CreA-independent carbon catabolite repression of cellulase genes by trimeric G-protein and protein kinase A in Aspergillus nidulans.

Cellulase production in filamentous fungi is repressed by various carbon sources. In our preliminary survey in Aspergillus nidulans, degree of de-repression differed depending on carbon sources in a mutant of creA, encoding the transcriptional repressor for carbon catabolite repression (CCR). To further understand mechanisms of CCR of cellulase production, we compared the effects of creA deletion with deletion of protein kinase A (pkaA) and G (ganB) genes, which constitute a nutrient sensing and signaling pathway. In plate culture with carboxymethyl cellulose and D-glucose, deletion of pkaA and ganB, but not creA, led to significant de-repression of cellulase production. In submerged culture with cellobiose and D-glucose or 2-deoxyglucose, both creA or pkaA single deletion led to partial de-repression of cellulase genes with the highest level by their double deletion, while ganB deletion caused de-repression comparable to that of the creA/pkaA double deletion. With ball-milled cellulose and D-glucose, partial de-repression was detected by deletion of creA but not of pkaA or ganB. The creA/pkaA or creA/ganB double deletion led to earlier expression than the creA deletion. Furthermore, the effect of each deletion with D-xylose or L-arabinose as the repressing carbon source was significantly different from that with D-glucose, D-fructose, and D-mannose. Consequently, this study revealed that PkaA and GanB participate in CreA-independent CCR and that contribution of CreA, PkaA, and GanB in CCR differs depending on the inducers, repressing carbon sources, and culture conditions (plate or submerged). Further study of CreA-independent mechanisms is needed to fully understand CCR in filamentous fungi.

Lack of the consensus sequence necessary for tryptophan prenylation in the ComX pheromone precursor.

ComX, an oligopeptide pheromone that stimulates the natural genetic competence controlled by quorum sensing in Bacillus subtilis and related bacilli, contains a prenyl-modified tryptophan residue. Since ComX is the only protein known to contain prenylated tryptophan, the universality of this unique posttranslational modification has yet to be determined. Recently, we developed a cell-free assay system in which the tryptophan residue in the ComX(RO-E-2) pheromone precursor derived from B. subtilis strain RO-E-2 can be geranylated by the ComQ(RO-E-2) enzyme. We report here our attempt to identify the consensus sequence surrounding the geranylated tryptophan residue by using the cell-free system with various ComX(RO-E-2) pheromone precursor analogs. We found that [47-58]ComX(RO-E-2), corresponding to the C-terminal 12-residue peptide of the pheromone precursor, contained a short sequence essential for geranylation. We also found that the length of the sequence between the tryptophan residue and the C-terminus was important for geranylation, and that some [47-58]ComX(RO-E-2) pheromone precursor amino acids were involved in the geranylation reaction. However, we could not identify a consensus sequence surrounding the geranylated tryptophan. Our evidence suggests that, like Rab which lacks a consensus sequence yet is geranylgeranyl-modified on a cysteine residue, the ComX pheromone and its precursor also lack a consensus sequence.

Xylose triggers reversible phosphorylation of XlnR, the fungal transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus oryzae.

XlnR is a transcription factor that mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. In order to clarify the molecular mechanisms underlying XlnR-mediated induction, Aspergillus oryzae XlnR was fused with the c-myc tag and examined by Western blotting. Phosphate-affinity SDS-PAGE revealed that XlnR was present as a mixture of variously phosphorylated forms in the absence of D-xylose, and that D-xylose triggered additional phosphorylation of the protein. D-Xylose-triggered phosphorylation was a rapid process occurring within 5 min prior to the accumulation of xynG2 mRNA, and removal of D-xylose caused slow dephosphorylation, leading to less-phosphorylated forms. At 30 min after removal, the phosphorylation status was almost identical to that in the absence of D-xylose, and the level of xynG2 mRNA started to decrease. These results indicate that XlnR is highly phosphorylated when it is active in transactivation, implying that D-xylose-triggered reversible phosphorylation controls XlnR activity.

Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits.

The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.

A novel ligand bound ABC transporter, LolCDE, provides insights into the molecular mechanisms underlying membrane detachment of bacterial lipoproteins.

The LolCDE complex of Escherichia coli belongs to the ABC transporter superfamily and initiates the lipoprotein sorting to the outer membrane by catalysing their release from the inner membrane. LolC and/or LolE, membrane subunits, recognize lipoproteins anchored to the outer surface of the inner membrane, while LolD hydrolyses ATP on its inner surface. We report here that ligand-bound LolCDE can be purified from the inner membrane in the absence of ATP. Liganded LolCDE represents an intermediate of the release reaction and exhibits higher affinity for ATP than the unliganded form. ATP binding to LolD weakens the interaction between LolCDE and lipoproteins and causes their dissociation in a detergent solution, while lipoprotein release from membranes requires ATP hydrolysis. Liganded LolCDE thus reveals molecular events brought about through ATP binding and hydrolysis. LolCDE is the first example of an ABC transporter purified with tightly bound native substrates. A single molecule of lipoprotein is found to bind per molecule of the LolCDE complex.

A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane-specific signals.

Lipoproteins in Gram-negative bacteria are anchored to the inner or outer membrane via fatty acids attached to the N-terminal cysteine. The residue at position 2 determines the membrane specificity. An ATP binding cassette transporter LolCDE complex releases lipoproteins with residues other than aspartate at position 2 from the inner membrane, whereas those with aspartate at position 2 are rejected by LolCDE and therefore remain in the inner membrane. For further understanding of this rejection mechanism, a novel strategy was developed to select mutants in which lipoproteins with aspartate at position 2 are released. The isolated mutants carried an alanine to proline mutation at position 40 of LolC, a membrane subunit of the LolCDE complex. A significant portion of an inner membrane lipoprotein, L10P(DQ), was localized to the outer membrane when the LolC mutant was expressed. Periplasmic chaperone LolA formed a complex with the released L10P(DQ), which was subsequently incorporated into the outer membrane in a LolB-dependent manner, indicating that neither LolA nor LolB rejects lipoproteins with aspartate at position 2. The amount of the LolC mutant co-purified with LolD and LolE after membrane solubilization was reduced significantly. Taken together, these results indicate that the mutation causes destabilization of the LolCDE complex and concomitantly prevents the accurate recognition of lipoprotein-sorting signals.