RESUMEN
Xylitol is transported by Streptococcus mutans via a constitutive phosphoenolpyruvate:fructose phosphotransferase system (PTS) composed of a IIABC protein. Spontaneous xylitol-resistant strains are depleted in constitutive fructose-PTS activity, exhibit additional phenotypes, and are associated with the caries-preventive properties of xylitol. Polymerase chain-reactions and chromosome walking were used to clone the fxp operon that codes for the constitutive fructose/xylitol-PTS. The operon contained three open reading frames: fxpA, which coded for a putative regulatory protein of the deoxyribose repressor (DeoR) family, fxpB, which coded for a 1-phosphofructokinase, and fxpC, which coded for a IIABC protein of the fructose-PTS family. Northern blot analysis revealed that these genes were co-transcribed into a 4.4-kb mRNA even in the absence of fructose. Inactivation of the fxpC gene conferred resistance to xylitol, confirming its function. The fxp operon is also present in the genomes of other xylitol-sensitive streptococci, which could explain their sensitivity to xylitol.
Asunto(s)
Proteínas Bacterianas/fisiología , Farmacorresistencia Bacteriana/genética , Genes Bacterianos , Proteínas de la Membrana/fisiología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Streptococcus mutans/enzimología , Streptococcus mutans/genética , Xilitol/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Paseo de Cromosoma , Proteínas Fimbrias , Silenciador del Gen , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Operón/fisiología , Reacción en Cadena de la Polimerasa , Transcripción Genética , Transformación BacterianaRESUMEN
The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria. Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species. In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon. In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria. Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins. Their synthesis is not coordinated and is influenced by environmental factors. The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl. Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/genética , Operón , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Animales , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.
Asunto(s)
Proteínas Bacterianas , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Streptococcus/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Manosa/metabolismo , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Monosacáridos/fisiología , Familia de Multigenes , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosforilación , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
An insertion sequence-like element, IS1139, was cloned and sequenced from Streptococcus salivarius ATCC 25975 chromosome. This insertion sequence-like element is 1168 bp long and is delimited by inverted repeats of 29 bp and by a duplicated sequence of 6 bp. This IS possesses an open reading frame that codes for a putative transposase of 339 amino acids which has, respectively, 94, 35, 33, and 30% amino-acid identity with the transposases of IS1161 from S. salivarius ATCC 25975, IS4351 from Bacteroides fragilis, IS30 from Escherichia coli, and IS1086 from Alcaligenes eutrophus. Sequence analysis revealed that these transposases may have evolved from a common ancestral gene. Southern hybridization of restriction endonuclease-digested genomic DNA from 21 strains of oral streptococci, using a probe specific to the transposase-encoding gene (tnpA), revealed that IS1139 is found in two strains of S. salivarius, ATCC 25975 and ATCC 13419, in eight and two copies, respectively.
Asunto(s)
Elementos Transponibles de ADN , Bacterias Gramnegativas/genética , Streptococcus/genética , Alcaligenes/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Bacteroides fragilis/genética , Secuencia de Bases , Clonación Molecular , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Familia de Multigenes , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta , Filogenia , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Streptococcus/clasificación , TransposasasRESUMEN
The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Toxinas Bacterianas/genética , Proteínas Portadoras/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Enterotoxinas/biosíntesis , Enterotoxinas/toxicidad , Escherichia coli/metabolismo , Técnicas In Vitro , Intestino Delgado/efectos de los fármacos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Plásmidos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
An indirect immunodot assay with rabbit antibodies raised against purified heat-stable enterotoxin type b (STb) and with a Western blotting (immunoblotting) detection system (ECL; Amersham International plc, Amersham, United Kingdom) was developed for the detection of STb toxin. Culture supernatants of 62 Escherichia coli isolates from pigs with diarrhea were blotted onto nitrocellulose and incubated with anti-STb serum. The chemiluminescence produced by the action of horseradish peroxidase with luminol and H2O2 was recorded by exposure of X-ray film. Over 90% correlation was observed between the rat or pig intestinal ligated loop assay and a radioactive DNA probe and the ECL immunodot assay for the detection of STb. In addition, using this new and sensitive technique, we could detect STb in the feces of a newborn pig inoculated with an STb-producing E. coli strain. Detection of STb-producing E. coli in pigs with diarrhea will be greatly facilitated by the use of this convenient and rapid diagnostic assay.
