In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.

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.

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.

Background:Bacillus thuringiensis is the most successful biological control agent, however, studies so far have shown that B. thuringiensis is very sensitive to environmental factors such as soil moisture and pH. Ultraviolet light from the sun had been considered as the main limiting factor for its persistence in soil and it has recently been shown that the antagonism exerted by other native soil organisms, such as Pseudomonas fluorescens, is a determining factor in the persistence of this bacterium under in vitro culture conditions.Objectives:The aim of the present investigation was to analyze the population dynamics of B. thuringiensis and its interaction with P. fluorescens using microbiological and molecular methods in soil, under different conditions, and to determinate the effect of nutrients and moisture on its interaction.Materials and Methods:The monitoring was performed by microbiological methods, such as viable count of bacteria, and molecular methods such as Polymerase Chain Reaction (PCR) and hybridization, using the direct extraction of DNA from populations of inoculated soil.Results:The analysis of the interaction between B. thuringiensis and P. fluorescens in soil indicated that the disappearance of B. thuringiensis IPS82 is not dependent on the moisture but the composition of nutrients that may be affecting the secretion of toxic compounds in the environment of P. fluorescens. The results showed that the recovered cells were mostly spores and not vegetative cells in all proved treatments. The molecular methods were effective for monitoring bacterial population inoculated in soil.Conclusions:Bacillus thuringiensis is very sensitive to the interaction of P. fluorescens, however is capable to survive in soil due to its capacity of sporulate. Some of the cells in the form of spores germinated and folded slightly and remained in a constant cycle of sporulation and germination. This confirms that B. thuringiensis IPS82 can germinate, grow and sporulate in soil.