Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

A análise de sequência de transposase é uma técnica importante para descobrir a presença de elementos transponíveis no genoma. Uma sequência potencial de transposase foi analisada no genoma do fitopatógeno Moniliophthora perniciosa, o agente causal da doença vassoura-de-bruxa no cacau. As comparações de sequência do peptídeo predito para a transposase indicam uma relação com transposases de elementos da superfamília Tc1-Mariner. A análise da distribuição desta sequência de transposase foi realizada através das técnicas de PCR e Southern blot nos diferentes isolados, pertencentes aos biótipos C, L, e S, coletados de várias áreas geográficas. O perfil de distribuição da transposase sugere a presença de cópias polimórficas entre os diferentes isolados do biótipo C. O perfil de hibridização do DNA total de cada isolado foi utilizado para o cálculo de distância genética e agrupamento pelo método de UPGMA. Os isolados do biótipo C coletados no estado da Bahia apresentaram dois perfis de hibridização para a sequência de transposase. Os dois diferentes padrões apresentados na análise de Southern podem ser relacionados com a presença de dois genótipos diferentes na Bahia.

Transposase sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative transposase sequence was analyzed in the genome of the phytopathogenic fungus Moniliophthora perniciosa, the causal agent of witches' broom disease of cocoa. Sequence comparisons of the predicted transposase peptide indicate a close relationship with the transposases from the elements of the Tc1-Mariner superfamily. The analysis of the distribution of transposase sequence was done by means of PCR and Southern blot techniques in different isolates of the fungus belonging to C-, L-, and S-biotypes and collected from various geographical areas. The distribution profile of the putative transposase sequence suggests the presence of polymorphic copies among the isolates from C-biotypes. The total DNA hybridization profile of each isolate was used to calculate genetic distance and group by the UPGMA method. C-biotype isolates colleted from of the Bahia showed two hybridization profiles for the transposase sequence. Thus the two different fingerprinting profiles for transposase sequence reported here by Southern analysis could also be correlated to the presence of two different genotypes in Bahia, Brazil.

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

A análise de sequências de transcriptase reversa (RT) é uma etapa importante para descobrir a presença de elementos transponíveis e investigar o seu papel na geração de variabilidade genética em C. perniciosa. Seqüências putativas de TR foram analisadas no genoma do fitopatógeno C. perniciosa, o agente causal da doença vassoura-de-bruxa no cacau. Um fragmento de 394 pb foi amplificado a partir do DNA genômico de diferentes isolados de C. perniciosa, pertencentes aos biótipos C, L e S e a distintas áreas geográficas. A clivagem dos produtos de PCR com diferentes enzimas de restrição e sequenciamento de vários fragmentos de TR indicou a presença de diferentes seqüências mostrando eventos de transição G:C para A:T. A análise por hibridização revelou alto número de sinais sugerindo a presença de cópias de TR com diferentes perfis entre os isolados dos biótipos C, S e L. As comparações de seqüências dos peptídeos preditos indicam uma relação próxima com a proteína TR de retrotransposons-LTR da família gypsy.

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

As bactérias recombinantes Escherichia coli KO11 e Klebsiella oxytoca P2 fermentaram sacarose a etanol. Em meio mínimo com 2% ou 12% de sacarose, KO11 apresentou, respectivamente, 75% e 41% do rendimento máximo teórico (0,54g de etanol/g de sacarose). No caldo Luria-Bertani (LB) com até 8% de sacarose, KO11 apresentou rendimento de aproximadamente 94-96% e com 12% de sacarose, KO11 apresentou cerca de 69% de rendimento (44,5g de etanol/L). A porcentagem do rendimento máximo teórico obtida com P2 em meio mínimo com 2% de sacarose foi de 55% e com 12% de sacarose foi de 47%. Em LB com 2 ou 4% de sacarose, P2 apresentou 94-95% do rendimento máximo teórico, porém somente cerca de 73% com 8% de sacarose (31,4g de etanol/L) e 58% com 12% de sacarose (37,5 g/L). A produtividade volumétrica em LB contendo 2% de sacarose foi de 0,41 g/L/h para KO11 e de 1,1 g/L/h para P2, enquanto que em LB com 12% de sacarose, a produtividade foi 0,96 g/L/h (KO11) e 1,4 g/L/h (P2). Durante a fermentação do caldo de cana, E. coli KO11 e K. oxytoca P2 produziram, respectivamente, 39,4 g de etanol/L e 42,1 g/L quando suplementado com 0,5% de extrato de levedura, micronutrientes e tiamina. No caldo de cana suplementado com os reagentes do meio LB, KO11 apresentou forte inibição da fermentação alcoólica, produzindo apenas 23,0 g de etanol/L, enquanto que P2 produziu 44,2 g/L. A produção de etanol por KO11 e P2, no caldo de cana suplementado com a) 0,2% de sulfato de amônio foi, respectivamente: 25,3 e 30,2 g/L, b) com sulfato de amônio e micronutrientes: 24,9 e 31,6 g/L, c) com sulfato de amônio, micronutrientes e tiamina: 25,6 e 37,5 g/L. Durante a fermentação do melaço, E. coli KO11 apresentou baixa produção de etanol e alta produção de ácido láctico. K. oxytoca P2 produziu 25 g de etanol/L a partir de melaço diluído 10X em água, com ou sem adição de 0,5% de extrato de levedura e 27,8 g/L com reagentes do caldo LB após 96h. P2 produziu 24,5, 26,9, e 28,0 g de etanol/L em melaço diluído 10X em vinhoto, vinhoto com 0,5% de extrato de levedura e com os reagentes do caldo LB, respectivamente.