Asunto(s)
Toxinas Bacterianas/análisis , Enterotoxinas/análisis , Immunoblotting/métodos , Animales , Bioensayo , Diarrea/diagnóstico , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/diagnóstico , Proteínas de Escherichia coli , Estudios de Evaluación como Asunto , Heces/microbiología , Mediciones Luminiscentes , Ratas , PorcinosRESUMEN
A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E. coli strains. Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA). STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops. Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STb-positive strains. A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique. The non-radioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe. In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb. The PCR was a highly specific and practical technique for the detection of STb-positive strains. All E. coli strains tested containing the STb gene produced the STb toxin.
Asunto(s)
Toxinas Bacterianas/aislamiento & purificación , Enterotoxinas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Sondas de ADN , Diarrea/etiología , Digoxigenina , Escherichia coli/química , Escherichia coli/clasificación , Proteínas de Escherichia coli , Reacción en Cadena de la PolimerasaRESUMEN
A total of 1,226 Escherichia coli strains isolated from 1979 to 1989 from pigs with diarrhea were examined for serogroup and fimbrial antigen F4 (K88) production. Four main patterns of isolation of the various serogroups were observed, depending on the ages of the pigs from which isolates were obtained and the production of F4. In pattern I, serogroups O8:K"S16", O9:K35, O9/O101:K30, O9/O101:K103, O9 (group), O20:K101, and O64:K"V142" were predominant in pigs aged 0 to 6 days (41.9% of isolates) and were less frequent in pigs aged 7 to 27 days (24.6% of isolates) but were rarely found in pigs aged 28 to 60 days (4.0% of isolates). In pattern II, the F4-associated serogroups O8:K"4627", O157:K"V17", O149:K91, and O147:K89 were predominant in pigs aged 7 to 27 days (29.8% of isolates) and in pigs aged 28 to 60 days (35.0% of isolates). In pattern III, serogroups O8 (group), O115:K"V165", and O147:K89 were rarely isolated from pigs aged 0 to 6 days but were equally distributed in pigs aged 7 to 27 days (10.1% of isolates) and in pigs aged 28 to 60 days (10.9% of isolates). In pattern IV, serogroups O138:K81, O139:K82, O141:K85ac, O45:K"E65", and O26:K60 were most frequently isolated in pigs aged 28 to 60 days (19.3% isolates). Over the period from 1979 to 1989, the proportion of isolates belonging to serogroups of pattern II and the proportion of F4 isolates within the serogroup O157:K"V17" declined, whereas the proportion of isolates of serogroups O147:K89, O8:K"S16", and O9:K35 increased. For 228 isolates selected from the most important serogroups, good agreement was observed between the results of gene probes and immunofluorescence for the detection of fimbrial antigens F4 (K88), F5 (K99), F6 (987P), and F41 and between the results of gene probes and biological assays for the detection of heat-labile enterotoxin (LT) and heat-stable enterotoxins a and b (STa and STb). The STa gene was mostly associated with isolates of pattern I serogroups, which had the F5, F6, and F41 genes alone or in various combinations. The LT and/or STb genes, with the F4 gene, mostly were observed in isolates of pattern II serogroups. The STb gene alone was observed mostly in isolates of pattern III serogroups, although isolates were negative for all fimbrial antigen genes. Similarly, isolates of pattern IV serogroups were negative for all fimbrial antigen genes and rarely positive for the enterotoxin genes. However, verotoxin production was associated with isolates of serogroups O138:K81 and O139:K82. The most important pathotypes among enterotoxigenic isolates in this study were F4:LT:STb, F5:STa, STb, F5:F41:STa, F4:STb, F6, STa, and LT.
Asunto(s)
Antígenos Bacterianos/genética , Toxinas Bacterianas/genética , Diarrea/veterinaria , Enterotoxinas/genética , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Fimbrias , Genes Bacterianos , Enfermedades de los Porcinos/microbiología , Factores de Edad , Animales , Animales Recién Nacidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Sondas de ADN , Diarrea/diagnóstico , Diarrea/microbiología , Enterotoxinas/inmunología , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/diagnóstico , Fimbrias Bacterianas/inmunología , Técnica del Anticuerpo Fluorescente , Fenotipo , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/diagnóstico , VirulenciaRESUMEN
Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay. The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I). Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains. Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E. coli strains. The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.