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Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms. José Vivas, 2004.Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized . We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout . In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks . The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water . When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A . hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state . Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity . Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A . hydrophila strains at low temperatures (6 and 16°C) . These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures . Operon Structure and Regulation of the nos Gene Region of Pseudomonas stutzeri, Encoding an ABC-Type ATPase for Maturation of Nitrous Oxide Reductase. Ulrike Honisch, 2003.The synthesis of a functional nitrous oxide reductase requires an assembly apparatus for the insertion of the prosthetic copper . Part of the system is encoded by maturation genes located in Pseudomonas stutzeri immediately downstream of the structural gene for the enzyme . We have studied the transcriptional organization and regulation of this region and found a nosDFYL tatE operon structure . In addition to a putative ABC transporter, consisting of NosD, NosF, and NosY, the operon encodes a Cu chaperone, NosL, and a component of the Tat translocon, TatE . The nosD operon was activated in response to anaerobiosis and nitrate denitrification . The membrane-bound regulator NosR was required for operon expression; in addition, DnrD, a regulator of the Crp-Fnr family, enhanced expression under anaerobic conditions . This establishes a likely signal transduction sequence of NO  DnrD
nosR/NosR
nosD operon . DnrD-dependent expression was also observed for the nnrS operon (located immediately downstream of the nosD operon), which encodes a putative heme-Cu protein (NnrS) and a member of the short-chain dehydrogenase family (ORF247) . The NosF protein, encoded within the nosD operon, exhibits sequence similarity to ABC-type ATPases . It was fused to the Escherichia coli maltose-binding protein and overexpressed in soluble form . The fusion protein was purified and shown to have ATPase activity . NosF is the first maturation factor for which a catalytic function has been demonstrated in vitro .
Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in Drosophila melanogaster. Hisashi Anbutsu, 2003.The endosymbiotic bacteria Spiroplasma spp . are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects . One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated . It is not known why NSRO-A does not kill males . In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique . In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same . Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 108 dnaA copy equivalents . However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns . The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 109 dnaA copy equivalents per insect, and then decreased . In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle . In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence . Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A . Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma . The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10-5 for NSRO and 3 x 10-4 for NSRO-A .
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