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1 Departement Osmorégulation chez les Bactéries, UMR-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, Av. du Général Leclerc, 35042 Rennes, France
2 Laboratoire de Biotechnologie et Chimie Marines, EA 3884, Université de Bretagne Sud, Lorient, France
Correspondence
Mohamed Jebbar
mohamed.jebbar{at}univ-rennes1.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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). One critical environmental parameter is the osmolality or the salinity of the extracellular medium. Both soil and saline habitats undergo frequent changes in this physico-chemical parameter because rainfall and evaporation cause drastic changes in the environmental osmolality. Despite variations in the osmotic pressure of their environment, bacteria maintain their cytoplasmic hydration level. This may be accomplished by accumulating solutes when extracellular osmolality rises and by rapidly releasing those solutes when extracellular osmolality declines (Wood et al., 2001). The vast majority of prokaryotes cope with increasing osmolality by uptake or synthesis of organic molecules; among these, those most compatible with cell physiology appear to be preferred (Wood et al., 2001). The spectrum of compatible solutes used by organisms from the three domains of life comprises only a limited number of compounds, which can be divided into two major groups: (i) sugars and polyols, and (ii) 
- and 
-amino acids and their derivatives, including methylamines (Roeßler & Müller, 2001). The preferred compatible solute for the majority of prokaryotes, and perhaps for the most widely utilized osmolyte, spanning both the plant and animal kingdoms, is the trimethylammonium compound glycine betaine (GB) (Bremer & Krämer, 2000; Robert et al., 2000; Sleator & Hill, 2002; Talibart et al., 1997).
Pseudomonas aeruginosa, a versatile Gram-negative bacterium, can grow in soil, marshes and costal marine habitats, as well as on plants and in animal tissues; it is an opportunistic pathogen that primarily affects cystic fibrosis patients and immuno-compromised patients. The investigation of osmoadaptative mechanisms in P. aeruginosa PAO1 showed that osmotically stressed cells synthesized and accumulated glutamate, trehalose and N-acetylglutaminylglutamine amide (NAGGN) in the absence of exogenously provided osmoprotectants, while they preferentially accumulated GB when this compound was available in the growth medium. Moreover, the accumulation of exogenous GB led to a dramatic reduction in the intracellular levels of the three endogenous osmolytes (D'Souza-Ault et al., 1993). GB and its precursors choline and carnitine serve as osmoprotectants, but only GB is accumulated as a cytosolic osmolyte under hyperosmolar conditions by P. aeruginosa (Lucchesi et al., 1995). In addition to their important role in osmoprotection, choline, carnitine and GB can also support the growth of P. aeruginosa at low osmolality, serving as both carbon and nitrogen sources (Lucchesi et al., 1995; Serra et al., 2002). A few reports have proposed a pathway for GB catabolism in bacteria such as Sinorhizobium meliloti (Smith et al., 1988) and Arthrobacter spp. (Meskys et al., 2001). Under aerobic conditions, bacterial degradation of GB often proceeds by three successive demethylations through the formation of dimethylglycine and sarcosine as intermediates; the resulting glycine is, in turn, converted to serine (Meskys et al., 2001; Smith et al., 1988). In P. aeruginosa, one important step towards the genetic and biochemical elucidation of the GB catabolic route was the identification of the GB transmethylase (Gbt), which catalyses the first demethylating step of GB (Serra et al., 2002). Many reports on P. aeruginosa showed a link between osmotic stress responses and the synthesis of virulence factors, such as alginate (Berry et al., 1989; Deretic et al., 1990) and phospholipase C (PLC) (Shortridge et al., 1992). GB and its precursors choline and carnitine induce the synthesis of PLC (Lucchesi et al., 1995); moreover, this osmoprotectant-dependent expression of PLC is subject to catabolite repression control (CRC) by succinate, but the global regulators Crc and Vfr are not involved in this mechanism (Sage & Vasil, 1997). Also, an active transport system induced by choline is repressed by succinate, the preferred source of carbon in P. aeruginosa (Salvano et al., 1989).
