Rett syndrome (RTT), a postnatal neurodevelopmental disorder, is caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Children with RTT display cognitive and motor abnormalities as well as autistic features. We studied mice bearing a truncated Mecp2 allele (Mecp2 308/Y mice) and found evidence of increased anxiety-like behavior and an abnormal stress response as evidenced by elevated serum corticosterone levels. We found increased corticotropin-releasing hormone (Crh) gene expression in the paraventricular nucleus of the hypothalamus, the central amygdala, and the bed nucleus of the stria terminalis. Finally, we discovered that MeCP2 binds the Crh promoter, which is enriched for methylated CpG dinucleotides. In contrast, the MeCP2 308 protein was not detected at the Crh promoter. This study identifies Crh as a target of MeCP2 and implicates Crh overexpression in the development of specific features of the Mecp2 308/Y mouse, thereby providing opportunities for clinical investigation and therapeutic intervention in RTT.autism ͉ corticotropin-releasing hormone ͉ methyl-CpG-binding protein 2

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

SummaryOuter surface lipoprotein (Osp) C is a virulence factor required for transmission of the Lyme disease agent, Borrelia burgdorferi. We have constructed an inducible promoter system to study the function and regulation of OspC by integrating regulatory elements from the Escherichia coli lac operon into the B. burgdorferi genome. An inducible promoter (flacp) was constructed by inserting a synthetic lac operator sequence between the transcriptional start site and the ribosomal binding site of the B. burgdorferi flgB promoter; flacp was then used to replace the native ospC and rpoS promoters in B. burgdorferi derivatives that constitutively express the E. coli Lac repressor protein (LacI). In vitro, the expression of ospC and rpoS from flacp was dependent on the inducer isopropyl b-D-thiogalactopyranoside and was unaffected by temperature or pH, conditions commonly used to mimic different aspects of the B. burgdorferi life cycle. Our results suggest that OspC is essential immediately upon injection into a mouse and OspC expression must be maintained during the early stages of infection. In addition, the mouse infectivity experiment indicates that this system can be used to regulate B. burgdorferi genes in vivo, within the context of an experimental tickmouse infectious cycle. RpoS is an alternative sigma factor that is required for ospC transcription. However, the role of other temperature-dependent factors has not previously been addressed. Our results with the inducible rpoS strain demonstrate that RpoS alone is sufficient to activate OspC expression, even at 23°C. This is the first functional inducible promoter system developed for use in B. burgdorferi and, for the first time, will provide researchers with the ability to artificially regulate the expression of genes in this pathogenic spirochaete.

SummaryOspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23 ∞ ∞ ∞ ∞ C compared with cultures grown at 35-37 ∞ ∞ ∞ ∞ C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A 1 , an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23 ∞ ∞ ∞ ∞ C to 35 ∞ ∞ ∞ ∞ C or after adding coumermycin A 1 . In addition, OspC synthesis was higher in a gyrB mutant than in wild-type B. burgdorferi . Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi , but not in Escherichia coli , as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.