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1 Microbiology/Membrane Physiology, University of Tübingen, Germany
2 Max Planck Institute for Developmental Biology, Department of Protein Evolution, Tübingen, Germany
Correspondence
V. Braun
volkmar.braun{at}tuebingen.mpg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-amylases (CC2285 and CC2286) and a periplasmic glucoamylase (CC2282). The TonB dependence together with the previously described ExbB ExbD dependence demonstrates energy-coupled maltose transport across the outer membrane. MalY is involved in maltose transport across the cytoplasmic membrane by a presumably ion-coupled mechanism.
A table of primers used for the construction of SL3508 is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nierman et al., 2001). In the crystal structures of outer-membrane transporters of Escherichia coli and Pseudomonas aeruginosa, the plug domain completely closes the pore formed by the β-barrel (Ferguson & Deisenhofer, 2004; Schalk et al., 2004; Wiener, 2005). In E. coli and other Gammaproteobacteria, TonB-dependent OMPs transport ferric ions incorporated into siderophores and haem, and vitamin B12 across the outer membrane (Andrews et al. 2003; Braun et al., 1998; Postle & Kadner, 2003; Braun & Mahren, 2007). This transport is energized by the electrochemical potential of the cytoplasmic membrane (White et al., 1973). Three proteins, TonB, ExbB and ExbD, are involved in transmitting energy from the cytoplasmic membrane to the outer-membrane transporters. TonB, ExbB and ExbD are inserted into the cytoplasmic membrane and extend into the periplasm. TonB interacts with the outer-membrane transporters, as shown by genetic (Heller et al., 1988; Schöffler & Braun, 1989), biochemical (Cadieux & Kadner, 1999; Ogierman & Braun, 2003) and crystallographic methods (Pawelek et al., 2006; Shultis et al., 2006). However, it is the release of substrates from their tight binding to the transporters and movement of the plug within the β-barrel of the transporters that requires an input of energy. Under the assumption that the 67 TonB-dependent proteins transport not only iron and vitamin B12 but also other substrates, we studied the uptake of potential nutrients into C. crescentus which might be available in nutrient-poor inshore waters in which C. crescentus thrives. We selected starch, the third most abundant carbon source in nature. We have shown that C. crescentus CB15, the strain whose genome has been sequenced (Nierman et al. 2001), does not grow on starch since it lacks an extracellular amylase, but it does grow on starch degradation products (maltodextrins) as sole carbon sources (Neugebauer et al., 2005). Uptake of maltodextrins across the outer membrane was mediated by the MalA protein, which is one of the 67 predicted TonB-dependent proteins in the bacterium. The initial transport rate of a malA insertion mutant was 1 % of the wild-type rate. However, growth of the malA mutant was stimulated on nutrient agar with maltose, maltotriose and maltotetraose as sole carbon sources, but not with maltopentaose. The slow uptake of the smaller maltodextrins was sufficient to support growth of the malA mutant.
Although C. crescentus belongs to the Alphaproteobacteria, analysis of its genome has identified genes which are 26, 40 and 46 % identical to the E. coli tonB, exbB and exbD genes, respectively. Inactivation of the exbBD genes impairs MalA-mediated maltose transport, but inactivation of the predicted tonB gene does not affect maltose, maltotriose and maltotetraose transport (Neugebauer et al., 2005). This finding was unexpected since in hitherto studied Gammaproteobacteria, ExbB and ExbD function only together with TonB. We have predicted the presence of a second TonB protein which was identified in the present study and have shown it to be essential for maltose transport.
