The hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202-312. hha mutants harbouring plasmid pANN202-312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various Y. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in cells harbouring plasmid pANN202-312. In addition, hha mutants show alterations in the level of plasmid DNA supercoiling. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novobiocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.
A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.
We constructed hha derivatives from both a clinical uropathogenic Escherichia coli isolate (strain FVL4) and a wild E. coli strain causing bovine diarrhea (strain CCB21) and analyzed the effect of the hha allele on the expression of the different virulence factors exhibited by these strains. Expression of hemolysin and of the Vir antigen was altered in hha mutants. Whereas production of hemolysin by strain FVL4 was repressed both at a low temperature and at high osmolarity, the hha allele accounted for a significant increase of hemolysin production under these conditions. Also, the low temperature-sensitive expression of the Vir adhesin was modified in hha mutants, which were able to express this adhesin at a low temperature. Expression of other virulence factors, such as cytotoxic necrotizing factor type 1 and 2 toxins, remained unmodified in hha derivatives of strains FVL4 and CCB21.
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