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ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

Microbiology/Membrane Physiology,¹ Cell Biology/Molecular Biology,² Proteom Centrum Tübingen, University of Tübingen, Tübingen, Germany³

Received 6 September 2005/ Accepted 29 September 2005

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 Fe³⁺ and vitamin B₁₂—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 [¹⁴C]maltodextrins from [¹⁴C]maltose to [¹⁴C]maltopentaose. [¹⁴C]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 µM, while the second transport had a K_d of 5 µM. 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 µM 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 [¹⁴C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe³⁺-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 Fe³⁺-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 B₁₂ 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 B₁₂ has been demonstrated.

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