Synthesis of MalA is induced by growing cells on maltodextrins which strongly increases the maltodextrin transport rate. A gene with sequence homology to lacI-type repressor genes is present in the mal locus of C. crescentus which we designated malI. Here we show that deletion of malI increases maltose transport to the level of the maltodextrin-induced rate and complementation of the mutant by wild-type malI represses maltose transport. In addition, we assigned a transport function to the malY gene in the mal operon which encodes a protein homologous to the LacY-type transport proteins in the cytoplasmic membrane.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All C. crescentus strains are derived from the sequenced CB15 strain. Cells of C. crescentus were grown at 30 °C in PYE rich medium or in M2 salt minimal medium (Ely, 1991) supplemented with 0.3 % glucose or 0.3 % maltose. C. crescentus did not grow on tryptone/yeast extract medium (Difco), which was used to test contamination of cultures of slow-growing C. crescentus cells by fast-growing bacteria. Cells of E. coli were grown at 37 °C on tryptone/yeast extract medium. Antibiotics were used at the following concentrations (µg ml–1) for C. crescentus (and in parentheses for E. coli): ampicillin, 50 on plates, 7.5 (50) in liquid culture; chloramphenicol, 2 (25); tetracycline, 2 on plates, 1 (12) in liquid culture; gentamicin, 50 on plates, 25 (50) in liquid culture; spectinomycin, 50 on plates, 25 (50) in liquid culture; streptomycin, 5 (30); kanamycin, (30); and nalidixic acid, (20). C. crescentus JS1003 is resistant to nalidixic acid, ampicillin and kanamycin. Growth promotion was tested on M2 salt agar plates on which 108 bacteria were seeded in 3 ml M2 salt soft agar. The nutrients to be tested (10 µl) were placed on sterile filter paper disks (6 mm in diameter). Bacterial growth was scored around the disks after 16 and 24 h incubation at 30 °C. Growth promotion was also determined on nutrient agar plates and in liquid culture with 0.1 and 0.3 % maltodextrins as sole carbon source. Maltose was purchased from Sigma-Aldrich Chemie and its purity was verified by HPLC analysis.
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). PCR of E. coli DNA was started with 3 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 2 min annealing at 54 °C, 3 min elongation at 72 °C, and then 10 min at 72 °C. The GC-rich DNA was amplified similarly, except that the annealing temperature started at 64 °C and was lowered in each cycle by 0.5 °C. The primers used for cloning by PCR are provided in Table S1 (available with the online version of this paper).
Cloning of genes and mutated genes.
The tonB1, malY and malI genes were amplified by PCR with appropriately designed primers from the C. crescentus chromosome and were then cloned into pBBR1-MCS2. Plasmid pBBtonB1 contains a 740 bp SpeI–EcoRI tonB1 fragment, plasmid pBBmalY a 1.6-kb SpeI–EcoRI malY fragment, pBBmalI a 1.1 kb EcoRI–SpeI malI fragment, plasmid pBBmalA a 3 kb EcoRV–SpeI malA fragment, and plasmid pBB2287 a 3 kb EcoRV–SpeI malA fragment with a TonB box mutation in pBBR1-MCS2. pSL2334a contains an 
insertion in cc2334a (
tonB1) cloned with SpeI in pNPTS138Tet. The constructed plasmids were transferred by conjugation into mutants lacking the plasmid-encoded genes on the chromosome.
Construction of chromosomal C. crescentus mutants.
The tonB1, malY and malI target genes were mutated by insertion of an resistance cassette (Alexeyev et al., 1995; Prentki & Krisch, 1984). The flanking genes of the target gene were amplified by PCR, ligated and cloned into the pDrive vector. The 2 kb SpcR–StrR fragment of pHP4 was cloned between the two flanking genes, and the DNA was amplified by PCR and cloned into pNPTS138Tet, which was derived from pNPTS138 by insertion of the 1.4 kb MluI TetR fragment of pBSL204. The following chromosomal insertion mutants were cloned with SpeI into pNPTS138Tet: pSL2334a tonB1 in cc2334a, pSL2283 malY in cc2283, pSL2284 malI in cc2284. These plasmids were introduced into E. coli S17-1 
pir by transformation and transferred by conjugation on PYE nutrient plates into C. crescentus UJ2602. Growth of the donor strain was inhibited by nalidixic acid, and integration of the plasmid into the recipient genome was selected with streptomycin and spectinomycin. A second recombination deleted the target gene along with sacB and the TetR gene by growing cells on 3 % sucrose. The deletion of the target gene was verified by Southern blotting and DNA sequencing.
