RESUMEN
Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.
Asunto(s)
Lacticaseibacillus rhamnosus , Probióticos , Animales , Ratones , Lacticaseibacillus , Lactobacillus , Células Caliciformes , Antiinflamatorios , Polisacáridos Bacterianos/farmacologíaRESUMEN
Gut dysbiosis has been strongly correlated with colorectal cancer (CRC) development and the use of probiotics to modulate this imbalance represents a potential and promising therapy to prevent and treat CRC. For this reason, the identification of novel probiotic strains from diverse origins has widely increased in recent years, including traditional fermented foods. In this work we describe a new strain previously isolated from pulque (a traditional Mexican beverage), Levilactobacillus brevis CNCM I-5321, which may represent an interesting probiotic candidate to prevent and treat cancer. Indeed, our results show that CNCM I-5321 displays significant and specific antiproliferative capacities in human intestinal cancer cell lines (HT-29, HTC-116 and Caco-2 cells), but not in normal cells (FH cells). In addition, CNCM I-5321 is able to induce: (1) a pro-inflammatory immune response through stimulation of interleukin (IL)-2, IL-6, IL-12 and IL-17 cytokines and (2) apoptosis via activation of caspase 8. On the other hand, a minimum inhibitory concentration (MIC) assay revealed phenotypic resistance of this strain to ampicillin and chloramphenicol. However, no known transferable determinants were found in the genome of CNCM I-5321, thus this probiotic candidate presents no risk of horizontal transfer to the intestinal bacterial population. Finally, the safety status of CNCM I-5321 was evaluated using an innovative model of chicken embryo chorioallantoic membrane (CAM) to assess undesirable and/or toxic effects. Overall, our results support that CNCM I-5321 strain is non-pathogenic and safe for potential use as an anti-cancer candidate in human and animal medicine.
Asunto(s)
Apoptosis , Proliferación Celular , Levilactobacillus brevis , Probióticos , Probióticos/farmacología , Humanos , Levilactobacillus brevis/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células CACO-2 , Citocinas/metabolismo , Pruebas de Sensibilidad Microbiana , Embrión de Pollo , Células HT29 , Pollos/microbiología , Neoplasias Colorrectales/tratamiento farmacológico , Células HCT116 , Línea Celular TumoralRESUMEN
BACKGROUND: Ribosomal RNA molecules are widely used for phylogenetic and in situ identification of bacteria. Nevertheless, their use to distinguish microorganisms within a species is often restricted by the high degree of sequence conservation and limited probe accessibility to the target in fluorescence in situ hybridization (FISH). To overcome these limitations, we examined the use of tmRNA for in situ identification. In E. coli, this stable 363 nucleotides long RNA is encoded by the ssrA gene, which is involved in the degradation of truncated proteins. RESULTS: Conserved sequences at the 5'- and 3'-ends of tmRNA genes were used to design universal primers that could amplify the internal part of ssrA from Gram-positive bacteria having low G+C content, i.e. genera Bacillus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Listeria, Streptococcus and Staphylococcus. Sequence analysis of tmRNAs showed that this molecule can be used for phylogenetic assignment of bacteria. Compared to 16S rRNA, the tmRNA nucleotide sequences of some bacteria, for example Listeria, display considerable divergence between species. Using E. coli as an example, we have shown that bacteria can be specifically visualized by FISH with tmRNA targeted probes. CONCLUSIONS: Features of tmRNA, including its presence in phylogenetically distant bacteria, conserved regions at gene extremities and a potential to serve as target for FISH, make this molecule a possible candidate for identification of bacteria.
Asunto(s)
Bacterias/genética , Bacterias/aislamiento & purificación , ARN Bacteriano/genética , Secuencia de Bases/genética , Evolución Molecular , Bacterias Grampositivas/genética , Bacterias Grampositivas/aislamiento & purificación , Hibridación Fluorescente in Situ , Lactococcus lactis/genética , Lactococcus lactis/aislamiento & purificación , Filogenia , Sondas ARN/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Análisis de Secuencia de ARN , Especificidad de la EspecieRESUMEN
In bacterial communities one bacterium can influence the growth of other members of the population. These interactions may be based on nutritional factors or may occur via bacterial signaling molecules that are released in the medium. We present an example, showing that in addition to the above means of interactions, muramidases, enzymes that specifically cleave peptidoglycan chains, can also mediate interactions between bacteria. Using fluorescent in situ hybridization we demonstrate that Lactococcus lactis muramidase AcmA can hydrolyze the cell wall of Streptococcus thermophilus, without affecting viability. This intercellular activity of the lactococcal muramidase results in chain disruption of streptococci in vivo. Our data lead us to propose that chains can give growth advantages to streptococci in aerobic conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lactococcus lactis/enzimología , Muramidasa/metabolismo , Streptococcus/crecimiento & desarrollo , Aerobiosis , Pared Celular/química , Pared Celular/metabolismo , Medios de Cultivo/química , Hibridación Fluorescente in Situ , Peptidoglicano/química , Peptidoglicano/metabolismo , Streptococcus/químicaRESUMEN
A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells.
