Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris.

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

Janus-type AT nucleosides: synthesis, solid and solution state structures.

Novel Janus-type nucleoside analogues (1a-d) were synthesized. Their pyrimido[4,5-d]pyrimidine base moiety has one face with a bidentate Watson-Crick donor-acceptor (DA) H-bond array of adenine and the other face with an acceptor-donor (AD) H-bond array of thymine. These nucleosides may self-associate through the self-complementary base pair. Indeed, in the solid state, compound 6d displayed a honeycomb-like supramolecular structure with tetrameric membered cavities formed through the combination of reverse Watson-Crick base pairs and aromatic stacking, in which the solvent molecules were accommodated. The result of temperature-dependent CD studies showed that the free nucleosides can form higher order chiral structures in aqueous solution.

A novel Janus-type AT nucleoside with benzoyl protecting groups forming a pleated-sheet structure.

The title compound, 5-amino-8-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione methanol monosolvate, C(32)H(25)N(5)O(9)·CH(4)O, which crystallized slowly from methanol, exhibits an anti conformation with a glycosyl-bond torsion angle of χ=-141.28 (17)°. The furanose moiety adopts an N-type sugar puckering ((3)T(4)). The corresponding pseudorotation phase angle and maximum amplitude are P=24.5 (2)° and τ(m)=38.3 (2)°, respectively. In the solid state, one methanol molecule acts as a bridge joining adjacent nucleoside molecules head-to-head, leading to a pleated-ribbon supramolecular structure, with the base moieties located in the centre of the ribbon and the sugar residues protruding to the outside of the layers, as in a DNA helix. The pleated-ribbon supramolecular structure is tethered together into a two-dimensional infinite pleated-sheet structure through aromatic stacking between the nucleobase planes and the benzene rings of the benzoyl protecting groups on the 5'-OH group of furanose.

Synthesis of Janus type nucleoside analogues and their preliminary bioactivity.

Novel Janus type nucleoside analogues 1a and 1b were synthesized in seven steps from 2-amino-4,6-dihydroxypyrimidine and 4,6-dihydroxypyrimidine. The base moiety of 1a has one face with a Watson-Crick donor-donor-acceptor (DDA) H-bond array of guanine and the other face with an acceptor-acceptor-donor (AAD) array of cytosine, which might lead to its base pairing with either cytosine or guanine due to the rotating of the glycosyl bond. This property may enable Janus type nucleoside analogues to act as an antiviral compound in a similar way to ribavirin. Both 1a and 1b were screened by a vitro HBV DNA replication inhibition test and indeed 1a showed a great potential with IC(50) = 10 µM and SI = 78.9 for antiviral drug development.