Gluconacin from Gluconacetobacter diazotrophicus PAL5 is an active bacteriocin against phytopathogenic and beneficial sugarcane bacteria.

AIMS: This study aimed to explore the possibility of using the Gluconacin from Gluconacetobacter diazotrophicus strain PAL5 in the biological control of diverse sugarcane phytopathogenic bacteria. METHODS AND RESULTS: An in silico analysis was first employed to determine the phylogenetic relationship between Gram-negative/positive bacteriocin producers and Gluconacin. The analysis showed that this trait is widespread among tested bacterial species and a well-conserved gene within the Acetobacteraceae family. The bacteriocin gene (GDI_0415) present in the genome of strain PAL5 was than cloned in pDEST™17 and expressed in Escherichia coli BL21-AI™. A bioassay showed growth inhibition of Xanthomonas albilineans by the recombinant bacteriocin. Subsequent bioassays indicated different levels of antagonistic activity against the majority of the sugarcane phytopathogenic bacteria (Xanthomonas axonopodis pv. vasculorum, Acidovorax avenae subsp. avenae, Pseudomonas syringae pv. syringae, Xanthomonas vasicola pv. vasculorum). In addition, the bacteriocin was also antagonistic to some beneficial bacterial strains belonging to G. diazotrophicus and endophytic Bacillus species, which also colonize sugarcane plants. CONCLUSIONS: The GDI_0415 gene, responsible for the production of Gluconacin, is well conserved within the Acetobacteraceae family and presented antagonistic activity against phytopathogenic and a few beneficial sugarcane bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of a recombinant protein, named Gluconacin, opens new avenues for the agro-biotechnology application in agriculture, mainly with regard to the sugarcane crop.

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes.

AIMS: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. METHODS AND RESULTS: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but beta-glucuronidase (GUS)-stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5-plant interaction. PAL5 could be isolated from the root surface (10(8) CFU g(-1)) and from surface-disinfected root and stem tissues (10(4) CFU g(-1)) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. CONCLUSION: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: These tools are of use to: (i) study PAL5 mutants affected in bacteria-plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast.

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited beta-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

Reactivity of anti-gp43 antibodies from Paracoccidioides brasiliensis antiserum with extracts from cutaneous lesions of Lobo's disease. Preliminary note.

We demonstrated through several immunochemical tests the presence of gp43 from P. brasiliensis in extracts of cutaneous lesions from Jorge Lobo's disease. This glicoprotein is one of the immunodominant antigens in this species, and is used to identify it. The demonstration of gp43 tissues infected by the agent of Jorge Lobo's disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboi.

Paracoccidioides cerebriformis Moore, 1935. Mycologic and immunochemical study.

The present study concern on mycologic and immunochemical data obtained from two samples of a fungus considered as belonging to the species Paracoccidioides cerebriformis described by Moore in 1935, and maintained since then on Sabouraud's agar in the mycology collection of the Instituto de Medicina Tropical de São Paulo. After 60 years, the samples exhibited the same characteristics described by MOORE (1935). However, experimental lesions did not resulted in guinea-pigs inoculated intratesticularly. The dominant antigen in Paracoccidioides brasiliensis, 43 kDa glicoprotein (gp43), could not be demonstrated by SDS PAGE and Western blotting. Immunoelectrophoresis did not demonstrated the E arch of cathodic migration using a policlonal anti gp43 serum. According to these findings, it is concluded that the fungus described by MOORE (1935) as P. cerebriformis does not belong to the genus Paracoccidioides. Paracoccidioidomycosis should therefore be considered as resulting from infection by a single species, Paracoccidioides brasiliensis (Splendore, 1912) as asserted by ALMEIDA (1930). Further studies, through molecular biology methods, could identify the mentioned fungus.

Paracoccidioides brasiliensis. A mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus).

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis--the glycoprotein with a molecular weight of 43 kDa--was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

Paracoccidioides brasiliensis. A mycologic and immunochemical study of two strains.