[14C]Maltose transport.
Cells were grown in M2 medium supplemented with 0.3 % maltose as carbon source, harvested by centrifugation and washed once in M2 medium. The pellet was resuspended in 1.3 ml M2 medium containing 0.3 % glucose, and the optical density at 578 nm was adjusted to 0.5 (5x108 cells ml–1). [14C]Maltose (specific activity 25.1 GBq mmol–1; Amersham Biosciences) was added to a final concentration of 0.2 µM. Samples of 0.2 ml were withdrawn after 0.2, 1, 2 and 3 min, collected on cellulose nitrate filters, washed twice with 5 ml M2 salt medium and dried. The radioactivity was determined in a liquid scintillation counter.
Sequence alignments.
The C. crescentus genome was analysed and compared with other genomes using the Comprehensive Microbial Resource (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi), and the programs FASTA (www.ebi.ac.uk/fasta33/proteomes.html) and WU-BLAST2 (www.ebi.ac.uk/blast2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The similar size of the encoded protein (240 residues) to the E. coli TonB protein (239 residues), the 26 % sequence identity with the E. coli TonB protein, and two features characteristic of E. coli TonB – a hydrophobic N-proximal sequence that could span the cytoplasmic membrane and the accumulation of proline residues in the central segment – supported the assignment (Fig. 1). However, inactivation of this gene did not affect maltodextrin transport (Neugebauer et al., 2005). We therefore re-examined the genome and identified an additional gene homologous to tonB that mapped adjacent to and was transcribed in the same direction as exbB exbD (Fig. 2a). The degree of amino acid sequence identity of the predicted protein to the E. coli TonB protein (Fig. 1) is low (23 %), but higher than that of the previously studied CC2327 TonB protein (19 %) (Fig. 1). The N-terminal fragment extending into the cytoplasm in C. crescentus TonB is longer (24 residues) than in E. coli TonB (11 residues). The hydrophobic, presumably transmembrane region contains a histidine residue which is conserved in the two C. crescentus TonB proteins; this residue (number 20) plays an essential role in E. coli TonB activity (Larsen et al., 2007, Larsen & Postle 2001; Traub et al., 1993). Since this newly identified tonB gene is at a locus that was previously assigned to another gene with opposite polarity (cc2334), we refer to it here as cc2334a (tonB1) (Fig. 2). The exbB exbD tonB1 genes are densely packed. The distance between exbB and exbD is 5 bp, and between exbD and tonB1 10 bp, suggesting that they are cotranscribed from a promoter upstream of exbB.
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insertion in C. crescentus SL2334a abolished maltose transport. Complementation of this mutant with the wild-type gene on plasmid pBBtonB1 restored transport (Fig. 3, Table 2). These results indicate that gene cc2334a is involved in maltose transport.
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). Inactivation of cc3508 and inactivation of both cc3508 and cc2327 together did not affect maltose transport (data not shown).
In E. coli, TonB binds to the TonB box of outer-membrane transporters (Pawelek et al. 2006; Shultis et al. 2006). The L8P replacement in the TonB box DTLVV of the BtuB transporter abolishes vitamin B12 transport (Heller et al., 1988), and the replacements I9P and V11D in the TonB box DTITV of the FhuA transporter abolish ferrichrome transport (Schöffler & Braun, 1989). To examine whether MalA interacts with TonB via its predicted TonB box EEVVIT similar to the interactions observed in E. coli, the valine at position 15 of MalA was replaced by proline (V15P), resulting in plasmid pBB2287. The chromosomal malA mutant HB2003 was transformed with pBB2287 malA(V15P). The transformant did not transport maltose (Table 2
). Complementation with plasmid-borne wild-type malA restored maltose transport to 63 % of that of the wild-type strain (Table 2). These results suggest an interaction of MalA with TonB, similar to the interactions between energy-coupled transporters and TonB in E. coli.
