Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter.

AIMS: To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST). METHODS AND RESULTS: The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract. CONCLUSIONS: The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.

First evidence of the presence of genomic islands in Escherichia coli P4, a mammary pathogen frequently used to induce experimental mastitis.

Mastitis pathogens belonging to Escherichia coli species are often considered as environmental opportunistic pathogens that invade the udder and are rapidly killed by the immune system of cows. However, several studies have reported that some of these strains are able to persist in the udder for prolonged periods or to adhere and invade mammary epithelial cells, suggesting that they might possess some specific properties or genes that could be involved in their capacity to provoke mastitis. The aim of this work was to search for such specific genes in the E. coli strain P4, which was isolated from a case of severe mastitis and is often used to induce experimental mastitis. We established that this strain belongs to phylogenetic group A of the E. coli species, and that its core genome is very similar to that of the commensal nonpathogenic strain E. coli K-12 MG1655. Seventeen transfer RNA loci, known to be frequently associated with genomic islands, were screened and an altered structure was detected for 7 of them. The partial characterization of 5 of these loci (asnT, leuX, pheV, serU, and thrW) and the complete characterization of 1 (argW) revealed the presence of genomic islands that differ from those already described in pathogenic or nonpathogenic E. coli strains.

Short communication: can the mammopathogenic Escherichia coli P4 strain have a direct role on the caseinolysis of milk observed during bovine mastitis?

During bacterial bovine mastitis, the quality of milk is altered because of caseinolysis. Endogenous potential actors in milk responsible for this caseinolysis have been well studied, unlike the exogenous bacterial ones. The aim of this study was to evaluate the direct role in caseinolysis of a mammopathogenic strain, Escherichia coli P4. Secretion of at least 4 extracellular bacterial caseinolytic enzymes was highlighted by zymography, in 3 different growth media, and at each bacterial growth state, suggesting that their expression was constitutive. Different experimental conditions to evaluate caseinolytic potential did not show any significant caseinolytic activity of E. coli P4 and of the 4 extracellular proteases detected, suggesting that the high caseinolysis observed during E. coli bovine mastitis does result from endogenous milk actors.

Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels.

alpha(S1)-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's alpha(S1)-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of alpha(S1)-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine alpha(S1)-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped.

Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon.

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine beta-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. beta-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that beta-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the beta-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10 degrees C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of beta-casein was achieved by mixing each phosphorylation isoform in its native state with the whole beta-casein fraction.

Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development.

In Streptomyces ambofaciens, genetic instability occurring during aerial mycelium development gives rise to white mutant papillae on colonies. Pig-pap mutants deriving from such papillae are unable to sporulate and devoid of the large genome rearrangement usually observed in the other Streptomyces mutants that genetic instability generated. Pig-pap mutants frequently harboured discrete mutations affecting the whiG gene. Furthermore, it has been established that the production of papillae dramatically increased when S. ambofaciens was grown under an amino acid limitation. In this work, we tested the implication of the stringent response, induced during an amino acid limitation, in the production of white papillae in Streptomyces coelicolor, a species which is phylogenetically close to S. ambofaciens. First, we showed that S. coelicolor produced mutant papillae and that this production was increased under an amino acid limitation. Secondly, we showed that the Pig-pap mutants generated both with and without amino acid limitation frequently exhibited mutations in whiH or whiG genes. Finally, we observed that a relA mutant of S. coelicolor, which was unable to elicit the stringent response under an amino acid limitation, was also unable to produce papillae. The restoration of the ability to elicit the stringent response also restored the papillae production. These papillae gave rise to Pig-pap mutants displaying the same characteristics as Pig-pap mutants spontaneously appearing on wild-type colonies. Altogether, these results show that whatever the underlying mechanism, the stringent response is involved in the production of white papillae in S. coelicolor.

Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity.

DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.

DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: evidence for developmental control.

In Streptomyces, a genomic instability results from frequent recombination events which occur at the ends of the linear chromosomal DNA. These events are believed to be responsible for the variability observed in these regions among Streptomyces species and strains. In order to identify functions able to control this type of genome plasticity, mutators as well as mutants produced at different stages of development have been characterized in S. ambofaciens. Their characterization suggests the existence of a relationship between genomic instability and colony development.

Identification and typing of Streptomyces strains: evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting.

The suitability of random amplified polymorphic DNA PCR for the detection of differences between Streptomyces species and strains was evaluated. For this purpose, a protocol of RAPD specific for Streptomyces DNA, i.e. suitable for DNA presenting a high G+C content, was developed using S. ambofaciens ATCC23877. Among the 30 primers tested, all containing 80% G+C, 17 gave a pattern with this strain. Six oligonucleotides were chosen to compare 12 strains belonging to six species of Streptomyces. These oligonucleotides were then used to determine whether these strains could be differentiated at the DNA level with this method. All fingerprints obtained with six primers differed from one species to another. We showed that the RAPD method could be used to reveal intraspecific and intraclonal polymorphisms. Thus, RAPD allows for the rapid, sensitive and specific detection of genetic diversity among species and strains of Streptomyces.

Amplification of a particular DNA sequence in Streptomyces ambofaciens RP181110 reversibly prevents spiramycin production.

Streptomyces ambofaciens RP181110 produces the macrolide antibiotic spiramycin. After treatment with ethidium bromide, 7 strains presenting an amplified sequence of DNA (ADS) were found in its progeny. These ADS were localized within the same amplifiable region of the RP181110 genome. It has been established that these amplified strains were non-producers (Spi-) and that the loss of one particular ADS was correlated with restoration of spiramycin production. Genome rearrangements such as deletions were detected on the same side of the amplifiable region in both amplified and deamplified strains.

