1 Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, 9750, AA Haren, The Netherlands
2 Department of Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, 9750, AA Haren, The Netherlands
Correspondence
Arnold J. M. Driessen
a.j.m.driessen{at}rug.nl
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ABSTRACT |
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Present address: Institute of Biochemistry, Biocenter, Goethe-Universität Frankfurt, Marie Curie Straße 9, D-60439 Frankfurt, Germany.
Present address: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
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INTRODUCTION |
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To date, only a few members of this class of transporters have been functionally described. One member is involved in the sodium- and pmf-dependent uptake of glutamate by Rhodobacter sphaeroides (Jacobs et al., 1996). The DctPQM transporter of Rhodobacter capsulatis transports the C4-dicarboxylates malate, succinate and fumarate (Forward et al., 1997
) and a similar system was found in Wolinella succinogenes (Ullmann et al., 2000
). More recently, TeaABC of Halomonas elongata was shown to transport the compatible solutes ectoine and hydroxyectoine (Grammann et al., 2002
). Consequently, these transporters may play roles both in the uptake of carbon sources and in the protection of the cell against non-favourable conditions. The Escherichia coli K-12 genome contains one binding-protein-dependent secondary transporter encoded by the yiaMNO genes, located in the yiaKLMNOPQRS gene cluster. yiaO encodes the periplasmic binding protein, while yiaM and yiaN encode the small and large membrane protein, respectively. Based on identity with the genes encoding TeaABC, YiaMNO has been suggested to be involved in the uptake of osmoprotectants (Ly et al., 2004
). However, experimental evidence indicates that YiaMNO catalyses the uptake of the rare pentose L-xylulose (L-threo-2-pentulose) (Plantinga et al., 2004a
). L-Xylulose is presumably metabolized by the enzymes encoded immediately downstream of the yiaMNO genes, since L-xylulose transport and metabolism is found only in cells that constitutively express the yiaK-S gene cluster. This coupling of the proposed carbohydrate transport and metabolism function is conserved in at least 26 bacterial genomes, most of which are human pathogens (Plantinga et al., 2004b
). However, L-xylulose does not induce expression of these genes and little is known about its utilization by E. coli K-12 (Ibañez et al., 2000
; Plantinga et al., 2004a
), indicating that L-xylulose might not be the sole substrate for the YiaMNO transporter.
This study focuses on the physiological role of the yiaMNO genes. We have characterized E. coli strain K-12 MC4100, which is widely used in gene expression work (Peters et al., 2003), and a
yiaMNO derivative thereof. Some striking phenotypical differences between the
yiaMNO mutant and parent strain were observed in our growth experiments.
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METHODS |
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Expression of the yiaM and yiaO genes.
Cells were grown aerobically in LB and LBG, samples were taken at various OD660 values and total RNA was extracted as described by Plantinga et al. (2004a). RT-PCR was performed on 1 µg total RNA using RT-PCR beads (Amersham Pharmacia Biotech) and primers directed against 474 and 632 bp fragments of the yiaM and secY genes, respectively (Plantinga et al., 2004a
). For Northern Blot analysis, 5 µg total RNA from each sample was run on a 1·2 % (w/v) agarose gel containing 6·7 % (v/v) formaldehyde and transferred to a positively charged nylon membrane (Zeta Probe; Bio-Rad) in 150 mM NaCl, 15 mM sodium citrate. For preparation of the probe, the yiaO gene was cloned via PCR using forward primer 5'-AATGGATCCATTAAAAGGAAAATATTATG-3' and reverse primer 5'-CCCTCTAGATTATTGCACCTCATCCAC-3', introducing BamHI and XbaI restriction sites, respectively. The fragment was ligated into vector pET401 (Van der Sluis et al., 2002
) for propagation and subsequently used as a template for labelling with [32P]dCTP using Klenow polymerase (Roche). The probe was purified using a PCR product isolation procedure (Qiagen) and hybridized overnight in 0·5 M sodium phosphate, pH 7·2, 1 % (w/v) blocking reagent (Roche) and 7 % (w/v) SDS at 65 °C. The membrane was washed in 50 mM sodium phosphate, pH 7·2, containing 1 % SDS, and the signal was recorded by autoradiography.
Promoter induction and -galactosidase assays.
