We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.Toxigenic strains of the Gram-negative bacterium Vibrio cholerae cause a life-threatening diarrheal disease that can kill its victims within hours of the onset of symptoms. The disease is endemic in the Indian subcontinent and continues to reemerge elsewhere in Asia, Africa, and the Americas with the incidence estimated to exceed 5 million cases each year (1-3). Although over 180 serogroups of V. cholerae exist, only the O1 and O139 serovars are known to cause epidemic cholera. The O1 serogroup can be divided further into two biotypes, classical and El Tor, based on various biochemical and phenotypic differences (4). Since 1817, seven cholera pandemics have occurred, of which the classical biotype is responsible for at least the fifth (1881-1896) and the sixth . The El Tor biotype is responsible for the seventh and current pandemic (1961-present). Toxigenic strains of O139 serovar appeared in India and Bangladesh in late 1992 as the first non-O1 serovar to cause epidemic cholera (5, 6). These strains are closely related and probably derived from toxigenic El Tor O1 strains (7). The factors that allowed El Tor biotype and O139 serotype to overtake the classical strains and establish pandemics remain a mystery.Work focusing on the pathogenesis of V. cholerae led to the identification of several key virulence factors, including the cholera toxin responsible for the diarrhea and the toxincoregulated pilus essential for colonizing the human small intestine (8, 9). Several groups tried to construct live, attenuated V. cholerae vaccines by deleting the genes for cholera toxin and several other putative accessory toxins (10-16). However, many of these strains remain reactogenic in human subjects, causing diarrhea or other significant symptoms (17).The virulence factors responsible for the residual reactogenicity remain to be identified.Bacterial genomics offers an opportunity to identify undiscov...

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds–Nα-aroyl-N-aryl-phenylalaninamides (AAPs)–that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.