Erschienen:
American Society for Microbiology, 2005
Erschienen in:
Journal of Bacteriology, 187 (2005) 24, Seite 8300-8311
Sprache:
Englisch
DOI:
10.1128/jb.187.24.8300-8311.2005
ISSN:
0021-9193;
1098-5530
Entstehung:
Anmerkungen:
Beschreibung:
ABSTRACTAnalysis of the genome sequence ofCaulobacter crescentuspredicts 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 aKdof 0.2 μM, while the second transport had aKdof 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 themalAgene diminished maltose transport to 1% of the wild-typemalAstrain and impaired transport of the larger maltodextrins. ThemalAmutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of twoC. crescentusgenes homologous to theexbB exbDgenes ofEscherichia coliabolished [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 atonBhomolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for thetonBmutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of theC. crescentus malAmutant was slower than permeation through the outer membrane of anE. coli lamBmutant, which suggests a low porin activity inC. crescentus. The pores of theC. crescentusporins are slightly larger than those ofE. coliK-12, since maltotetraose supported growth of theC. crescentus malAmutant but failed to support growth of theE. coli lamBmutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins withKdvalues 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B12and 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 B12has been demonstrated.