The 1000 bp upstream region of yiaM was cloned via PCR. The forward primer (5'-ATGGTGGATCCGATGATGAGGGCA-3') introduced a BamHI site and the reverse primer (5'-TGAATTCATAGCTATTCCTTGAGGC-3') introduced an EcoRI site. The fragment was translationally fused to a lacZ-reporter gene in vector pBC3 (Meijer et al., 1991). This vector, termed pP1000, was transformed into strain MC4100. Heat and cold shock were applied by shifting liquid cultures to 42 and 10 °C, respectively. High salt, sucrose and spent medium (prepared as described below) effects were determined by harvesting the cells and resuspending them in the respective media. Induction by L-xylulose was tested by harvesting the cells and resuspending them in minimal medium containing 0·4 % L-xylulose. Following 30 min of incubation, the cells were harvested and
-galactosidase assays were performed as described by Miller (1992)
. Values were expressed in Miller units (MU).
Spent medium growth experiments.
MC4100 and TP001 cells were grown aerobically in LB or LBG, 50 ml samples were taken at OD660 values of 0·5, 1·5, 2·0 and 2·5, and harvested (4000 g, 10 min, 4 °C). The supernatant was filtered to remove all cells. Fresh LB or LBG cultures of MC4100 and TP001 were grown to an OD660 of 0·5, harvested and resuspended in equal volumes of pre-warmed (37 °C) spent medium. Aerobic growth was continued and monitored over time.
NMR analysis.
MC4100 and TP001 were grown to an OD660 of 0·5 in a total of 8 l LBG containing 0·9 M NaCl per strain. Cells were harvested and freeze-dried overnight. Ethanol extracts for determination of intracellular solutes were prepared as described elsewhere (Martins & Santos, 1995). 13C-NMR spectra were measured by Dr A. Ramos at the Instituto Technologia Química e Biológica of the University of Lisbon, Portugal, using a Bruker DRX500 spectrometer as described by Martins & Santos (1995)
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Biofilms.
Cells were plated on LB-agar and grown overnight at 37 °C. Colonies were picked and resuspended in M63 minimal medium and diluted into untreated polystyrene 96-wells plates (Costar) containing M63 0·5 % (w/v) glucose. Plates were incubated at 37 °C for 60 h and biofilm formation was quantified using crystal violet, as described by O'Toole et al. (1999).
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RESULTS |
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Deletion of the yiaMNO genes affects growth
MC4100 and its yiaMNO derivative TP001 were previously used in a search for the substrate of the YiaMNO transporter (Plantinga et al., 2004a). A striking phenotypical difference between mutant and parent strain was observed in growth experiments. When grown aerobically in LBG batch culture, i.e. containing glucose, both strains grew at nearly identical rates during the exponential phase, with the relatively sharp transition from exponential to stationary phase typical for a carbon-limited batch culture (Mason & Egli, 1993
). However, for TP001 this transition was delayed, reproducibly yielding a higher final OD660 at the stationary phase (Fig. 2
). This was supported by c.f.u. numbers (data not shown). This difference in growth was not observed in LB (Fig. 2
). When the cells were growing in minimal media supplied either with glucose or other carbon sources, a similar difference in final OD660 was observed (data not shown; compounds listed in Plantinga et al., 2004a
). These findings suggest that, under specific conditions, the deletion of the three structural genes encoding the YiaMNO transporter affects growth, and in particular the ability of the cells to enter stationary phase.
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DISCUSSION |
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Although expression of the yiaK-S gene cluster in strain JA134 is subject to carbon catabolite repression via the cyclic AMP receptor protein (CRP) (Ibañez et al., 2000), in strain MC4100 expression does not appear to be repressed by glucose (Fig. 1
). Interestingly, transcription of the yiaK promoter is highly upregulated in the mouse pathogen Salmonella enterica serovar Typhimurium SL1344 during colonization of the caecum of a murine enteritis model, and to a lesser extent in a murine typhoid fever model (C. Rollenhagen and D. Bumann, personal communication). Moreover, the promoter is also active in an in vitro model mimicking the conditions in the gut, i.e. low oxygen and increased NaCl concentration (C. Rollenhagen and D. Bumann, personal communication). These recent findings therefore support the notion that the YiaMNO transporter may play a role in scavenging scarce substrates under the limiting conditions encountered by this pathogen inside a eukaryotic host. Indeed, in E. coli strain MC4100, we detect the highest expression levels in cells that had been in stationary phase for 24 h (Fig. 1
). Since L-xylulose does not induce expression of the YiaMNO transporter, while expression does not appear to be repressed by glucose, a major role of this system in carbon source uptake and utilization is not evident at this time.
