Identification of an operon required for ferrichrome iron utilization in Vibrio cholerae.

Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to Escherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhu operon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.

Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage.

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.

Brain ECF pH and central chemical control of ventilation during anoxia in turtles.

The role of changes in brain extracellular fluid [H+] in the control of breathing during anoxia was studied in unanesthetized turtles, Chrysemys scripta. Ventilation, [minute ventilation (VE), tidal volume (VT), and breathing frequency (f)], cerebral extracellular fluid (ECF) pH, and arterial blood gases were measured at 25 degrees C during a 30-min control period (room air), 30 min of anoxia (100% N2 breathing), and 60 min of recovery (room air). ECF pH was measured in the cerebral cortex with a glass microelectrode (1-2 micron tip diam). Large changes in ventilation, ECF [H+], and arterial blood gases were observed. The predominant ventilatory response was an increase in f with a slight increase in VT. A correlation was observed between ECF [H+] and f, which suggested that central chemoreceptor stimulation was involved in the ventilatory response.

Effect of hypocapnia on ventral medullary blood flow and pH during hypoxia in cats.

Ventral medullary blood flow was measured in 33 chloralose-urethan anesthetized cats during 60 min of isocapnia-hypoxia, mild hypocapnia-hypoxia, or severe hypocapnia-hypoxia. In an additional group of six animals we measured ventral medullary extracellular fluid (ECF) pH during mild hypocapnia-hypoxia. The increase in blood flow during hypoxia was reduced by mild hypocapnia and eliminated by severe hypocapnia. With the exception of an initial decrease in ECF [H+], which occurred during the first 10 min of mild hypocapnia-hypoxia, ECF [H+] increased progressively throughout the exposure and recovery periods and was significantly elevated from the control value by the first 10 min of the recovery period. The results suggest that hypocapnia affects the hypoxic cerebrovascular response of the ventral medulla and that this phenomenon could affect the regulation of ventral medullary ECF [H+].