In this paper, we show by a proteomic approach that 21 proteins were induced by GB and most of them were involved in GB degradation. The synthesis of all these proteins was not induced in the presence of a mixture of GB and succinate, but occurred for most of them when glucose was supplied with GB. This is corroborated by the fact that GB, while improving growth at inhibitory osmolality, was accumulated stably in succinate-grown cells, while it was accumulated only transiently in glucose-grown cells. These data suggest that GB utilization in P. aeruginosa is controlled by a succinate CRC mechanism at both low and high osmolalities.
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METHODS |
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) and its derivatives PAO8020 (PAO1 
crc; Tcr) (MacGregor et al., 1996), PAO9001 [PAO1 (‘orfX, vfr, trpC’) aacC1; Gmr] (Suh et al., 2002) and PAO9002 (PAO1 vfr101 : : aacC1; Gmr) (Suh et al., 2002). These strains were grown aerobically at 37 °C with constant shaking (175 r.p.m.) in LB and defined M63 media (Miller, 1972). Glucose (10 mM), succinate (20 mM) and GB (10 mM) were added to M63 media as the carbon source. The osmotic strength of the media was increased by the addition of NaCl or sucrose as necessary. The antibiotics tetracycline and gentamicin were used in solid media at final concentrations of 50 µg ml–1. Bacterial growth was monitored by OD570 measurements.
Extraction and analysis of cellular solutes.
The pellets of freshly harvested and washed cells were extracted in 80 % (v/v) ethanol with stirring at room temperature for 30 min. After centrifugation, the supernatant (ethanol-soluble fraction) was evaporated to dryness at 40 °C, dissolved into distilled water and stored at –20 °C until needed for further determination. This fraction was analysed by paper chromatography, TLC and paper high-voltage electrophoresis as described by Bernard et al. (1993). For spectroscopic analysis, the fraction was evaporated to dryness, and the dry residue was dissolved into 1 ml D2O; the 13C NMR spectrum was recorded as described previously (Bernard et al., 1993; Jebbar et al., 1997).
Radiolabelling assays.
Transport and metabolic assays were done with [methyl-14C]GB (2·07 GBq mmol–1), which was prepared from [methyl-14C]choline chloride (2·07 GBq mmol–1) as described previously (Bernard et al., 1993; Jebbar et al., 1992). Transport assays were carried out as described by Choquet et al. (2005); the GB concentration in the uptake assay was 10 µM. To study the fate of GB or choline, cells were cultured in M63 with or without 0·5 M NaCl, and supplemented with glucose (10 mM) or succinate (20 mM) as the carbon source, and 1 mM [14C]GB (5·5 MBq mmol–1) or 1 mM [14C]choline (5·5 MBq mmol–1). After two to five generations, 1 ml cell culture was harvested by centrifugation (5000 g for 10 min) and washed twice with carbon-free M63 containing the same NaCl or sucrose concentration as the growth medium. After extraction in 80 % ethanol, the radioactive components of the ethanol-soluble fraction was analysed by paper chromatography and/or electrophoresis (Bernard et al., 1993). The radioactive spots were visualized using a Packard Instant Imager. The radioactivity of the ethanol-insoluble fraction and the CO2 trapped on filter paper soaked with 6 M KOH was also determined.
GB transmethylase assays.
P. aeruginosa PAO1 strain was grown either in M63 with 10 mM GB as the carbon source, or in M63 with 10 mM glucose or 20 mM succinate as the carbon source, and with or without 1 mM GB. Cultures (100 ml) were incubated at 37 °C, with shaking (175 r.p.m.), and harvested by centrifugation at mid-exponential phase (OD570 0·7). The pellets were washed twice in carbon-free M63, once with sodium phosphate buffer (10 mM, pH 7·5), and finally resuspended in the same buffer to 100 OD570 units ml–1. Bacterial cells were disrupted with glass beads, and cell debris were removed by centrifugation; the clarified cell lysates were then used to carry out GB transmethylase assays using a procedure adapted from that performed by Serra et al. (2002). Briefly, the standard assay contained 10 mM Na+-phosphate buffer (pH 7·5), 50 µM L-homocysteine, 20 µM [methyl-14C]GB (2·83 GBq mmol–1) and 0·5 mg protein in a final volume of 100 µl. The remaining substrate and the resulting products of this enzymic assay were extracted in 80 % ethanol and analysed by TLC using aqueous phenol (80 % w/v) as the mobile phase. The radioactive spots were visualized and quantified using a Packard Instant Imager.
