The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT

The Gram-negative, curved rod Vibrio cholerae causes the severe diarrhoeal disease cholera. The two major virulence factors produced by V. cholerae during infection are the cholera toxin (CT) and the toxin-coregulated pilus (TCP). Transcription of the genes encoding both CT and the components of the TCP is directly activated by ToxT, a transcription factor in the AraC/XylS family. ToxT binds upstream of the ctxAB genes, encoding CT, and upstream of tcpA , the first gene in a large operon encoding the components of the TCP. The DNA sequences upstream of ctxAB and tcpA that contain ToxT binding sites do not have any significant similarity other than being AT-rich. Extensive site-directed mutagenesis was performed on the region upstream of tcpA previously shown to be protected by ToxT, and we identified specific base pairs important for activation of tcpA transcription by ToxT. This genetic approach was complemented by copper-phenanthroline footprinting experiments that showed protection by ToxT of the base pairs identified as most important for transcription activation in the mutagenesis experiments. Based on this new information and on previous work, we propose the presence of a ToxT-binding motif – the ‘toxbox’– in promoters regulated by ToxT. At tcpA , two toxbox elements are present in a direct repeat configuration and both are required for activation of transcription by ToxT. The identity of only a few of the base pairs within the toxbox is important for activation by ToxT, and we term these the core toxbox elements. Lastly, we examined ToxT binding to a mutant having 5 bp inserted between the two toxboxes at tcpA and found that occupancy of both binding sites is retained regardless of the positions of the binding sites relative to each other on the face of the DNA. This suggests that ToxT binds independently as a monomer to each toxbox in the tcpA direct repeat, in accordance with what we observed previously with the inverted repeat ToxT sites between acfA and acfD .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75755/1/j.1365-2958.2006.05053.x.pd

Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross-linking: Wzz is able to oligomerise.

The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination. Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of WzzSF. Some of the WzzSF variants resulted in O-antigen modal chain lengths much shorter than those of wild-type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O-antigen chain length distribution. The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins. A carboxy-terminal-truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino-terminal region is sufficient for the Wzz-Wzz interaction observed. Shortened WzzSF proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization. Cross-linking of WzzSF proteins with mutationally altered residues showed that loss of WzzSF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects WzzSF-WzzSF dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis

Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

The common cushion moss Grimmia laevigata (Bridel) Bridel grows on bare rock in a broad range of environments on every continent except Antarctica. As such, it must harbor adaptations to a remarkably broad set of environmental stresses, the extremes of which can include very high temperatures, prolonged nearly complete desiccation, and high ultraviolet B (UVB) exposure. Yet, like many mosses, G. laevigata shows very little morphological variability across its cosmopolitan range. This presents an evolutionary puzzle, the solution to which lies in understanding the phylogeographic structure of this morphologically simple organism. Here we report the results of an analysis of amplified fragment length polymorphisms (AFLPs) in G. laevigata, focusing on individuals from the California Floristic Province. We found evidence that populations within California constitute two distinct geographically overlapping cryptic species. Each clade harbors multiple private alleles, indicating they have been genetically isolated for some time. We suggest that the existence of cryptic species within G. laevigata, in combination with its life history, growth habits, and extreme desiccation tolerance, makes this moss an ideal research tool and a candidate for a biological indicator of climate change and pollution