MalY is required for maltose transport
The gene cluster encoding the maltose transport system (Fig. 2b) contains a gene (cc2283) homologous to sugar permease genes (Busch & Saier, 2002), such as the lacY permease gene of E. coli. Therefore, we named this gene malY. Inactivation of malY by insertion in C. crescentus SL2283 abolished maltose transport, and complementation of this mutant with plasmid pBBmalY largely restored maltose transport (Table 2). We conclude that malY encodes a permease that catalyses transport of maltose across the cytoplasmic membrane.
MalI functions as a repressor of maltose gene transcription
We have previously shown that synthesis of the MalA protein and transport of maltose, maltotriose and maltotetraose are induced when C. crescentus is grown in a medium containing maltose as sole carbon source (Neugebauer et al., 2005). Analysis of the mal locus identified a gene (cc2284) that encodes a protein with homology to the LacI repressor family of proteins. We therefore assumed that a repressor was involved in the regulation of maltose transport. We named this gene malI and inactivated it by insertion. In the absence of maltose in the growth medium, the C. crescentus malI mutant SL2284 transported maltose at the same rate as the wild-type after induction with maltose (Table 2). Complementation of the malI mutant by wild-type malI encoded on the medium-copy plasmid pBBmalI repressed maltose transport (Table 2). The complemented mutant did not transport maltose even when maltose was added to the medium, and it did not grow on maltose, which indicates that repression by overproduced MalI could not be overcome by maltose. The overproduced repressor probably completely inhibited mal transport gene transcription, resulting in insufficient amounts of maltose in the cells to inactivate the repressor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), has a rather high sequence homology to tonB1 (proteins are 41 % identical). It is likely that it displays a TonB function in TonB-dependent uptake systems other than maltose transport and therefore we have named it tonB2 (TonB2). More than one functional tonB gene was identified in a number of organisms; some of these tonB genes can replace each other, whereas others display specific functions (Blanvillain et al., 2007; Chu et al., 2007; Wandersman & Delepelaire, 2004; Wyckoff et al., 2007; Zhao & Poole, 2000).
We have proposed that many Gram-negative bacteria take up a variety of scarce substrates other than ferric siderophores and vitamin B12 by energy-coupled transport across the outer membrane (Neugebauer et al.; 2005). The 67 predicted outer-membrane transporters of C. crescentus probably transport a large variety of substrates under the nutrient-poor conditions in the freshwater lakes in which C. crescentus thrives. Tight binding to outer-membrane transporters extracts these substrates from the medium, but this also requires a structural, energy-consuming change in the transporters to release the substrates from their binding sites and movement of the plug within the β-barrel to enable diffusion of the substrates through the opened pore into the periplasm. In fact, MalA tightly binds maltose with a Kd of 0.2 µM (Neugebauer et al.; 2005), which is 1000-fold lower than the Kd of the LamB porin of E. coli (Benz et al., 1987).
Maltose uptake by C. crescentus is mechanistically different from the well-studied maltose uptake system in E. coli. In C. crescentus, maltodextrins are actively transported by MalA across the outer membrane. In E. coli, maltodextrins enter the periplasm by facilitated diffusion through the LamB protein. In C. crescentus, maltodextrin transport across the cytoplasmic membrane is mediated by a protein that is predicted to be energized by proton motive force. In E. coli, maltodextrin transport across the cytoplasmic membrane is mediated by an ABC transporter consisting of four proteins. In C. crescentus, transcription of the maltose transport genes is controlled by a repressor, whereas in E. coli it is controlled by an activator. Two maltose-cleaving -amylases encoded in the mal loci occur in the cytoplasm and are common to both organisms, but their mode of action might differ. We did not find a function for the periplasmic MalS protein which is predicted to be a glucoamylase. Inactivation of malS by insertion did not reduce the growth rate of C. crescentus on maltose, maltotriose and maltotetraose (data not shown). For maltodextrins up to this size, degradation by MalS to maltose is not required. It may degrade larger maltodextrins provided that they are transported into the periplasm by MalA for which uptake of maltodextrins up to maltohexaose has been shown (Neugebauer et al. 2005).
