ABSTRACT
Gallibacterium anatis has the ability to hemagglutinate rabbit erythrocytes; however, no bacterial component has yet been associated with this function. In the present work, a protein of approximately 65 kDa with hemagglutinating activity for glutaraldehyde-fixed chicken erythrocytes was purified by ion interchange chromatography from G. anatis F149(T) secreted proteins. The protein was recognized by a rabbit polyclonal serum against a hemagglutinin from Avibacterium paragallinarum. The 65 kDa purified protein presented identity with a G. anatis filamentous hemagglutinin by mass spectrometric analysis. As well, the bacterial surface of G. anatis was labeled by immune gold assays using a polyclonal serum against the 65-kDa protein. A similar protein was recognized in four other G. anatis strains by immunoblots using the same antiserum. The protein binds sheep or pig biotinylated fibrinogen, suggesting an interaction with basement membrane eukaryotic cells components, and the protein is present in G. anatis biofilms. Overall, the results suggest that the 65 kDa hemagglutinin is a common antigen and a potential virulence factor in G. anatis.
Subject(s)
Hemagglutinins/metabolism , Pasteurellaceae/physiology , Animals , Biofilms , Carbohydrates/pharmacology , Chickens , Erythrocytes/immunology , Erythrocytes/metabolism , Hemagglutination/drug effects , Hemagglutination Tests , Hemagglutinins/chemistry , Hemagglutinins/genetics , Hemagglutinins/isolation & purification , Pasteurellaceae/classification , Pasteurellaceae/ultrastructure , PhylogenyABSTRACT
We undertook the study of the decay process of the cry1Aa mRNA of Bacillus thuringiensis expressed in B. subtilis. The cry1Aa transcript is a 3.7-kb mRNA expressed during sporulation whose transcriptional control has previously been studied in both B. subtilis and B. thuringiensis. We found that the cry1Aa mRNA has a half-life of around 9 min and that its decay occurs through endoribonucleolytic cleavages which result in three groups of high-molecular-weight mRNA intermediates ranging in size from 2.7 to 0.5 kb. A comparative study carried out with Escherichia coli showed a similar pattern of degradation intermediates. Primer extension analysis carried out on RNA from B. subtilis revealed that most cleavages occur within two regions located toward the 5' and 3' ends of the mRNA. The most prominent processing site observed for the cry1Aa mRNA isolated from B. subtilis is only two bases away from that occurring on RNA isolated from E. coli. Most cleavage sites occur at seemingly single-stranded RNA segments rich in A and U nucleotides, suggesting that a common and conserved mechanism may process the cry1Aa mRNA.
Subject(s)
Bacillus subtilis/genetics , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins , Endotoxins/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Bacillus subtilis/metabolism , Bacillus thuringiensis/metabolism , Bacillus thuringiensis Toxins , Base Composition , Blotting, Northern , Cloning, Molecular , Endoribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Hemolysin Proteins , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , Spores, BacterialABSTRACT
The stability of integration and amplification of an integrational plasmid in Bacillus subtilis was analyzed. A cat-containing plasmid was constructed that could be integrated into the amy locus to facilitate measurement of excision events. Pulse-field gel electrophoresis was used to measure the copy number in strains that were resistant to different levels of chloramphenicol. The stability of the amplified unit in strains containing from 2 to 18 tandem copies of the amplicon in the presence and absence of chloramphenicol and through different generation times was then determined. Our results demonstrate that, for any given strain, the copy number of the amplicon remains stable. Furthermore, this stability is maintained when a clone containing an amplicon of defined size is cultured through as many as 100 generations in the absence of selective pressure.
