Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.

Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae.

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

A new set of bioinformatics tools for genome projects.

A new tool called System for Automated Bacterial Integrated Annotation--SABIA (SABIA being a very well-known bird in Brazil) was developed for the assembly and annotation of bacterial genomes. This system performs automatic tasks of assembly analysis, ORFs identification/analysis, and extragenic region analyses. Genome assembly and contig automatic annotation data are also available in the same working environment. The system integrates several public domains and newly developed software programs capable of dealing with several types of databases, and it is portable to other operational systems. These programs interact with most of the well-known biological database/softwares, such as Glimmer, Genemark, the BLAST family programs, InterPro, COG, Kegg, PSORT, GO, tRNAScan and RBSFinder, and can also be used to identify metabolic pathways.

A System for Automated Bacterial (genome) Integrated Annotation--SABIA.

UNLABELLED: A web-based software suite, SABIA (System for Automated Bacterial Integrated Annotation), is described that provides a comprehensive computational support for the assembly and annotation of whole bacterial genomes from the data derived from sequencing projects. AVAILABILITY: Both SABIA and supplementary materials are available at http://www.sabia.lncc.br

Carlos Chagas Filho (1910) / Carlos Chagas Filho (1910)

Introduz algumas notas biográficas sobre Carlos Chagas Filho e apresenta a entrevista por ele concedida a Darcy F. de Almeida, que focaliza sua atuaçäo no Instituto de Biofísica e sua passagem por Manguinhos.(MAM)

Peptides 14VIDLL18 and 96FEAAAL101 defined as epitopes of antibodies raised against amino acid sequences of enterotoxigenic Escherichia coli colonization factor antigen I fused to Salmonella flagellin.

Antibodies raised against four hybrid Salmonella flagellins carrying amino acid sequences derived from the fimbrial subunit of the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC), i.e. hybrid flagellins Fla I (aa 1-15), Fla II (aa 11-25), Fla III (aa 32-45) and Fla IV (aa 88-102), were not able to inhibit the in vitro binding of CFA/I-expressing ETEC bacteria to enterocyte-like Caco-2 cells. However, one of the hybrid flagellins (Fla II) was recognized by a previously described anti-CFA/I subunit mAb (S-CFA/I 17:8) which was able to block adhesion of CFA/I-expressing bacteria to Caco-2 cells and to bind to the amino acid sequences 15IDLLQ19 of the CFA/I fimbrial subunit. Pepscan analysis of antibodies raised against the hybrid flagellins Fla II and Fla IV showed that they were specific for the sequences 14VIDLL18 and 96FEAAAL101, respectively, of the CFA/I fimbrial subunit. Thus, the discrepancy in the abilities of the anti-Fla II serum and the mAb S-CFA/I 17:8 to block binding might be ascribed to their slightly different fine specificity for epitopes.

Penicillin-binding proteins of pathogenic Yersinia species

As PBPs de Yersinia pestis, Y. enterocolitica e Y. pseudotuberculosis crescidas a 28ºC ou a 37ºC foram detectadas após marcaçäo com [3H]-benzilpenicilina e fluorografia dos géis de poliacrilamida. Cada amostra apresentou um perfil único de PBPs composto por 3 a 6 proteínas com peso molecular variando entre 120.000 e 43.000. Incubaçäo a 37ºC resultou em mudanças significativas nos perfis de PBPs das 3 espécies estudadas. As possíveis implicaçöes destes resultados na açäo dos antibióticos ß-lactâmicos e na fisiologia destas bactérias

Carlos Chagas Filho (1910) / Carlos Chagas Filho (1910)

Introduz algumas notas biográficas sobre Carlos Chagas Filho e apresenta a entrevista por ele concedida a Darcy F. de Almeida, que focaliza sua atuaçäo no Instituto de Biofísica e sua passagem por Manguinhos.(MAM)

Site-directed mutagenesis of the foot-and-mouth disease virus RNA-polymerase gene

O gene da RNA-polimerase do vírus da febre aftosa foi mutagenizado dentro do seu sítio de atividade. Utilizando o cDNA da cepa viral CS8 C1 - Santa Pau, o gene foi digerido com Pst I gerando um fragmento contendo a seqüência crítica de mutagênese. Este fragmento foi subclonado em M13mp8 e quatro mutaçöes foram geradas in vitro através do método de mutagênese sítio-dirigida por oligonucleotídeo. O gene da polimerase foi entäo reconstruído e subclonado em pUC19. Estes mutantes seräo usados em estudos de estrutura e atividade da enzima e no desenvolvimento da técnica de "imunizaçäo intracelular" em células eurcariontes