Chemicals.
The radiolabelled compound [methyl14C]choline was purchased from NEN-Dupont. Glucose and succinate used in this study as carbon sources were purchased from Sigma-Aldrich. The osmoprotectants, GB, choline, L-carnitine, D, L-pipecolate, L-proline, sucrose and trehalose, were also purchased from Sigma-Aldrich. Ectoine was purchased from Bitop. Dimethylsulfonioacetate and dimethylsulfoniopropionate were synthesized in the laboratory as described previously (Pichereau et al., 1998).
Separation of proteins by two-dimensional electrophoresis (2DE) and MALDI-TOF MS analysis.
After harvesting by centrifugation, bacterial cells were washed in sodium phosphate buffer (10 mM, pH 7·5), then resuspended in the same buffer and disrupted by three passages through a French press. Cells debris was removed by centrifugation at 4 °C and 12 000 g for 30 min. The protein concentration of the supernatant fraction was determined according to the Lowry method. The lysate was precipitated with 10 % trichloroacetic acid, 80 % acetone, and then solubilized in the rehydration buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0·24 % Triton X-100, 20 mM DTT, 0·48 % Biolytes pH 3–10 and traces of bromophenol blue). For analytical and preparative two-dimensional (2D) gels, 200–300 µg crude protein extract was analysed by 2DE as previously described (Jebbar et al., 2005). Proteins were identified by tryptic digestion of the polypeptides isolated from the Coomassie-stained control gels followed by peptide mass fingerprinting with a MALDI-TOF mass spectrometer (Voyager TM Elite; Perspective Biosystems) as described previously (Jebbar et al., 2005). Peptide mass fingerprints were identified by comparison with theoretical values for proteins in the National Center for Biotechnology Information database or the P. aeruginosa protein database. The P. aeruginosa PAO1 protein database was created after downloading the whole-genome protein sequence of P. aeruginosa in FASTA format, available at the Pseudomonas genome project BLAST server (http://www.pseudomonas.com/). The proteins were identified by searching, in the above-cited databases, with the MS-FIT software of ProteinProspector (http://prospector.ucsf.edu/) and Mascot from Matrix Science (http://www.matrixscience.com/search_form_select.html). The search parameters allowed for oxidation of methionine, carbamidomethylation of cysteine and a maximum of one missed cleavage site within the peptides.
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RESULTS |
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show that succinate is the best carbon source at low salinity, but when medium osmolality rises glucose progressively becomes a better carbon source than succinate. At 0·75 M NaCl, PAO1 failed to grow on succinate, but it still grew on glucose. Addition of GB to M63 without NaCl did not modify the growth parameters, but it did improve growth when medium salinity increased from 0·3 to 0·75 M NaCl. The osmoprotective effect of GB on the growth rate was more obvious when succinate was used as the carbon source at 0·75 M NaCl in comparison to glucose. Various osmoprotectants were assayed to assess their ability to protect PAO1 cells from the detrimental effect of high salinity, when grown on succinate or glucose in M63 with 0·5 M NaCl. Only dimethylsulfoniopropionate, dimethylsulfonioacetate, ectoine and proline stimulated the growth as equally well as GB and its precursors choline and carnitine did (Lucchesi et al., 1995), but other bacterial osmoprotectants such as pipecolate, sucrose and trehalose failed to protect PAO1 cells against osmotic stress (Table 2). At similar osmolalities the replacement of NaCl by the non-electrolyte sucrose did not induce significant changes in the growth pattern, but in the presence of GB, the GB osmoprotective effect was observable only at 0·96 M sucrose, a concentration that develops an osmolality equivalent to that of 0·75 M NaCl (data not shown).