Escherichia coli KO11 and Klebsiella oxytoca P2 recombinants fermented sucrose to ethanol. In minimal medium with 2% or 12% added sucrose KO11 produced 75% and 41%, respectively, of the maximum theoretical yield (0.54g ethanol/g sucrose). In Luria-Bertani (LB) broth with up to 8% sucrose, KO11 presented a 94-96% yield and with 12% sucrose, KO11 presented about 69% yield (44.5g ethanol/L). P2 presented 55% of the theoretical maximum yield in minimal medium supplemented with 2% sucrose and 47% of the maximum in 12% sucrose. In LB broth with 2 or 4% sucrose, P2 presented 94-95% of the theoretical maximum yield, which fell to 73% with 8% added sucrose (31.4g ethanol/L) and 58% with 12% sucrose (37.5 g/L). Volumetric productivity in LB broth containing 2% sucrose was 0.41 g/L/h for KO11 and 1.1 g/L/h for P2, while in LB broth with 12% added sucrose, productivity was 0.96 g/L/h (KO11) and 1.4 g/L/h (P2). During fermentation of sugar cane juice by E. coli KO11 and K. oxytoca P2 produced 39.4 g/L and 42.1 g/L ethanol when supplemented with 0.5% yeast extract, micronutrients and thiamine. In sugar cane juice supplemented with LB broth ingredients, KO11 ethanol fermentation was inhibited with production of only 23.0 g ethanol/L, while P2 produced 44.2 g/L. Ethanol production by KO11 and P2, respectively, in sugarcane juice was a) 25.3 and 30.2 g/L with 0.2% ammonium sulfate, b) 24.9 and 31.6 g/L with ammonium sulfate and micronutrients, c) 25.6 and 37.5 g/L with ammonium sulfate, micronutrients and thiamine. During molasses fermentation E. coli KO11 presented low ethanol production and high lactic acid production. K. oxytoca P2 produced 25 g ethanol/L in molasses diluted 10-fold in water, with or without addition of 0.5% yeast extract, and 27.8 g/L with addition of LB broth ingredients after 96h. P2 produced 24.5, 26.9, and 28.0 g ethanol/L in molasses diluted 10-fold in vinasse, vinasse with 0.5% added yeast extract and with LB broth ingredients, respectively.

A variabilidade genética de 20 isolados de Fusarium oxysporum, nove não-patogênicos e 11 patogênicos ao feijoeiro (Phaseouls vulgaris), foi determinada com base na distribuição do elemento transponível impala. A presença de impala das subfamílias D e E foi determinada por experimentos de PCR, empregando oligonucleotídeos específicos para cada subfamília. Foi observada a presença de representantes das duas subfamílias na maioria dos isolados, sugerindo, portanto, que impala é um antigo componente do genoma de F. oxysporum f. sp. phaseoli. A hibridização do DNA total de cada isolado, clivado com a enzima EcoRI, com um fragmento do elemento impala da subfamíla E, mostrou uma variação nos padrões de bandas dos isolados não-patogênicos, indicando a possível atividade desses elementos. No entanto, no caso dos isolados patogênicos, foram observados padrões de bandas mais homogêneas e alguns isolados apresentaram o mesmo perfil de bandas, indicando que se trata de cópias de impala que, possivelmente, não são mais capazes de sofrer transposição. Estas cópias inativas são excelentes marcadores genéticos. Um dos isolados patogênicos, Fus4, não apresentou cópias endógenas de impala, o que torna esse isolado um candidato para experimentos de mutagênese insercional usando o vetor pNI160, que possui o elemento impala ativo interrompendo o gene niaD, que codifica a enzima nitrato redutase.

The genetic variability of 20 isolates of Fusarium oxysporum, nine nonpathogenic and 11 causing common bean (Phaseouls vulgaris) wilt, was analyzed on the basis of the distribution of the transposable element impala. The presence of transposable elements belonging to subfamilies D and E of impala was determined through polymerase chain reaction (PCR) using specific primers for each subfamily. The presence of members of the two subfamilies was observed in most of the isolates, suggesting that it is an old component in the F. oxysporum f. sp. phaseoli genoma. Hybridization of total DNA of each isolate, digested with EcoRI, with impala fragments of subfamily E produced a highly variable band pattern in the nonpathogenic isolates, indicating the possible activity of these elements. On the other hand, in pathogenic isolates, the band patterns were more homogeneous and some isolates showed very similar patterns, indicating that these impala copies have lost their capacity to transpose. These inactive copies are suitable as genetic markers. Among the pathogenic isolates, endogenous copies of impala were not detected in Fus4; therefore, this isolate could be used in experiments of insertional mutagenesis with the pNI160 plasmid, which harbors the active W impala element disrupting the niaD (nitrate reductase) gene.

Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp.), and Laccaria (L. bicolor and L. amethystea). Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

The pectinolytic system of Penicillium griseoroseum has been studied as a model to investigate aspects of gene organization in filamentous fungi. Here we show that the endopolygalacturonase-coding genes previously isolated exist as single copies in the fungus genome. DNA blot analysis revealed the presence of corresponding genes in other Penicillium species, although only one or two genes were found in opposition to the endoPG gene family reported for other filamentous fungi. The nucleotide and amino acid sequences of Penicillium PG genes of retrieved from data banks were compared for intron length and number, codon usage, and consensus sequences for translation initiation sites. The introns are conserved in the same position, although there was no conservation of their nucleotide sequences. Other sequence features resemble those seen in Aspergillus and Neurospora genes.