The loss of the YiaMNO transporter clearly affects growth of strain MC4100, in particular the transition from exponential to stationary growth on LBG (Fig. 2). In the yiaMNO mutant this transition was delayed, yielding a higher final OD660 during stationary phase. Strikingly, this may be related to a phenomenon observed in Ralstonia sp. TFD14. After 1000 generations of experimental evolution, all evolved populations showed an increased fitness compared to the ancestor strain (Korona et al., 1994
) and 71 out of 72 evolved populations had lost the 2·4 kb genomic fragment containing the Ralstonia yiaMNO-homologues (Nakatsu et al., 1998
). The size of the complete deletion was not determined, but our data now clearly demonstrate that this growth advantage may be attributed to the yiaMNO deletion. The observed increased fitness may be related to a defective quorum sensing circuit, in which the transporter is involved in the uptake of a signalling molecule that negatively affects growth. However, although our data suggest that strain MC4100 produces an autoinducer-like activity, the YiaMNO transporter is not required for transport of the autoinducer or a precursor thereof (Fig. 3
).
In the evolved populations of Ralstonia sp. the extracellular polysaccharide (EPS) had disappeared, leading to changes in bile salt sensitivity and adhesion properties (Riley et al., 2001). Although the yiaMNO mutant is clearly more sensitive to high salt, we were unable to identify a possible transported substrate involved in osmoprotection. The major EPS of E. coli K-12, colanic acid (Rick & Silver, 1996
), is required for development of biofilm architecture (Prigent-Combaret et al., 1999
; Danese et al., 2000
). It has been shown that deletion of the producing genes delays, but does not abolish, biofilm formation, with nearly identical amounts of surface-attached biomass at times
45 h (Danese et al., 2000
). However, we still observe major differences in biofilm formation after 60 h (Fig. 5
), therefore colanic acid biosynthesis most likely is not affected by the yiaMNO deletion. Nevertheless, the possible (indirect) involvement of the YiaMNO transporter in EPS biosynthesis does provide a basis for future experiments.
The yiaMNO derivatives of two additional strains were used in the biofilm experiments to test whether the observed effects are strain-specific. No effect of the deletion on biofilm formation was found in strain JA134, which, as its parent ECL1, formed significantly higher amounts of biofilm than strain MC4100 in the assay (data not shown). The effect of the deletion may therefore have been obscured in this genetic background (see above). Deletion of the yiaMNO transporter in the sequenced K-12 strain MG1655, yielding strain TP010, did however reduce biofilm formation (data not shown). In this regard, a negative effect similar to that observed in strain TP001 was observed during growth of strain TP010 on LB(G) in the presence of high salt (data not shown). Although significant genomic differences have been detected between both strains (Peters et al., 2003) these do not localize to the region encoding the yiaMNO genes and our findings do not appear to be strain-specific.
Concluding remarks
The phenotypic effects we have observed in the yiaMNO mutant of E. coli K-12 strain MC4100 are diverse and the pathways that underlie these phenomena are complex. However, the observations made with Ralstonia sp. support our finding that a localized deletion may have drastic effects on growth of the organism. In particular, the possible role of the transporter and its transported substrate in EPS formation deserves further experimental investigation. In addition, the recent findings in Salmonella support the assumption that the YiaMNO transporter functions under limiting substrate conditions, where L-xylulose is an intermediate in eukaryotic metabolism. The co-localization of carbohydrate transport and metabolism functions is found in a range of pathogenic bacteria (Plantinga et al., 2004b), suggesting these systems may play important roles in survival during infection. Further studies are required to establish the physiological role of the binding-protein-dependent secondary transporter encoded by the E. coli K-12 yiaMNO genes.
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ACKNOWLEDGEMENTS |
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Received 22 December 2004;
revised 7 February 2005;
accepted 11 February 2005.
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