Serum from Myalgic encephalomyelitis/chronic fatigue syndrome patients causes loss of coherence in cellular circadian rhythms.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling disorder characterized by disrupted daily patterns of activity, sleep, and physiology. Past studies in ME/CFS patients have examined circadian rhythms, suggested that desynchronization between central and peripheral rhythms may be an important pathological feature, and identified associated changes in post-inflammatory cytokines such as transforming growth factor beta (TGFB). However, no previous studies have examined circadian rhythms in ME/CFS using cellular models or studied the role of cytokines on circadian rhythms. In this study, we used serum samples previously collected from ME/CFS patients (n = 20) selected for the presence of insomnia symptoms and matched controls (n = 20) to determine the effects of serum factors and TGFB on circadian rhythms in NIH3T3 mouse immortalized fibroblasts stably transfected with the Per2-luc bioluminescent circadian reporter. Compared to control serum, ME/CFS serum caused a significant loss of rhythm robustness (decreased goodness of fit) and nominally increased the rate of damping of cellular rhythms. Damping rate was associated with insomnia severity in ME/CFS patients using the Pittsburgh Sleep Quality Index (PSQI). Recombinant TGFB1 peptide applied to cells reduced rhythm amplitude, caused phase delay and decreased robustness of rhythms. However, there was no difference in TGFB1 levels between ME/CFS and control serum indicating the effects of serum on cellular rhythms cannot be explained by levels of this cytokine. Future studies will be required to identify additional serum factors in ME/CFS patients that alter circadian rhythms in cells.

The beta-ketoadipate pathway of Acinetobacter baylyi undergoes carbon catabolite repression, cross-regulation and vertical regulation, and is affected by Crc.

The degradation of many structurally diverse aromatic compounds in Acinetobacter baylyi is accomplished by the beta-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 beta-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.

Aromatic degradative pathways in Acinetobacter baylyi underlie carbon catabolite repression.

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.

Multiple-level regulation of genes for protocatechuate degradation in Acinetobacter baylyi includes cross-regulation.

The bacterium Acinetobacter baylyi uses the branched beta-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 (cross-regulation). 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.