The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIA, isolated from soil in the municipality of IBIA (Minas Gerais) by Silva-Vergara et al. (1996, 1998), and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas Da Silva (1996). Both strains showed cotton-like (M) and yeast-like (Y) forms and were pathogenic for testicularly inoculated guinea pigs, producing granulomatous and/or suppurative orchitis. Immunochemically was demonstrated the presence of gp43 by double immunodiffusion, immunoelectrophoresis and immunoblotting.

Molecular characterization of a virus from the family Luteoviridae associated with cotton blue disease.

Cotton blue disease is an aphid-transmitted cotton disease described in Brazil in 1962 as Vein Mosaic "var. Ribeirão Bonito". At present it causes economically important losses in cotton crops if control measures are not implemented. The observed symptoms and mode of transmission have prompted researchers to speculate that cotton blue disease could be attributed to a member of the family Luteoviridae, but there was no molecular evidence supporting this hypothesis. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the family Luteoviridae. Sequence analysis of the entire capsid and a partial RdRp revealed a virus probably belonging to the genus Polerovirus. Based on our results we propose that cotton blue disease is associated with a virus with the putative name Cotton leafroll dwarf virus (CLRDV).

Highly specific and sensitive, immunoblot-detected 54 kDa antigen from Fonsecaea pedrosoi.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by a group of different dematiaceous fungi, first described by Rudolph in 1914. In Brazil there is a clear predominance of Fonsecaea pedrosoi. Sixty sera samples obtained from patients with F. pedrosoi-caused CBM were analysed. Sera obtained from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and from 48 blood donors (HBD) were used as control. F. pedrosoi metabolic antigen was obtained from F. pedrosoi sample no. 884 (Instituto de Medicina Tropical de São Paulo Collection). IE reaction disclosed an anodic migrating arch, which was eluted and used as antigen. Both metabolic and eluate F. pedrosoi antigens were submitted to SDS-PAGE and two fractions, weighing approximately 54 and 66 kDa were identified. The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (15.3%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM sera (96.7% sensitivity) and was not recognized by HBD, SPT nor CL sera (100% specificity). Such high sensitivity and specificity levels suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on F. pedrosoi-caused CBM.

Co-suppression mediated virus resistance in transgenic tobacco plants harboring the 3'-untranslated region of Andean potato mottle virus.

Nicotiana tabacum plants were transformed with the 3'-untranslated region of the Andean Potato Mottle Virus (APMoV) genome RNA-2. Three strategies were used: the introduction of this region in sense and in antisense orientation and of a fragment comprising the entire 3'-untranslated region from RNA-2 and part of the CP22 coat protein sequence. The transgenic lines were inoculated with the virus and different responses were observed, ranging from susceptibility to APMoV to complete immunity to virus infection, in which neither the virion nor viral RNA was detected in the inoculated leaf and leaves that emerged after inoculation. The R1 progeny from different R0 lines also showed an array of virus resistance phenotypes, which was not correlated with the zygotic state of the transgene. Resistance was positively correlated with low levels of transgene mRNA accumulation, indicating a co-suppression-mediated mechanism towards the transgenic transcripts and APMoV viral RNA.

Lack of reactivity of paracoccidioidomycosis sera in the double immunodiffusion test with the gp43 antigen: report of two cases.

Sera from two patients with chronic active paracoccidioidomycosis yielded negative double immunodiffusion results with a culture filtrate antigen from Paracoccidioides brasiliensis routinely used in our laboratory. Complement fixation tests were positive for both sera using a polysaccharide-rich antigen. This study reports the results of a more extensive serological investigation of these two sera. Both a somatic antigen and a saline extract from the fungus yielded positive results in the double immunodiffusion. However, the immunodominant 43 kDa glycoprotein antigen showed negative results, although it was recognized by both sera in the Western blot assay. The value of the double immunodiffusion as a single serological test in paracoccidioidomycosis diagnosis is discussed.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374