ToxR is required in Vibrio cholerae for transcriptional activation of the toxT gene, the protein product of which activates numerous genes involved in virulence. Although ToxR cannot activate the toxT promoter in Escherichia coli, the products of the tcpPH operon are shown here to activate the toxT promoter, and co‐expression with ToxRS enhances activation. An identical pattern was seen in a ΔtcpPΔtoxR strain of V. cholerae when TcpPH or ToxRS was expressed from plasmids. Although overexpression of the TcpP/H proteins in V. cholerae partially complemented both a ΔtoxR strain and a ΔtcpPΔtoxR double mutant for toxin production and toxT–lacZ activation, the presence of ToxR greatly increased their expression. Analysis of a toxT–lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR binding site. This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, which showed that TcpP interacts with the promoter region from −51 to −32, whereas ToxR protected a region from −100 to −69. In addition, membranes containing endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP. Characterization of a number of tcpP substitution mutants revealed one derivative (TcpP‐H93L) that, when overexpressed, was markedly defective for toxT activation, cholera toxin and TcpA (toxin co‐regulated pilus) production and DNA binding; however, toxT activation by TcpP‐H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation. Two additional mutant derivatives, TcpP‐W68L and TcpP‐R86A, failed to activate toxT or direct toxin and TcpA production in the presence or absence of ToxR. Both TcpP‐W68L and TcpP‐R86A, like TcpP‐H93L, were defective for DNA binding. Finally, a ToxR mutant derivative, ToxR‐G80S, served to separate the different roles of ToxR on different promoters. Although ToxR‐G80S was inefficient at activating the ompU promoter in V. cholerae (ompU encodes an outer membrane porin regulated by ToxR), it was fully capable of activating the toxT promoter. These data suggest that ToxR is not a direct activator in the toxT expression system but, instead, enhances the activity of TcpP, perhaps by recruiting it to the toxT promoter under conditions in which expression levels of TcpP are too low for it to activate toxT efficiently on its own.

SummaryCo-ordinate expression of many virulence genes in the human pathogen Vibrio cholerae is under the direct control of the ToxT protein, including genes whose products are required for the biogenesis of the toxin-co-regulated pilus (TCP) and cholera toxin (CTX). This work examined interactions between ToxT and the promoters of ctx and tcpA genes. We found that a minimum of three direct repeats of the sequence TTTTGAT is required for ToxT-dependent activation of the ctx promoter, and that the region from -85 to -41 of the tcpA promoter contains elements that are responsive to ToxT-dependent activation. The role of H-NS in transcription of ctx and tcpA was also analysed. The level of activation of ctx-lacZ in an E. coli hns -strain was greatly increased even in the absence of ToxT, and was further enhanced in the presence of ToxT. In contrast, H-NS plays a lesser role in the regulation of the tcpA promoter. Electrophoretic mobility shift assays demonstrated that 6¥ His-tagged ToxT directly, and specifically, interacts with both the ctx and tcpA promoters. DNase I footprinting analysis suggests that there may be two ToxT binding sites with different affinity in the ctx promoter and that ToxT binds to -84 to -41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT-dependent gene expression, ToxT binds to AT-rich promoters that may have a specific secondary conformation, displaces H-NS and stimulates RNA polymerase resulting in transcription activation.

SummaryMyxococcus leaves a trail of slime on agar as it moves. A filament of slime can be seen attached to the end of a cell, but it is seen only at one end at any particular moment. To identify genes essential for A motility, transposon insertion mutations with defective A motility were studied. Fifteen of the 33 mutants had totally lost A motility. All these mutant cells had filaments of slime emerging from both ends, indicating that bipolar secretion prevents A motility. The remaining 18 A motility mutants, also produced by gene knockout, secreted slime only from one pole, but they swarmed at a lower rate than A + and are called 'partial' gliding mutants, or pgl. For each pgl mutant, the reduction in swarm expansion rate was directly proportional to the reduction in the coefficient of elasticotaxis. The pgl mutants have a normal reversal frequency and normal gliding speed when they move. But their probability of movement per unit time is lower than pgl + cells. Many of the pgl mutants are produced by transposon insertions in glycosyltransferase genes. It is proposed that these glycosyltransferases carry out the synthesis of a repeat unit polysaccharide that constitutes the slime.

Centrosomes serve as microtubule‐organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule‐dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C‐terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C‐terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild‐type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole‐induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.

Coordinate expression of many virulence genes in the human pathogenVibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activatetoxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of toxT transcription. We tested a model whereby activation of toxT by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the tcpA promoter reads through a proposed terminator between the tcpF and toxT genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxTΔpro ) resulted in the abolishment oftoxT transcription, as predicted. Deletion of thetcpA promoter (tcpAΔpro ) had no effect on subsequent detection of the smaller toxT primer extension product, but the larger toxT product was not detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. ThetoxTΔpro strain produced little detectable CTX, while the tcpAΔpro strain produced CTX levels intermediate between those of the wild-type andtoxTΔpro strains. Dependence oftoxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectabletoxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest thattoxT transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.