruginosa from settling and forming a biofilm. This hypothesis is strengthened by the very recent report that rhamnolipid production is involved in the maintenance of biofilm architecture (Davey et al., 2003
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bollinger, N., Hassett, D. J., Iglewski, B. H., Costerton, J. W. & McDermott, T. R. (2001). Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation. J Bacteriol 183, 19901996.
Burger, M. M., Glaser, L. & Burton, R. M. (1963). The enzymatic synthesis of a rhamnose-containing glycolipid by extracts of Pseudomonas aeruginosa. J Biol Chem 238, 25952602.
Campos-García, J., Caro, A. D., Nájera, R., Miller-Maier, R. M., Al-Tahhan, R. A. & Soberón-Chávez, G. (1998). The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent -ketoacyl reductase which is specifically involved in rhamnolipid synthesis. J Bacteriol 180, 44424451.
Davey, M. E., Caiazza, N. C. & O'Toole, G. A. (2003). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185, 10271036.
Déziel, É., Paquette, G., Villemur, R., Lépine, F. & Bisaillon, J.-G. (1996). Biosurfactant production by a soil Pseudomonas strain growing on polycyclic aromatic hydrocarbons. Appl Environ Microbiol 62, 19081912.[Abstract]
Déziel, E., Lépine, F., Dennie, D., Boismenu, D., Mamer, O. A. & Villemur, R. (1999). Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene. Biochim Biophys Acta 1440, 244252.[Medline]
Déziel, E., Lépine, F., Milot, S. & Villemur, R. (2000). Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP. Biochim Biophys Acta 1485, 145152.[Medline]
Déziel, E., Comeau, Y. & Villemur, R. (2001). Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with the emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming and twitching motilities. J Bacteriol 183, 11951204.
Eberl, L., Molin, S. & Givskov, M. (1999). Surface motility of Serratia liquefaciens MG1. J Bacteriol 181, 17031712.
Fraser, G. M. & Hughes, C. (1999). Swarming motility. Curr Opin Microbiol 2, 630635.[CrossRef][Medline]
Guerra-Santos, L. H., Käppeli, O. & Fiechter, A. (1984). Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Appl Environ Microbiol 48, 301305.[Medline]
Guerra-Santos, L. H., Käppeli, O. & Fiechter, A. (1986). Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Appl Microbiol Biotechnol 24, 443448.
Harshey, R. M. (1994). Bees aren't the only ones: swarming in Gram-negative bacteria. Mol Microbiol 13, 389394.[Medline]
Hauser, G. & Karnovsky, M. L. (1957). Rhamnose and rhamnolipid biosynthesis by Pseudomonas aeruginosa. J Biol Chem 224, 91105.
Ishimoto, K. S. & Lory, S. (1989). Formation of pilin in Pseudomonas aeruginosa requires the alternative factor (RpoN) subunit of RNA polymerase. Proc Natl Acad Sci U S A 86, 19541957.[Abstract]
Itoh, S., Honda, H., Tomita, F. & Suzuki, T. (1971). Rhamnolipids produced by Pseudomonas aeruginosa grown on n-paraffin (mixture of C12, C13 and C14 fractions). J. Antibiot 24, 855859.[Medline]
Jain, D. K., Collins-Thompson, D. L., Lee, H. & Trevors, J. T. (1991). A drop-collapsing test for screening surfactant-producing microorganisms. J Microbiol Methods 13, 271279.[CrossRef]
Jarvis, F. G. & Johnson, M. J. (1949). A glycolipid produced by Pseudomonas aeruginosa. J Am Chem Soc 71, 41244126.
Johnson, M. K. & Boese-Marrazzo, D. (1980). Production and properties of heat-stable extracellular hemolysin from Pseudomonas aeruginosa. Infect Immun 29, 10281033.[Medline]
Köhler, T., Curty, L. K., Barja, F., Van Delden, C. & Pechère, J.-C. (2000). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182, 59905996.
Kownatzki, R., Tummler, B. & Doring, G. (1987). Rhamnolipid of Pseudomonas aeruginosa in sputum of cystic fibrosis patients. Lancet 1, 10261027.
Lang, S. & Wullbrandt, D. (1999). Rhamnose lipids biosynthesis, microbial production and application potential. Appl Microbiol Biotechnol. 51, 2232.
Lazazzera, B. A., Solomon, J. M. & Grossman, A. D. (1997). An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell 89, 917925.[Medline]
Lépine, F., Déziel, E., Milot, S. & Villemur, R. (2002). Liquid chromatographic/mass spectrometric detection of the 3-(3-hydroxyalkanoyloxy)alkanoic acid precursors of rhamnolipids in Pseudomonas aeruginosa cultures. J Mass Spectrom 37, 4146.[CrossRef][Medline]
Lindum, P. W., Anthoni, U., Christophersen, C., Eberl, L., Molin, S. & Givskov, M. (1998). N-acyl-L-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1. J Bacteriol 180, 63846388.