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. As described by D'Souza-Ault et al. (1993), the 13C NMR spectra showed that in the absence of exogenously provided osmoprotectant, cells synthesized and accumulated glutamate and the dipeptide NAGGN as the major organic osmolytes, and trehalose as a minor one (Fig. 1a). Similar results were obtained both with succinate-grown and glucose-grown cells, regardless of their growth stage. The spectral analysis of stressed cells in the presence of GB showed that when glucose was used as a carbon source, stressed cells from the late exponential growth phase accumulated GB and trehalose as minor organic osmolytes, and L-glutamate and NAGGN as the major ones (Fig. 1b). At stationary growth phase, signals from GB were undetectable, whereas those from trehalose, glutamate and NAGGN revealed high intracellular levels of these solutes (data not shown). When succinate was used as the carbon source, only minor amounts of glutamate and NAGGN, and no trehalose were detected; interestingly, exogenously supplied GB was by far the dominant cytoplasmic osmolyte at both exponential (Fig. 1c) and stationary growth phases (data not shown).
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Since 13C NMR spectroscopy can detect accumulated compounds only qualitatively, we determined by radiotracing experiments, through the whole growth cycle, the fate of 1 mM GB in P. aeruginosa cells cultivated at low (0 M NaCl) and high (0·5 M NaCl) salinities with glucose or succinate as the carbon source. Growth experiments showed that, in the presence of GB, cells grew better with succinate than with glucose at low salinity, as well as at high salinity (data not shown). As shown by the absence of both evolved radioactive CO2 and insoluble materials, no GB degradation activity occurred when succinate was used as the carbon source at both low and high salinities during the exponential growth phase (Fig. 2b, d). Only a slight GB catabolic activity was detected at low salinity, when cells had reached the late stationary phase (Fig. 2b, d). Very low levels of GB were detected in the cells at low salinity, but at high salinity cells rapidly accumulated GB up to 560 nmol (OD unit)–1 in early exponential growth phase. This level decreased to 470 nmol (OD unit)–1 in late exponential phase. Less than 40 % of the total supplied radioactivity was taken up by the cells. The amount of GB decreased progressively to reach 280 nmol (OD unit)–1, 2 h after the entry of the cells into the stationary growth phase. In parallel, the radioactivity (of GB) in the supernatant increased at the entry of the cells into the stationary growth phase (Fig. 2a). Thus, a significant decrease in intracellular accumulated GB was not due to a start in a catabolic activity of this solute but rather to a GB efflux activity.
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). At elevated salinity, the radioactivity in the medium decreased linearly and concomitantly with increasing cell density (Fig. 2a). The radioactivity incorporated by the cells was found in CO2, and in ethanol-insoluble and ethanol-soluble fractions. After two generations, more than 50 % of the total radioactivity was taken up by the cells and the radioactivity was mostly found in ethanol-soluble fraction and in 14CO2 (Fig. 2b, c). Chromatographic and electrophoresis analyses of the ethanol-soluble fraction revealed that GB was the sole radiolabelled osmolyte, the maximal level reaching 520 nmol (OD unit)–1 at early exponential growth phase; this level decreased to about 140 nmol (OD unit)–1 in late exponential growth phase. Much more 14CO2 was produced from GB at high salinity than at low salinity (Fig. 2b); the remaining radiocarbon was incorporated into insoluble materials, and this fraction was much more labelled at low salinity than at high salinity (Fig. 2d). The GB precursor choline was also assayed and the radiotracing experiments carried out showed that choline was actively taken up and converted into GB at high salinity; the GB produced was subjected to CRC only when succinate but not glucose was used as the carbon source (data not shown). Unexpectedly, choline uptake was almost inactive at low salinity under our growth conditions, independent of the carbon source used (data not shown).
To determine if GB degradation activity was under the control of global regulators like Crc or Vfr, the fate of GB was also determined in crc and vfr mutant derivatives of wild-type P. aeruginosa PAO1. The results showed that GB degradation activity in these mutants was similar to that observed in PAO1, and this activity was repressed by succinate (data not shown).
