Design of new promoters and of a dual-bioreporter based on cross-activation by the two regulatory proteins XylR and HbpR.

The HbpR protein is the sigma54-dependent transcription activator for 2-hydroxybiphenyl degradation in Pseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the PhbpC and Pu promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays. XylR measurably activated the PhbpC promoter in the presence of the effector m-xylene, both in Escherichia coli and Pseudomonas putida. HbpR weakly stimulated the Pu promoter in E. coli but not in P. azelaica. Poor HbpR-dependent activation from Pu was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on PhbpC were gradually changed into the XylR binding sites of Pu by site-directed mutagenesis. Inducible luciferase expression from mutated promoters was tested in E. coli on a two plasmid system, and from mono copy gene fusions in P. azelaica and P. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR-dependent transcription than from Pu itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type PhbpC, while keeping the same maximal induction level. On the basis of these results, a dual-responsive bioreporter strain of P. azelaica was created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently with m-xylene and 2-hydroxybiphenyl

Exploring Spatiotemporal Organization of SCN Circuits

Suprachiasmatic nucleus neuroanatomy has been a subject of intense interest since the discovery of the SCN’s function as a brain clock and subsequent studies revealing substantial heterogeneity of its component neurons. Understanding the network organization of the SCN has become increasingly relevant in the context of studies showing that its functional circuitry, evident in the spatial and temporal expression of clock genes, can be reorganized by inputs from the internal and external environment. Although multiple mechanisms have been proposed for coupling among SCN neurons, relatively little is known of the precise pattern of SCN circuitry. To explore SCN networks, we examine responses of the SCN to various photic conditions, using in vivo and in vitro studies with associated mathematical modeling to study spatiotemporal changes in SCN activity. We find an orderly and reproducible spatiotemporal pattern of oscillatory gene expression in the SCN, which requires the presence of the ventrolateral core region. Without the SCN core region, behavioral rhythmicity is abolished in vivo, whereas low-amplitude rhythmicity can be detected in SCN slices in vitro, but with loss of normal topographic organization. These studies reveal SCN circuit properties required to signal daily time