Asunto(s)
Escherichia coli/aislamiento & purificación , Hibridación Fluorescente in Situ , Plásmidos , ARN/genética , Escherichia coli/genética , Sondas de OligonucleótidosRESUMEN
Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.
Asunto(s)
Pared Celular/fisiología , Bacterias Grampositivas/fisiología , Hibridación Fluorescente in Situ , Lactobacillus/fisiología , Lactococcus lactis/fisiología , Amidohidrolasas/metabolismo , Bacteriófagos/fisiología , Colorantes Fluorescentes/metabolismo , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/genética , Peroxidasa de Rábano Silvestre/metabolismo , Lactobacillus/genética , Lactobacillus/virología , Lactococcus lactis/genética , Lactococcus lactis/virología , Muramidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Sondas de Oligonucleótidos , Permeabilidad , ARN Ribosómico/genética , Sensibilidad y Especificidad , Activación ViralRESUMEN
The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.
Asunto(s)
Metilasas de Modificación del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Haemophilus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Metilasas de Modificación del ADN/metabolismo , ADN Bacteriano , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli , Vectores Genéticos , Haemophilus/genética , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.
Asunto(s)
Bacterias/enzimología , Enzimas de Restricción-Modificación del ADN/fisiología , Plásmidos/metabolismo , Bacillus subtilis/enzimología , Bacterias/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas de Restricción del ADN/metabolismo , Escherichia coli/enzimologíaRESUMEN
A genetic system enabling the in vivo selection of genes encoding the DNA-modifying enzymes was developed. A gene library is transformed into a strain harboring the restriction-modification (R-M) system which a recognition sequence is a subset of the target sequence of the DNA methyltransferase (MTase) to be cloned. If the residing MTase is temperature sensitive, the inability of transformants to grow at 42 degrees C provides a simple and convenient procedure for the isolation of new MTase-encoding genes. The feasibility of this procedure has been demonstrated by the isolation of the ppu21IM gene from a Pseudomonas putida RFL21 gene library.
Asunto(s)
Proteínas Bacterianas , Metilasas de Modificación del ADN/biosíntesis , Genes Bacterianos , Metiltransferasas/biosíntesis , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Clonación Molecular/métodos , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Biblioteca de Genes , Hidroxilamina , Hidroxilaminas , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutagénesis , Plásmidos , Mapeo RestrictivoRESUMEN
We report the organization of the HpaII restriction and modification (R-M) system from Haemophilus parainfluenzae (recognition sequence: 5'...CCGG...3'), the sequence of the gene coding for the HpaII restriction endonuclease, and the sequence of the upstream flanking DNA. The HpaII system comprises two genes, hpaIIM, coding for the methyltransferase (MTase; 358 amino acids (aa), 40.4 kDa: product, Cm5CGG), and hpaIIR, coding for the restriction endonuclease (ENase; 358 aa, 40.9 kDa: product, C'CGG). The genes are adjacent, they have the same orientation, and they occur in the order hpaIIM then hpaIIR. The ENase bears little as sequence similarity to the isoschizomeric R.BsuFI and R.MspI ENases. Upstream of, and partly overlapping hpaIIM is the coding sequence for a 141-aa protein that resembles the very-short-patch-repair endonuclease (Vsr) of Escherichia coli. Upstream of that is the coding sequence for a protein that resembles valyl-tRNA synthetase (ValS).
Asunto(s)
ADN-Citosina Metilasas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Haemophilus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , ADN-Citosina Metilasas/metabolismo , Desoxirribonucleasa HpaII , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Haemophilus/genética , Datos de Secuencia Molecular , Mutación , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Two strains carrying metE::Tn10 insertions (upstream of the udp gene) were used to isolate mutants of Escherichia coli overexpressing udp. These strains differ in their gene order; one contains an inversion between the rrnD and rrnE rRNA operons. Selection was based on the ability of overexpressed Udp to complement thymine auxotrophy. Chromosomal rearrangements that connect the udp gene and promoters of different rrn operons were obtained by this selection. Seven of 14 independent mutants selected in one of the initial strains contained similar inversions of the metE-rrnD segment of the chromosome (about 12% of its length). Another mutant contained traces of a more complicated event, inversion between rrnB and rrnG operons, which was followed by reinversion of the segment between metE and the hybrid rrnG/B operon. Similar inversions (udp-rrn) in a strain already carrying an rrnE-rrnD inversion flip the chromosomal segment between metE and rrnD/E in the opposite direction. In this case, inversions are also accompanied by duplications of the chromosomal region between the rrnA and hybrid udp-rrnD/E operons. PCR amplification with a set of oligonucleotides from the rrn, Tn5, and met genes was used for more detailed mapping. Amplified fragments of the rearranged chromosomes connecting rrnD sequences and insertion elements were sequenced, and inversion endpoints were established.
