Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk

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.

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao

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.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

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.

Ethanolic fermentation of sucrose, sugarcane juice and molasses by Escherichia coli strain ko11 and Klebsiella oxytoca strain P2

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 ...

Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum

Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

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

Structural organization of polygalacturonase-encoding genes from Penicillium griseoroseum

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