Purification and characterization of the membrane adenosine triphosphatase complex from the wild-type and N,N''-dicyclohexylcarbodiimide-resistant strains of Streptococcus faecalis

We have purified the F1-F0 adenosine triphosphatase complex from wild-type Streptococcus faecalis ATCC 9790 and an N,N'-dicyclohexylcarbodiimide (DCCD)-resistant mutant strain, SF-dcc-8. For preliminary purification of the complex, reconstituted F1-F0, prepared from isolated F1 adenosine triphosphatase and depleted membranes, was extracted with sodium deoxycholate and fractionated by salt precipitation. By means of two-dimensional gel electrophoresis, the F1-F0 complex was purified as a single, catalytically active band in the first dimension and then resolved into constituent subunits under denaturing conditions in the second dimension. The electrophoretic purification of F1-F0 removed a delta-less form of F1 as well as other impurities, including lipoteichoic acid. Both the DCCD-sensitive and the DCCD-resistant F1-F0 adenosine triphosphatase appeared to consist of eight proteins, five of which corresponded to the F1 subunits alpha, beta,, gamma, delta, and epsilon. The F0 sector proteins, designated M27, M15, and M6, had Mr values of 27,000, 15,000, and 6,000, respectively. There appear to be multiple copies of M6 in the complex. [14C]DCCD reacted specifically and covalently with M6 in the wild-type F1-F0 but failed to label the M6 protein in the complex from the DCCD-resistant strain. It is suggested that DCCD resistance in the SF-dcc-8 mutant may be due to a modification of the M6 protein which hinders access of DCCD to the reactive site.     

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.     

Carbodiimide-resistant mutant of Escherichia coli: suppression of resistance to dicyclohexylcarbodiimide by growth on glucose or glycerol.

We have previously reported on the isolation of a mutant strain of Escherichia coli, RF-7, that has a dicyclohexylcarbodiimide (DCCD)-resistant, membrane-associated adenosine triphosphatase (ATPase) activity (R. H. Fillingame, J. Bacteriol. 124:870--883, 1975). We report here that the DCCD resistance of the ATPase of this mutant varies significantly, depending upon the carbon source used for growth. When strain RF-7 was grown aerobically on either glycerol or glucose or anaerobically on glucose rather than on a combination of succinate, acetate, and malate, ATPase activity was more sensitive to inhibition by DCCD because the carbodiimide-reactive proteolipid reacted more readily with DCCD.     

Thymine transport in Lactobacillus casei

Abstract A transport system for thymine was investigated in a Lactobacillus casei mutant lacking thymidine phosphorylase activity (the first enzyme required for thymine utilization). Transport was dependent on an energy supply; transport was inhibited by the uncouplers carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4 dinitrophenol (DNP), and also by sodium azide and N,N'-dicyclohexylcarbodiimide (DCCD). Thymine transport was inhibited by some uracil and adenosine derivatives and by thymidine, but was not affected by guanosine, deoxycytidine or azacytidine. Inhibition by p-chloromercurybenzoate (PCMB) was reversed by dithiothreitol (DTT).     

Different effects of inhibitors on two mutants of Escherichia coli K12 affected in the Fo portion of the adenosine triphosphatase complex.

The effects of the inhibitors dicyclohexyl-carbodiimide (DCCD), bathophenanthroline and tertiary octylcatechol, on some enzyme activities in membranes from strains of Escherichia coli carrying mutations in the uncB or uncC genes have been studied. Membranes prepared from uncC mutants retain a normal DCCD-sensitive Mg2+-stimulated adenosine triphosphatase (Mg-ATPase) activity whereas in uncB mutants this enzyme activity is insensitive to DCCD. The membrane-bound Mg-ATPase activity from the uncC mutant strain, as compared with that from the normal strain, is only partially sensitive to the inhibitors bathophenanthroline or tertiary-octylcatechol. Both of these inhibitors stimulate the membrane-bound Mg-ATPase from uncB mutant strains. A DCCD-insensitive Mg-ATPase activity is found in the cytoplasmic fraction following cell disruption of either the uncB or the uncC mutants. The lipophilic chelators bathophenanthroline and tertiary-octylcatechol stimulate the activity of the 'soluble' Mg-ATPase in the uncB mutant but partially inhibit the activity in the uncC mutant. The NADH oxidase activities in membranes from both mutant and normal strains are strongly inhibited by tertiary-octylcatechol and bathophenanthroline but not by DCCD.     

Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation.

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.     

Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.