Growth patterns of P. aeruginosa on GB plus succinate or glucose
In order to determine whether GB and glucose (or succinate) are used simultaneously or successively as carbon and energy sources, PAO1 cells were pre-cultured in minimal medium M63 with 20 mM succinate, and then used to inoculate M63 medium with 20 mM succinate, 5 mM succinate or a mixture of 5 mM succinate and 10 mM GB. A preculture in M63 with 10 mM glucose was used to inoculate M63 medium with 10 mM glucose, 2·5 mM glucose, or a mixture of 2·5 mM glucose and 10 mM GB. As a control, a pre-culture in M63 with 10 mM GB, was used to inoculate M63 medium with the same carbon source. As shown in Fig. 3(a, b), the growth rate was slightly higher with succinate than with glucose. The maximal OD570 reached was about 0·45 when succinate and glucose were used, at concentrations of 5 and 2·5 mM, respectively, while it reached 1·5 and 1·7 with 10 mM glucose and 20 mM succinate, respectively. The growth rate and growth yield in M63 with 10 mM GB as the sole carbon source were lower than those observed with 20 mM succinate and 10 mM glucose (Fig. 3a). These data showed that, in P. aeruginosa, the compatible solute GB was a less efficient carbon and energy source than succinate and glucose were. As expected, cells grown on a mixture of 2·5 mM succinate and 10 mM GB showed identical growth parameters as cells grown on 2·5 mM succinate without GB (Fig. 3b); but the cells grown on a mixture of succinate and GB restarted to grow and used GB after a lag of about 10 h, and after a short exponential growth phase the cells reached a maximal OD570 of about 1 (data not shown). These data suggest that there was a long diauxic lag phase between the consumption of succinate and the initiation of GB utilization as a carbon source. However, growth on a mixture of glucose and GB resulted in a biphasic pattern characterized by two distinct exponential phases separated by a short lag phase of about 2 h (Fig. 3b). Thus, these results suggest that succinate repressed GB assimilation, but glucose had no effect even if we observed a diauxic-like lag.
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. By comparing the protein profile of GB-grown cells to the protein profiles of succinate- and glucose-grown cells, we observed that more than 20 proteins showed induced expression in the presence of GB (Fig. 4d). The 2DE gels obtained with succinate (20 mM) plus GB (1 mM) (Fig. 4a) and with succinate (20 mM) alone (not shown) were identical, and all proteins induced by GB in Fig. 4(d) were repressed in the presence of succinate. However, we observed a significant difference between 2DE gels from cells grown on glucose (10 mM) (Fig. 4b) and cells grown on glucose (10 mM) plus GB (1 mM) (Fig. 4c). A total of 11 of the GB-induced proteins, out of 21, were detected in Fig. 4(c). All of these protein spots were excised from the gels and examined by MALDI-TOF MS to generate mass fingerprints. The putative identities based on peptide mass fingerprint homology are given in Table 3. Among the identified polypeptides were SoxD (sarcosine oxidase 
subunit), GlyA1 and GlyA2 (serine-hydroxymethyltransferase), FdhA (glutathione-independent formaldehyde dehydrogenase), PurU2 (formyltetrahydrofolate deformylase), SdaB (L-serine dehydratase) and AdhC (glutathione-dependent formaldehyde dehydrogenase). The genes glyA1, soxD, purU2 and fdhA encoding GB-induced proteins are organized in a gene cluster, which also contains soxB, soxA and soxG (http://www.pseudomonas.com/). All these enzymes belong to a probable sarcosine catabolism pathway similar to that described in Arthrobacter spp. (Meskys et al., 2001).
