Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-kappaB signaling.

Because NF-kappaB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappaB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappaB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IkappaB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappaB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappaB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappaB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappaB signaling.

Immunological detection of Nitrospira-like bacteria in various soils.

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.

[Application of immunologic methods to the analysis of bio-leaching bacteria]. / Aplicación de métodos inmunológicos al análisis de bacterias biolixiviadoras.

Pure cultures of Thiobacillus ferrooxidans and mixed cultures of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans isolated from the Matahambre mine (Cuba) were used to fit immunodiffusion and immunoelectron microscopy to the study of iron oxidizing bacteria. The possibilities, advantages and limits of those techniques have been studied from both the identification and the serological characterization points of view. Finally, the efficiency of these methods was tested by applying them to the identification of microorganisms from acidic waters from the mine.

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Immunological comparison of ribosomal proteins from archaebacteria.

Antisera were raised in rabbits against ribosomal proteins of Methanobacterium bryantii and used to analyze immunological relationships to ribosomes from other archaebacteria, from eubacteria, and from yeasts. Cross-reaction could be detected within the methanogens and with a member of the extreme halophiles; the degree of immunological similarity reflected the relationship delineated by 16S ribosomal ribonucleic acid oligonucleotide analysis (Fox et al., Science 209:457-463, 1980). With the methods and the anti-total-protein sera employed, there was no detectable cross-reaction with ribosomal proteins or ribosomes from Sulfolobus sp., eubacteria, or yeast.

Serological studies on lithotrophic, ammonia oxidizing bacteria.

Rabbit antisera were prepared against living cells of six different ammonia oxidizing nitrifying bacteria. They were examined as to cross-reactivity in the agglutination test (Microtiter-system) with 24 nitrifier strains, including members of all known genera. Usually distinct cross-reactions were obtained only within the genera, but some exceptions were noticed. There was stated a clear cross-reaction between the two anti-Nitrosospira-antisera and the four tested Nitrosolobus strains. In some cases cross-reactions between cells of the Nitrosovibrio strains and the anti-Nitrosospira- as well as the anti-Nitrosococcus-antisera could be observed. The interpretation of the results obtained with the Nitrosomonas group was complicated by the fact that all strains showed positive zero titers with the control sera. In seven cases lipopolysaccharides were isolated and tested in the passive hemagglutination test to their cross-reactivity with the above mentioned antisera. Hemagglutination could only be observed in the homologous system, cross-reactivity was never expressed.