NagA-Dependent Uptake ofN-Acetyl-Glucosamine andN-Acetyl-Chitin Oligosaccharides across the Outer Membrane ofCaulobacter crescentus

Abstract

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-␤-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a ␤-barrel composed of 22 antiparallel ␤-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc) 3 and (GlcNAc) 5 requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.TonB-dependent outer membrane proteins actively transport Fe 3ϩ siderophores, heme, Fe 3ϩ , and vitamin B 12 across the outer membrane of gram-negative bacteria (4,6,39,40,52,54). It is thought that energy is required for two steps: (i) release of the tightly bound substrates from the transporters and (ii) movement of the hatch to open a pore in the ␤-barrel (10, 53). The energy is provided by the proton motive force of the cytoplasmic membrane. A protein complex composed of TonB, ExbB, and ExbD is involved in the energy transfer from the cytoplasmic membrane to the outer membrane. TonB interacts with the TonB box in the N-terminal region of the transporters (9, 36, 37, 48). In Escherichia coli, eight such transporters have been related to the transport of specific substrates.Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins (32, 33). We have identified the tonB, exbB, and exbD genes, required for the transport of maltose and larger maltodextrins (29, 32). Transport is mediated by the outer membrane protein MalA (32). malA, tonB, exbB, and exbD gene insertion mutants transport maltose at 2% of the wild-type rate. This was the first description of TonB-dependent energy-coupled transport across the outer membrane for substrates other than Fe 3ϩ , Fe 3ϩ chelates, and vitamin B 12 .In nature, maltodextrins are derived from abundant starch. It is reasonable ...

TheCaulobacterTol-Pal Complex Is Essential for Outer Membrane Integrity and the Positioning of a Polar Localization Factor

“…Thus, TolA and TolB are essential for viability. It was reported previously that genes encoding two other components of the Caulobacter Tol-Pal complex, tolQ and tolR, are essential (17).…”

TonB-Dependent Receptors in Nitrogen-Fixing Nodulating Bacteria

“…Later, some TBDRs were shown to be responsible for the import of non-Fe compounds (44), such as vitamin B12 (20), sugars (4,11,30), and non-Fe cations (43,52). Compared to porins, which allow the passive transport of molecules, TBDRs exhibit high affinity to their ligands: the Kd of SuxA for sucrose is 330 nM (4) while that of FepA for ferric enterobactin is 24 nM (35).…”

CrfA, a Small Noncoding RNA Regulator of Adaptation to Carbon Starvation in Caulobacter crescentus

“…The sequence of the C. crescentus genome revealed that this bacterium possesses an unusually large number of TonB-dependent receptors, outer membrane proteins that bind to diverse substrates and facilitate their transport across the outer membrane and ultimately into the cytoplasm (28). It has been hypothesized that this diverse group of TonB-dependent receptors enables C. crescentus to diversify its nutrient uptake mechanisms during periods of nutrient deprivation, and two of these TonB-dependent receptors have been implicated in the uptake of the carbon sources maltodextrin (17) and N-acetylglucosamine (5). In addition to TonB-dependent receptors, C. crescentus also employs the intracellular second messenger ppGpp to adapt to carbon starvation (16).…”