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), an outer-membrane protein and a phosphorylcholine phosphatase enzyme (Fig. 4d, Table 3), which has been described as an enzyme produced in the presence of choline, GB, carnitine or dimethylglycine in P. aeruginosa (Massimelli et al., 2005). Among the GB-induced proteins, there was a putative GB/proline transport substrate-binding protein (PA5378), a component of an ABC-type transporter homologue to ProU from Escherichia coli (Lucht & Bremer, 1994) and OpuC from Bacillus subtilis (Kappes et al., 1996). This system was renamed Opu, for osmoprotectant uptake, and the genes pa5378, pa5377 and pa5376, composing this system, were renamed opuA, opuB and opuC, respectively. Furthermore, many other proteins with undefined biochemical functions were synthesized by the cells in the presence of GB (Table 3). When glucose was used as the carbon source, the addition of GB allowed the induction of some GB-induced proteins that did not comprise SdaB, SoxD, PurU2 and AdhC proteins (Fig. 4c); this suggests that these enzymes are not implicated in GB degradation in the presence of glucose, and that the other proteins present on 2D gels could be sufficient for the GB catabolism activity described above in Fig. 2. In contrast, all GB-induced proteins were undetectable in the presence of both succinate and GB. These data confirm the radiotracing data described above and demonstrate that succinate represses the synthesis of enzymes involved in GB degradation.
GB transmethylase activity
To assay in vitro the GB transmethylase activity, cells were grown in M63 with 10 mM glucose, 10 mM glucose plus 1 mM GB, 20 mM succinate, 20 mM succinate plus 1 mM GB, or 10 mM GB. Cultures were harvested at mid-exponential phase, and crude extracts were prepared and checked for their ability to catabolize [methyl-14C]GB in the presence of L-homocysteine. Curiously, no GB catabolic activity was detected in crude protein extracts from cells grown in M63 with glucose as a carbon source and 1 mM GB (Fig. 5). Such an activity was observed only in the crude extract from culture grown in M63 with 10 mM GB as a carbon source (Fig. 5). Surprisingly, we were able to detect not only radioactive methionine and dimethylglycine, which are produced by a GB transmethylase (Gbt) activity reported in P. aeruginosa (Serra et al., 2002), but also catabolic products such as sarcosine and serine. These data showed that enzymes such as sarcosine oxidase and serine hydroxymethyltransferase, in addition to Gbt, are still active in vitro.
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DISCUSSION |
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; Serra et al., 2002; Smith et al., 1988). However, the alfalfa symbiont S. meliloti can use GB as both an osmoprotectant and a source of carbon and energy (Talibart et al., 1997), leading the dissimilative pathway for GB to be operative at both high and low osmolalities in this bacterium (Talibart et al., 1997). In this study, we showed that besides GB itself (D'Souza-Ault et al., 1993), only its immediate precursors (choline and carnitine) (Lucchesi et al., 1995), two of its sulfonium structural analogues (dimethylsulfonioacetate and dimethylsulfoniopropionate), ectoine and proline were efficient osmoprotectants for P. aeruginosa PAO1 (Table 2). Even pipecolate, which is known to protect efficiently various bacteria against hyperosmotic constraints (Choquet et al., 2005; Gouesbet et al., 1994; Gouffi et al., 2000), has no beneficial effect on growth of P. aeruginosa at high salinity (Table 2). As described previously in S. meliloti (Talibart et al., 1997), we showed that GB was only transiently accumulated in P. aeruginosa. Moreover, the ability of PAO1 to degrade GB was repressed in the presence of succinate at both low and high osmolalities. P. aeruginosa exhibits diauxic growth in media containing both GB and succinate; although in this case succinate was utilized first, while GB catabolism occurred several hours after complete succinate consumption. The presence of a long and unusual lag phase could be explained by a need to induce enzymes involved in GB degradation, and to adapt the cellular machinery to produce energy from catabolic products resulting from GB degradation. When used as a carbon and energy source, GB could support growth but not as efficiently as succinate and glucose did (Fig. 3a). In addition, when M63 containing 10 mM GB (as the carbon source) was inoculated with pre-cultures grown on succinate or glucose, PAO1 cells started to grow after a lag phase of at least 7 h (data not shown). This long lag phase was apparently required to initiate the catabolic and energy production machinery necessary to assimilate GB. P. aeruginosa PAO1 is known to exhibit diauxic growth when it is cultured on succinate and other tricarboxylic acid cycle intermediates in the presence of carbohydrate compounds such as glucose, mannitol, gluconate, glycerol, glycerate or fructose (Collier et al., 1996). In addition to enzymes of the carbohydrate catabolic pathways of P. aeruginosa, succinate mediates repression of several other catabolic pathways involved in the utilization of histidine, methylamine and arginine (Collier et al., 1996). The Crc protein, which is involved in the catabolic repression by succinate of the expression of several genes implicated in the metabolism of some carbohydrates and nitrogenous compounds, is not often necessary for mediating CRC of all genes and operons in P. aeruginosa (Collier et al., 1996). Succinate, via a CRC mechanism that does not involve Crc protein, directly regulates osmoprotectant-dependent expression of plcH, encoding the haemolytic PLC (Sage & Vasil, 1997; Sage et al., 1997). Since P. aeruginosa utilizes GB, and its precursors choline and carnitine, as sole carbon and energy sources, it seems possible that succinate represses the degradation of GB (this study), which results in a repression of osmoprotectant-dependent plcH expression in P. aeruginosa PAO1 (Sage & Vasil, 1997).