Evolution of the linear chromosomal DNA in Streptomyces: is genomic variability developmentally modulated?

Genome rearrangements are responsible for the variability observed at the ends of the chromosome among Streptomyces species. The characterization of mutators, which are stimulated for genome plasticity, and of mutants produced at different stages of development support the idea that genome instability is developmentally modulated.

Generation of a genetic polymorphism in clonal populations of the bacterium Streptomyces ambofaciens: characterization of different mutator states.

In Streptomyces ambofaciens, colony pigmentation is an unstable character. Very unstable mutants selected from twelve wild type (WT) subclones of S. ambofaciens ATCC23877 were investigated. This research showed that the polymorphism in colony pigmentation had distinct features. The first aspect is the coexistence of four types of colonies: pigmented colonies (Pig+), pigment-defective colonies (Pigcol-), pigmented colonies harboring pigment-defective sectors (Pigsec+) or pigment-defective papillae (Pigpap+). The second feature was revealed by the study on Pigpap+ colonies. We showed that WT progeny after 14 days of growth consisted almost totally of Pigpap+ colonies. Pigpap+ colonies were also found to be genetically different from each other. Characterization of twelve colonies presenting more than 20 papillae (Hyperpap colonies) led to the isolation of twelve mutator strains which produced at high frequency Pigcol- and Hyperpap colonies. Each exhibited a specific mutator phenotype and were distinct from each other. Such strains constituted a part of the polymorphism observed in each of the WT progeny and also generated a high variability. Finally, we showed that pigment-defective papillae were mutants and constituted a new form of genetic instability.

Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: evidence for a high degree of heterogeneity of the wild type clones.

In Streptomyces ambofaciens a genetic instability generates a high degree of polymorphism consisting of four main phenotypes: pigmented colonies (Pig(+) qualified as WT phenotype), pigment-defective colonies, pigmented colonies with pigment-defective sector and pigmented colonies with pigment-defective papillae. Molecular analysis of Pig(col)(-) and Pig(sec)(-) (pigment-defective mutant derived from a colony and a sector, respectively) produced by genetic instability and isolated in five Pig(+) subclones progenies revealed a new aspect of polymorphism in S. ambofaciens ATCC23877. Frequencies of Pig(col)(-) and Pig(sec)(-) mutants deleted at the chromosome ends varied from one WT progeny to another. Two main types of deleted mutants were observed: deleted for one or both chromosomal extremities. The relative proportion of these two categories differed according to the WT progeny. These results argue for heterogeneity of the WT clones, i.e., Pig(+) colonies, originated from S. ambofaciens ATCC23877.

[Cloning of a nosiheptide-resistant gene from Streptomyces actuosus]. / Clonage d'un gène de résistance au nosiheptide de Streptomyces actuosus.

The cloning of the nosiheptide resistance gene (nos) from S. actuosus, which produces this antibiotic, in plasmid pS10147 was carried out in S. lividans. The nosipheptide resistant clones contained the plasmid pNS1. It deletion derivative plasmid, pNS2, confers nosiheptide resistance to both S. ambofaciens and S. lividans.

Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants.

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205(+) Spi-). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205(-) Spi+). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.

Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression.

Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse-labelled proteins separated by two-dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or overexpressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.

An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes.

Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0.93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.

Carcinoma gástrico tipo difuso em paciente de 16 anos / diffuse type gastric cancer in a 16 year-old man

Apresentamos um caso de um jovem de 16 anos de idade, com queixa de dor epigástrica e vômitos após as alimentaçöes. A gastroscopia evidenciou onde estava situad a lesäo ulcerada com 3,0cm de diâmetro. O diagnóstico histopatológico foi de carcinoma gástrico tipo difuso, com moderada quantidade de células em anel de sinete, PAS positivas. O paciente foi operado quatro semanas após o diagnóstico, com quadro de semi-obstruçäo. A peça apresentava invasäo tumoral no limite superior da resecçäo cirúrgica e em profundidade correspondendo a lesäo ulcerada, comprometendo a serosa. O paciente evoluiu satisfatoriamente bem após a cirurgia permanecendo com restriçäo dietética, voltando a apresentar quadro de semi-obstruçäo, vindo falecer quatro meses depois. A incidência no Ceará da neoplasia gástrica é apresentada. O objetivo principal é chamar atençäo para a possibilidade de diagnóstico desse tipo de neoplasia, em pacientes dessa faixa etária, principalmente em regiöes de média a alta incidência de neoplasia gástrica

A preliminary study of the suprarenal gland in canine visceral leishmaniasis of the state of Ceara, Brazil

Por examen histopatológico de muestras de 11 perros (canis familiaris) sometidos a necropsia y naturalmente infectados por leishmania donovani, fué posible demostrar intenso parasitismo de las suprarrenales, lo que indujo a los autores a un análisis pormenorizado de los aspectos morfológicos de esas glándulas alteradas. En las muestras de 7 perros fué encontrado parasitismo en el interior de macrófagos de la zona reticularis, en tanto que la zona fasciculata se encontraba parasitada en 1 y la región medular en 4. Infiltrados inflamatorios fueron observados en las regiones cortical y medular; contenían células plasmáticas, linfocitos y fagocitos mononucleares. En 3 casos, numerosos neutrófilos se encontraban diseminados entre las células inflamatorias; en 1 de esos casos, los neutrófilos eran predominantes y originaban microabscesos, lo cual es una observación que no ha sido relatada en la literatura disponible