Aurovertins B and D inhibited the adenosine triphosphatase (ATPase) activity of soluble Escherichia coli coupling factor ATPase (BF1) isolated from wile-type E. coli K-12. Half inhibition was obtained with 2 microns aurovertin B and 0.9 microns aurovertin D. Aurovertins B and D had no inhibitory effect on BF1 isolated from the aurovertin-resistant E. coli mutant MA12. Acetylation or saponification of aurovertin D yielded a derivative which was devoid of inhibitory effect on BF1. Citreoviridin also inhibited wild-type BF1 but with much less efficiency (half inhibition at 60 microns) than aurovertin. Citreoviridin had no effect on the aurovertin-resistant BF1. The fluorescence intensity of aurovertins B and D was markedly enhanced upon addition to purified BF1. There was no enhancement of fluorescence when the aurovertins were added to BF1 isolated from the aurovertin-resistant mutant. The fluorescence of the aurovertin-BF1 complex was enhanced by adenosine 5'-diphosphate and by low concentrations of adenosine 5'-triphosphate. The adenosine 5'-diphosphate-enhanced fluorescence of the aurovertin-BF1 complex was quenched by high concentrations of adenosine 5'-triphosphate or by MG2+. Aurovertin bound selectively to the beta subunit of BF1 isolated from wile-type cells. By complementation assays in vitro, using a reconstituted system made of subunits isolated from wild-type and aurovertin-resistant BF1, it was shown that the altered peptide in aurovertin-resistant BF1 was the beta subunit.     

Oxidative phosphorylation in Escherichia coli K 12. Mutations affecting magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.     

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.     

Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli.

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca2+, Mg2+-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca2+, Mg2+-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome-containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under under anaerobic conditions is energized by ATP through the Ca2+, Mg2+-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.     

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.5-7.5 was also measured. The elevated H+ conductance and lowered H+ flux were shown for cells growing in acidic pH and under anaerobic conditions, when bacteria were fermenting glucose. The atp mutant, which is deprived of the F0F1- adenosine triphosphatase, had less Gm H+ independent of growth conditions. In contrast with wild-type or precursor strain, a remarkable difference in Gm H+ for atp mutant was observed between aerobic and anaerobic conditions; such a difference was significant at pH 4.5. These results could indicate distinguishing pathways determining Gm H+ under anaerobic conditions after the fermentation of glucose at different pH and an input of the F0F1-adenosine triphosphatase in Gm H+. In addition, the effect of osmotic stress was demonstrated with grown cells. Gm H+ and H+ efflux both were increased after hyperosmotic stress at pH 7.5, and these changes were inhibited by N,N\'-dicyclohexylcarbodiimide, whereas these changes were lower in atp mutant. A role of the F0F1-adenosine triphosphatase in osmo-sensitivity of bacteria was confirmed under fermentative conditions.     

On the control mechanism of bacterial growth by cyclic adenosine 3',5'-monophosphate

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.     

Na+ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate-grown cells was 40 nmol mg-1 min-1 and was resistant to the uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), but was inhibited by the complex I-specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1-3 nmol mg-1 min-1), and rotenone was without effect.     

Inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by dicyclohexylcarbodi-imide.

The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.     

Mutant of Escherichia coli defective in response to colicin K and in active transport.

A mutant of Escherichia coli has been isolated that grows poorly on succinate and exhibits a markedly reduced sensitivity to colicin K. This mutant is also deficient in the respiration-linked transport of proline and thiomethyl-beta-D-galactoside but appears normal for the adenosine triphosphate-dependent transport of glutamine and arginine. A temperature-conditional revertant of the mutant grows on succinate and is sensitive to colicin K at 27 C, but fails to grow on succinate and is insensitive to colicin K at 42 C. Proline transport in the temperature-conditional revertant is reduced at 42 C when either glucose or succinate is used as energy source. Glutamine transport, on the other hand, is normal at 42 C with glucose as energy source, but is reduced with succinate, although not to the same extent as is proline transport. The lack of growth on succinate and the deficiencies in transport at 42 C are not due to a temperature-dependent lesion in either the electron transport chain or in Ca2+, Mg2+-activated adenosine triphosphatase activity. Membrane vesicles prepared from the temperature-conditional revertant are impaired in proline transport at both 27 and 42 C. These findings suggest the existence in the cytoplasmic membrane of E. coli cells of a component, presumably protein, that is required for colicin K action and that functions in respiration-linked and, to a lesser degree, in adenosine triphosphate-dependent active transport systems. This protein may serve as the primary target of colicin K action.     

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.     

Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.     

ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose-mineral-salts medium, and membrane preparations do not have Mg(2+)-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg(2+)-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA(-)), which forms an inactive membrane-bound Mg(2+)-stimulated adenosine triphosphatase aggregate.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.