Growth on a combination of GB and glucose showed a diauxic-like profile; from this observation, one could speculate that glucose may exert CRC control on GB utilization. This could not be the case, however, because the delay necessary for the initiation of growth on GB is slightly longer than that allowing the full utilization of glucose. Furthermore, the radiotracing experiments (Fig. 2) showed that cells degraded GB at low and high salinities in the presence of glucose; they also showed that for the crude extracts prepared from cells grown on a combination of glucose and GB as carbon and energy sources (presenting a diauxic-like growth profile), a GB degradation activity occurred from a lag and second exponential growth phase (Fig. 3b) but not from the first exponential growth phase (data not shown). In contrast, the absence of GB-demethylating activity in crude extracts from cells grown in M63 with glucose as the carbon source and 1 mM GB (Fig. 5) could be linked to the absence of 10, out of 21, proteins induced by GB (Fig. 4c, d). The other explanations are that (i) at 1 mM, in comparison to 10 mM, GB is probably not sufficient to induce enough GB catabolic enzymes necessary to detect GB-demethylating activity in vitro, or (ii) all GB catabolic enzymes (necessary when GB is used as carbon source) are probably not required to degrade 1 mM GB in the presence of a carbon and energy source such as glucose. This implies that the glucose catabolic pathway(s) helps to assimilate GB under these conditions.
Our proteomic study showed that GB, when used as a carbon and energy source, induced proteins involved in its own degradation, and that the induction of these proteins was subjected to CRC generated by succinate. The radiotracing studies allowed detection of radiolabelled dimethylglycine, methionine, sarcosine and serine (Fig. 5), which are the catabolic products of [methyl-14C]GB. Thus, GB was demethylated in three steps to produce glycine, and dimethylglycine and sarcosine as intermediates; sarcosine oxidase and other undetected enzymes, such as Gbt and dimethylglycine demethylase or dehydrogenase, are involved in this catabolic pathway. Glycine could be further transformed, probably through the action of hydroxymethyltransferase (GlyA1 and GlyA2) into serine, which in turn could give pyruvate through serine dehydratase (SdaB) activity. The P. aeruginosa PAO1 genome also contains sdaA (PA2443), a gene encoding another serine dehydratase that, curiously, was not detected on our 2DE; sdaA belongs to the same gene cluster as glyA2 (PA2444) whose product was detected as being a protein accumulated when GB was used as a bacterial growth substrate. Sarcosine oxidase characterized both in Arthrobacter spp. (Meskys et al., 2001) and Corynebacterium sp. P-1 (Chlumsky et al., 1995) is a heterotetrameric enzyme composed of , , and 
subunits encoded, respectively, by genes soxBDAG that are organized in an operon with glyA and purU2 genes, themselves encoding a serine hydroxymethyltransferase and a formyltetrahydrofolate deformylase, respectively. The genetic organization of this cluster composed of genes encoding enzymes for the catabolism of sarcosine to glycine in Arthrobacter spp. and Corynebacterium sp. P-1 is found to be similar to that of P. aeruginosa (Stover et al., 2000), but different in that the glutathione-independent formaldehyde dehydrogenase (FdhA) gene was observed downstream of purU2 in P. aeruginosa (Stover et al., 2000).