Asunto(s)
Inversión Cromosómica , Elementos Transponibles de ADN/fisiología , Escherichia coli/genética , ARN Bacteriano/genética , ARN Ribosómico/genética , Uridina Fosforilasa/genética , Secuencia de Bases , Desoxirribonucleasas de Localización Especificada Tipo II , Escherichia coli/enzimología , Expresión Génica/genética , Genes Bacterianos , Datos de Secuencia Molecular , Mutación/genética , Operón/genética , Reacción en Cadena de la Polimerasa , Uridina Fosforilasa/biosíntesisRESUMEN
The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.
Asunto(s)
Cisteína/biosíntesis , Escherichia coli/genética , Genes Bacterianos , Aerobiosis , Alelos , Secuencia de Aminoácidos , Amoníaco/metabolismo , Secuencia de Bases , Cisteína/genética , ADN Bacteriano/química , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/fisiología , Datos de Secuencia Molecular , Mutación , Fenotipo , Transcripción GenéticaRESUMEN
An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Mutagénesis Insercional , Alelos , Bacteriófago lambda , Genes Bacterianos , Plásmidos , Transducción GenéticaRESUMEN
An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.
Asunto(s)
Bacteriófago lambda/genética , Análisis Mutacional de ADN , Elementos Transponibles de ADN , ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Clonación Molecular/métodos , Plásmidos , Recombinación Genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Transduction of genetic markers phoA1::Tn5 and lacZ::Tn10 was used to show the high frequency of occurrence of duplication (5-8%) carrying the above mentioned genes without application of selection for the increase of their function. Genetic tests were used to show that duplicated segments vary in size and in some cases comprise the whole lacZ-phoA region of Escherichia coli K-12 chromosome.
Asunto(s)
Cromosomas Bacterianos , Escherichia coli/genética , Genes Bacterianos/genética , Familia de Multigenes/genética , Mapeo Cromosómico , Marcadores Genéticos/genética , Fenotipo , Transducción Genética/fisiologíaRESUMEN
Experiments on transformation of Escherichia coli K-12 cells by plasmids carrying RM systems with different recognition sites containing 5-methylcytosine have shown that the gene mcrB determines the function of restriction. The data obtained made it possible to believe that E. coli possesses no restriction system recognizing specifically cytosine methylated in position 4.
Asunto(s)
Citosina/análogos & derivados , Enzimas de Restricción del ADN/genética , Escherichia coli/genética , Plásmidos , Transformación GenéticaRESUMEN
Using the set of transducing lambda phages the gpp gene, responsible for pppGpp to ppGpp conversion, was localized between rep and trxA genes on 85 min of the Escherichia coli genetic map. Taking advantage of the Tn10 transposon inserted into the adjacent ilvY locus, we deleted the region of E. coli chromosome covering ilvC, rep and gpp genes. The metabolism of (p)ppGpp in the deletion-containing cells confirms that the product of the gpp gene, guanosine pentaphosphatase, is not the only enzyme, responsible for pppGpp degradation and ppGpp synthesis.
Asunto(s)
Deleción Cromosómica , Escherichia coli/genética , Genes Bacterianos , Nucleótidos de Guanina/biosíntesis , Guanosina Tetrafosfato/biosíntesis , Bacteriófago lambda/genética , Mapeo Cromosómico , Cromosomas Bacterianos , Escherichia coli/metabolismo , Guanosina Tetrafosfato/genética , Transducción GenéticaRESUMEN
We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.
Asunto(s)
Inversión Cromosómica , Cromosomas Bacterianos , Escherichia coli/genética , Escherichia coli/enzimología , Marcadores Genéticos , Operón , Transducción Genética , beta-Galactosidasa/genéticaRESUMEN
The Escherichia coli strain PLK1427 (Henson, Kopp, Kuempel, 1984) was used in this work. It carries a deletion of 60 thousand pairs of nucleotides in the chromosomal region 30-31 min and a partially deleted prophage lambda rev cI875Sam7 integrated into the 30 min region, instead of the rac prophage. Among the mutants of PLK1427 strain selected for resistance to 42 degrees C, deletions extending about 4 min and affecting the loci nirR (29.3 min), zdc235::Tn10 (32.3 min) and zdd230::Tn9 (33.3 min) were found. Although the deletion mutants obtained affect the region of replication termination (terC) of the chromosome, they have no alterations in the growth rate. It was demonstrated that some deletions may be transferred and are capable of recombination, giving the wild type in transductional experiments with the mutant phage T4.
Asunto(s)
Deleción Cromosómica , Cromosomas Bacterianos , Escherichia coli/genética , Genes Bacterianos , Replicón , Bacteriófago lambda/genética , Mapeo Cromosómico , Marcadores Genéticos , Mutación , Transducción GenéticaRESUMEN
Deletions in the argD, crp, cysG genes (73-74 min of the Escherichia coli genetic map) were obtained by heat induction of the phage lambda c1857 b221 rex::Tn5 integrated previously into the cysG gene by homologous recombination in the cysG::Tn5 mutant. Properties of the deletions obtained suggest the gene order: argD-crp-cysG.