Maier, R. M. & Soberón-Chávez, G. (2000). Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl Microbiol Biotechnol 54, 625633.[CrossRef][Medline]
Matsuyama, T. & Nakagawa, Y. (1996). Surface-active exolipids: analysis of absolute chemical structures and biological functions. J Microbiol Methods 25, 165175.[CrossRef]
Matsuyama, T., Murakami, T., Fujita, M., Fujita, S. & Yano, I. (1986). Extracellular vesicle formation and biosurfactant production by Serratia marcescens. J Gen Microbiol 132, 865875.
Matsuyama, T., Kaneda, K., Ishizuka, I., Toida, T. & Yano, I. (1990). Surface-active novel glycolipid and linked 3-hydroxy fatty acids produced by Serratia rubidaea. J Bacteriol 172, 30153022.[Medline]
Matsuyama, T., Kaneda, K., Nakagawa, Y., Isa, K., Hara-Hotta, H. & Yano, I. (1992). A novel extracellular cyclic lipopeptide which promotes flagellum-dependent and -independent spreading growth of Serratia marcescens. J Bacteriol 174, 17691776.[Abstract]
Matsuyama, T., Bhasin, A. & Harshey, R. M. (1995). Mutational analysis of flagellum-independent surface spreading of Serratia marcescens. J Bacteriol 177, 987991.[Abstract]
McClure, C. D. & Schiller, N. L. (1996). Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo. Curr Microbiol 33, 109117.[CrossRef][Medline]
Mendelson, N. H. & Salhi, B. (1996). Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms. J Bacteriol 178, 19801989.[Abstract]
Merrick, M. J. & Edwards, R. A. (1995). Nitrogen control in bacteria. Microbiol Rev 59, 604622.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mireles, J. R., 2nd, Toguchi, A. & Harshey, R. M. (2001). Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation. J Bacteriol 183, 58485854.
Mulligan, C. N. & Gibbs, B. F. (1989). Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa. Appl Environ Microbiol 55, 30163019.[Medline]
Ochsner, U. A. & Reiser, J. (1995). Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92, 64246428.[Abstract]
Ochsner, U. A., Fiechter, A. & Reiser, J. (1994a). Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 269, 1978719795.
Ochsner, U. A., Koch, A. K., Fiechter, A. & Reiser, J. (1994b). Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J Bacteriol 176, 20442054.[Abstract]
Ochsner, U. A., Hembach, T. & Fiechter, A. (1995). Production of rhamnolipid biosurfactants. Adv Biochem Eng Biotechnol 53, 89118.
Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 57565767.[Abstract]
Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. (1997). Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 31273132.[Abstract]
Rahim, R., Ochsner, U. A., Olvera, C., Graninger, M., Messner, P., Lam, J. S. & Soberon-Chávez, G. (2001). Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis. Mol Microbiol 40, 708718.[CrossRef][Medline]
Rashid, M. H. & Kornberg, A. (2000). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97, 48854890.
Read, R. C., Roberts, P., Munro, N. & 7 other authors (1992). Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating. J Appl Physiol 72, 22712277.
Rehm, B. H. A., Krüger, N. & Steinbüchel, A. (1998). A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. The phaG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme A transferase. J Biol Chem 273, 2404424051.
Rehm, B. H. A., Mitsky, T. A. & Steinbüchel, A. (2001). Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by Pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli. Appl Environ Microbiol 67, 31023109.
Sharma, M. & Anand, S. K. (2002). Swarming: a coordinated bacterial activity. Curr Science 83, 707715.
Siegmund, I. & Wagner, F. (1991). New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5, 265268.
Singh, P. K., Parsek, M. R., Greenberg, E. P. & Welsh, M. J. (2002). A component of innate immunity prevents bacterial biofilm development. Nature 417, 552555.[CrossRef][Medline]
Smith, A. W. & Iglewski, B. H. (1989). Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17, 10509.[Medline]
Solomon, J. M., Lazazzera, B. A. & Grossman, A. D. (1996). Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev 10, 20142024.[Abstract]
Toguchi, A., Siano, M., Burkart, M. & Harshey, R. M. (2000). Genetics of swarming motility in Salmonella enterica serovar Typhimurium: critical role for lipopolysaccharide. J Bacteriol 182, 63086321.
Totten, P. A., Lara, J. C. & Lory, S. (1990). The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J Bacteriol 172, 389396.[Medline]
Received 27 November 2002;
revised 3 March 2003;
accepted 15 April 2003.