Carbon catabolite repression is an important mechanism allowing efficient carbon source utilization. In the soil bacterium Acinetobacter baylyi, this mechanism has been shown to apply to the aromatic degradative pathways for the substrates protocatechuate, p-hydroxybenzoate and vanillate. In this investigation, transcriptional fusions with the gene for luciferase in the gene clusters for the degradation of benzyl esters, anthranilate, benzoate, hydroxycinnamates and dicarboxylates (are, ant, ben, hca and dca genes) were constructed and established in the chromosome of A. baylyi. The respective strains revealed the presence of strong carbon catabolite repression at the transcriptional level. In all cases, succinate and acetate in combination had the strongest repressing effect, and pyruvate (or lactate in case of the ben and hca genes) allowed the highest expression when these carbon sources were supplied together with the respective inducer. The pattern of repression for the different cosubstrates was similar for all operons investigated and was also observed in the absence of the respective inducing compounds, indicating a mechanism that is independent of the respective specific regulators. Repression by acetate and succinate varied between 88 % for the hca genes and 99 % for the pca genes.

Responsiveness in the cochlear nucleus complex and inferior colliculus of the mouse to tonal stimulation is labelled via immunocytochemically stained Fos protein that is expressed by c-fos gene activation in excited neurons. The locations of Fos-positive neurons closely reproduce the tonotopic maps in the dorsal cochlear nucleus and inferior colliculus. Thus, the c-fos method can demonstrate stimulus-related local neuronal activation on a single-cell level and may be useful to complement other mapping techniques such as electrophysiological recording or 2-deoxyglucose autoradiography.

The bacterium Acinetobacter baylyi uses the branched ␤-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the pca-qui operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter pcaIp, namely protocatechuate-dependent activation of pca-qui operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (Escherichia coli) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the pca-qui operon. The increase of the pcaU gene copy number leads to a decrease of the basal expression at pcaIp, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of pcaIp when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (crossregulation). The same repression pattern was observed for promoter pcaUp. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (pobAp upstream of pobA, pcaIp, and pcaUp) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on pobA expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of pobA expression.

The b-ketoadipate pathway of Acinetobacter baylyi undergoes carbon catabolite repression, crossregulation and vertical regulation, and is affected by Crc The degradation of many structurally diverse aromatic compounds in Acinetobacter baylyi is accomplished by the b-ketoadipate pathway. In addition to specific induction of expression by certain aromatic compounds, this pathway is regulated by complex mechanisms at multiple levels, which are the topic of this study. Multiple operons feeding into the b-ketoadipate pathway are controlled by carbon catabolite repression (CCR) caused by succinate plus acetate. The pathways under study enable the catabolism of benzoate (ben), catechol (catA), cis,cis-muconate (catB,C,I,J,F,D), vanillate (van), hydroxycinnamates (hca), dicarboxylates (dca), salicylate (sal), anthranilate (ant) and benzyl esters (are). For analysis of CCR at the transcriptional level a luciferase reporter gene cassette was introduced into the operons. The Crc (catabolite repression control) protein is involved in repression of all operons (except for catA), as demonstrated by the analysis of respective crc strains. In addition, cross-regulation was demonstrated for the vanA,B, hca and dca operons. The presence of protocatechuate caused transcriptional repression of the vanA,B-and hca-encoded funnelling pathways (vertical regulation). Thus the results presented extend the understanding both of CCR and of the effects of Crc for all aromatic degradative pathways of A. baylyi and increase the number of operons known to be controlled by two additional mechanisms, cross-regulation and vertical regulation. INTRODUCTIONThe bacterium Acinetobacter baylyi is a soil organism known to be able to use aromatic substances through the b-ketoadipate pathway (Harwood & Parales, 1996). Numerous more complex aromatic compounds can be converted into the two central starting compounds of the b-ketoadipate pathway, protocatechuate (PCA) and catechol, by additional short metabolic pathways (funnelling pathways). The expression of all the respective operons is thoroughly controlled by specific inducers. Furthermore, to cope with an array of environmental changes, the bketoadipate pathway and its funnelling pathways are controlled by a regulatory network the complexity of which is only beginning to be elucidated (Vaneechoutte et al. 2006;Gerischer, 2008;Williams & Kay, 2008). One part of this network is carbon catabolite repression (CCR) (Cánovas & Stanier, 1967;Tresguerres et al., 1970;Dal et al., 2005;Fischer et al., 2008). The molecular mechanisms of CCR are well understood in Escherichia coli and Gram-positive bacteria such as Bacillus subtilis, but not in bacteria belonging to the genera Pseudomonas and Acinetobacter. In these bacteria, organic acids such as succinate and acetate, as well as the protein Crc (catabolite repression control), play an important role in CCR (Wolff et al., 1991;Zimmermann et al., 2009). A. baylyi Crc has been shown to be involved in the degradation of the pcaqui transcript, which encodes enzymes ...

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