TonB-abhängige Substrataufnahme bei dem Gram-negativen Bakterium Caulobacter crescentus (English shop)

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The genome sequence of Caulobacter crescentus predicts 67 TonB-dependent membrane receptors which, in addition to iron and vitamin B12 supply, are also responsible for the uptake of maltose, maltooligosaccharides, N-acetyl-ß-D-glucosamine (GlcNAc) and its higher homologues. Maltodextrins and GlcNAc each induce the expression of an outer membrane protein, designated MalA and NagA, respectively. An energy-dependent uptake of these substrates could be demonstrated by means of exbB/D deletion mutants. In contrast, the absence of the tonB gene predicted in the database showed no effect on growth or on substrate transport of these compounds. In this work, an attempt was made to demonstrate TonB-dependent substrate uptake in C. crescentus.

MalA possesses the TonB box sequence EEVVIT. When the amino acid valine at position 15 in the TonB box was replaced by the amino acid proline, the bacterium was no longer able to take up maltose. By means of sequence analyses, a further tonB gene could be found in the genome of C. crescentus, which was deleted. Growth tests of this chromosomal tonB deletion mutant proved that a TonB protein is responsible for the uptake of maltodextrins. Smaller maltodextrins such as maltose, maltotriose and maltotetraose can enter the cell via other receptor proteins, whereas the larger maltodextrins such as maltopentaose and maltohexaose are taken up in a MalA- and TonB-ExbB/D-dependent manner. For the uptake of GlcNAc and its higher homologues, no TonB-dependent uptake could be demonstrated. Presumably, NagA constitutes a specific pore for GlcNAc and a transporter for larger chitin oligosaccharides.

For the characterization of the gene cluster around malA (cc2287), chromosomal deletion mutants of the neighbouring genes were generated. The MalY protein (CC2283) encodes a transport protein in the cytoplasmic membrane which accomplishes the transport of maltodextrins via an ion-coupled mechanism. The repression of the mal locus could be attributed to the protein MalI (CC2284). The function of the MalS protein (CC2282) could not be unambiguously determined. Absent amylase activity in the medium and a predicted signal sequence point to an action of this protein in the periplasm.