Recently, an explanation has been given for the long-known observation that C. crescentus forms much longer stalks in phosphate-limiting media (Wagner et al., 2006). The stalks serve to collect phosphate, which then diffuses into the common periplasm of the stalk and the cell body where, in the cell body, the ABC transporter for phosphate resides. However, no phosphate-specific energy-coupled transporter has been identified in the outer membrane of the stalk, and the ExbB protein is not found in the cytoplasmic membrane of the stalk, but rather in the cytoplasmic membrane of the cell body (Wagner et al., 2006). These results suggest that phosphate diffuses through the outer membrane of the stalk. Diffusion is favoured by the long and thin shape and the small volume of the stalk. This seems to be an adaptation to an environment where nutrient uptake is limited by diffusion, along with the adaptations of strong binding to outer-membrane transporters and subsequent energy-coupled transport across the outer membrane. In the experiments reported in this paper cells may have formed stalks. It is unlikely that maltose porins exist in the outer membrane of stalks since maltose transport in a malA mutant is only 1 % of the malA wild-type.
An energized outer-membrane transport for nickel ions has been characterized in Helicobacter pylori (Schauer et al., 2007). Ni2+ uptake at pH 5 was strongly reduced in an frpB4 mutant (frpB4 encodes an OMP). Likewise Ni2+ uptake at pH 5 was strongly reduced in an exbB exbD tonB mutant. Transcription of frpB4 and exbB exbD tonB was under the control of the Ni2+-responsive NikR repressor (Davis et al., 2006; Ernst et al. 2006).
Additional evidence for energy-coupled transport of nutrients other than ferric siderophores and vitamin B12 across the outer membrane comes from the analysis of genomes which, for example, predict 107 paralogues of SusC, an OMP required for growth of Bacteroides thetaiotaomicron on maltodextrins. SusC is predicted to consist of a β-barrel with a plug inside (Reeves et al., 1996), similar to the crystal structures of energy-coupled outer-membrane transporters of E. coli and P. aeruginosa. For Xanthomonas campestris, 48 TonB-dependent outer-membrane transporters are predicted, and it has been shown that for sucrose transport, the SuxA OMP is required (Blanvillain et al., 2007). Synthesis of SuxA is induced by growing cells on sucrose. The assumed TonB dependence could not be verified because individual knockout of eight potential tonB genes did not affect sucrose transport; this might be explained by cross-complementation by one or more tonB genes.
The maltodextrin transport system is the only transport system in C. crescentus for which the outer- and inner-membrane transport proteins have been identified and for which its mode of action has been characterized to such an extent that energization via the proton motive force can reasonably be derived. We propose that this system is representative of many substrate transport systems and supports the prediction of 67 predicted outer-membrane transporters in C. crescentus.
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ACKNOWLEDGEMENTS |
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Edited by: T. Abee
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Neugebauer, H., Herrmann, C., Kammer, W., Schwarz, G., Nordheim, A. & Braun, V. (2005). ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus. J Bacteriol 187, 8300–8311.
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Received 5 February 2008;
revised 11 March 2008;
accepted 18 March 2008.
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, C. crescentus UJ2602; 
, tonB1 mutant SL2334a; 
, SL2334a complemented by plasmid-encoded wild-type tonB1. Cells were grown on maltose.