It has been demonstrated that sarcosine oxidase from Corynebacterium sp. P-1 and Arthrobacter spp. catalyses the oxidative demethylation of sarcosine to yield glycine, H2O2 and 5,10-methylenetetrahydrofolate, in the presence of tetrahydrofolate and O2 (Chlumsky et al., 1995; Meskys et al., 2001). In the absence of tetrahydrofolate, the same rate of sarcosine oxidation is observed and the oxidized methyl group is released as formaldehyde (Chlumsky et al., 1995; Meskys et al., 2001). Sarcosine oxidase enzymes from P. aeruginosa, Corynebacterium sp. P-1 (Chlumsky et al., 1995) and Arthrobacter spp.(Meskys et al., 2001) each contain a putative folate binding domain. Also, enzymes that utilize folates as substrates, such as PurU2, a 10-formyltetrahydrofolate deformylase, and GlyA1 and GlyA2, two hydroxymethyltransferase enzymes, were induced during growth of P. aeruginosa on GB. These observations further suggest that in P. aeruginosa tetrahydrofolate is involved in GB catabolism as a C1 acceptor. In this study, different enzymes that could use formaldehyde as substrate were induced in P. aeruginosa in the presence of GB (Table 2, Fig. 4d). It seems that GB degradation, via a sarcosine oxidase activity, could produce formaldehyde, which in turn could be used by FdhA (PA5421) and AdhC (PA3629) to produce formate. Tracing experiment using radiolabelled GB showed that high amounts of radioactive CO2 were produced, suggesting that formate was transformed into CO2 and H2O by a probable formate dehydrogenase, which was not detected as an accumulated protein on our 2D gels. Serine dehydratase (SdaB), cited above, is an enzyme encoded by sdaB gene. This gene, while oriented divergently, is adjacent to opuA (pa5378), which encodes a putative binding protein belonging to a GB/proline transport system that was also induced in the presence of GB. The adjacent localization of the two genes suggests that their simultaneous induction by GB could be dependent on the same regulatory mechanism. The opuA gene belongs to an opu operon composed of three genes (opuABC) encoding components of an ABC-type transporter. Usually, the ABC-type transporters of osmoprotectants are induced by elevated osmolality but not by their substrates (Bremer & Krämer, 2000; Choquet et al., 2005), except Hut and Ehu transporters from S. meliloti, which are induced by histidine and ectoines (ectoine and hydroxyectoine), respectively (Boncompagni et al., 2000; Jebbar et al., 2005).
All these data led us to propose a reconstructed GB catabolic pathway in P. aeruginosa (Fig. 6), which is similar to that proposed in Arthrobacter spp.(Meskys et al., 2001) and S. meliloti (Smith et al., 1988).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 November 2005;
revised 18 January 2006;
accepted 19 January 2006.
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,
) or 20 mM succinate (
,
), without (filled symbols) or with 0·5 M NaCl (open symbols). Culture aliquots were withdrawn periodically to determine the remaining radioactivity in the supernatant (a), the radioactivity in the CO2 (b), the radioactivity in the cytosol GB (c) and the radioactivity in the insoluble cellular macromolecules (d). Results were expressed as nmoles (OD unit)–1 or nmoles ml–1 GB for cytosol GB (c) and supernatant (a), respectively. For the radioactivity recovered in CO2 (b) and macromolecules (d), nmoles (OD unit)–1 GB corresponds to the amount of catabolized GB allowing the production of a number of d.p.m. of radiolabelled carbons that were incorporated into macromolecules or released as 14CO2. Results are the means of triplicate experiments; SE did not exceed 10 %.
) 10 mM glucose, (
) 10 mM GB. (b) Cells were grown in M63 medium supplemented with 2·5 mM glucose (
), 5 mM succinate (
), or a mixture of 5 mM succinate and 10 mM GB (
