RESUMEN
The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial "molecular mimicry" of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.
Asunto(s)
Genoma Bacteriano , Streptococcus pyogenes/genética , Bacteriófagos/aislamiento & purificación , Regulación de la Expresión Génica , Transferencia de Gen Horizontal , Datos de Secuencia Molecular , Filogenia , Transducción de Señal , Streptococcus pyogenes/patogenicidad , Streptococcus pyogenes/virología , Virulencia/genéticaRESUMEN
The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production.
Asunto(s)
Antígenos CD , NAD+ Nucleosidasa/genética , NAD+ Nucleosidasa/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/enzimología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/química , Aplysia/enzimología , Humanos , Glicoproteínas de Membrana , Datos de Secuencia Molecular , Mutación , NAD+ Nucleosidasa/química , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Streptococcus pyogenes/genéticaRESUMEN
In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307-311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766-1771, 1995). In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Proteínas de Unión al ADN , Escherichia coli/genética , Proteínas de la Membrana , Mutación , Novobiocina/farmacología , Inhibidores de Topoisomerasa II , Alanina-ARNt Ligasa/genética , Amdinocilina/farmacología , Arginino-ARNt Ligasa/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Microbiana/genética , Inhibidores Enzimáticos , Proteínas de Escherichia coli , Isoleucina-ARNt Ligasa/genética , Leucina-ARNt Ligasa/genética , Ligasas/metabolismo , ARN Polimerasa Sigma 54 , Factor sigma/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaAsunto(s)
Reordenamiento Génico , Genoma Bacteriano , Streptococcus pyogenes/genética , Secuencia de Bases , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Modelos Genéticos , Reacción en Cadena de la PolimerasaRESUMEN
cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos/genética , Proteínas de la Membrana , Novobiocina/farmacología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Escherichia coli/enzimología , Prueba de Complementación Genética , Genotipo , Mutación , Fosfatidiletanolaminas/análisis , Fosfatidilgliceroles/análisisRESUMEN
In a previous study, we demonstrated the existence of a gene locus, nov, which affects resistance of Escherichia coli K-12 to the gyrase inhibitor novobiocin and, to a lesser degree, coumermycin (J. Rakonjac, M. Milic, D. Ajdic, D. Santos, R. Ivanisevic, and D. J. Savic, Mol. Microbiol. 6:1547-1553, 1992). In the present study, sequencing of the nov gene locus revealed one open reading frame that encodes a protein of 54,574 Da, a value. found to be in correspondence with the size of the Nov protein identified in an in vitro translation system. We also located the 5' end of the nov transcript 8 bp downstream from a classical sigma70 promoter. Transcription of the gene is in the counterclockwise direction on the E. coli chromosome. Transposon mutagenesis of nov followed by complementation analyses and replacement of chromosomal alleles with mutated nov confirmed our previous assumption that the nov gene exists in two allelic forms and that the Novr gene is an active allele while the Novs gene is an inactive form. After comparing nucleotide sequences flanking the nov gene with existing data, we conclude that the gene order in this region of the E. coli K-12 map is att phi 80-open reading frame of unknown function-kch (potassium channel protein)-nov-opp. Finally, the possible identity of the nov gene with cls, the gene that codes for cardiolipin synthase, is also discussed.
Asunto(s)
Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Genes Bacterianos , Novobiocina/farmacología , Inhibidores de Topoisomerasa II , Transcripción Genética , Alelos , Secuencia de Bases , Análisis Mutacional de ADN , Elementos Transponibles de ADN , Escherichia coli/genética , Prueba de Complementación Genética , Datos de Secuencia MolecularRESUMEN
We report on a significant difference between the published nucleotide sequence of the Escherichia coli K12 histidine operon and our sequencing results repeatedly obtained from a number of different E. coli K12 strains. The discrepancies include 39 base-pair changes and one addition located predominantly in the proximal portion of the operon. Our data also suggest that neutral and near-neutral mutations do not accumulate to a significant extent in the histidine operon of E. coli strains harbouring strong mutator alleles.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos/genética , Histidina/genética , Operón/genética , Secuencia de Bases , Mutación/genética , Salmonella typhimurium/genética , Análisis de Secuencia de ADNRESUMEN
We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos/genética , Novobiocina/farmacología , Alelos , Aminocumarinas , Transporte Biológico/genética , Permeabilidad de la Membrana Celular/genética , Mapeo Cromosómico , Cumarinas/farmacología , ADN Bacteriano/genética , ADN Superhelicoidal/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Genes Bacterianos/fisiología , Genes Supresores/fisiología , Mutación , Novobiocina/metabolismoRESUMEN
Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Novr) mutants: 10% of Novr mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.
Asunto(s)
Acetiltransferasas , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Novobiocina , Acriflavina/farmacología , Aminocumarinas , Supervivencia Celular/efectos de los fármacos , Cumarinas/farmacología , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Proteínas de Escherichia coli , Mutación , Rifampin/farmacología , Serina O-AcetiltransferasaRESUMEN
This paper presents the first detailed structural analysis of termini of an inversion mediated by recombination between Escherichia coli native IS elements. The complete nucleotide sequence of the inversion termini in the lactose region of Escherichia coli K-12 confirms our previous suggestion that the inversion occurred by homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements (D. J. Savic, J. Bacteriol. 140:311-319, 1979; D. J. Savic, S. Romac, and S. D. Ehrlich, J. Bacteriol. 155:943-946, 1983). The data show a slight structural divergence of alpha 3 beta 3 and beta 5 alpha 5 elements, but they do not reveal new sequences within recombined IS3 elements that could influence the expression of nearby genes.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Secuencia de Bases , Clonación Molecular , Genes Bacterianos , Variación Genética , Vectores Genéticos , Genotipo , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150,000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5'-GCTGGCTGGCTGGCTG-3'. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong mutator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5'-GTGG-3' sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.
Asunto(s)
ADN Polimerasa I/genética , Escherichia coli/genética , Histidina/genética , Mutación , Secuencia de Bases , ADN Polimerasa I/metabolismo , ADN Bacteriano/genética , Modelos Genéticos , Datos de Secuencia Molecular , Operón , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Escherichia coli possesses three well-established DNA polymerases, I, II, and III. DNA polymerase I (Pol I) is the main repair polymerase in E. coli and also has a minor but important role in chromosomal replication. A major advantage of Pol I as an experimental system is its simplicity; unlike other replication enzymes, it is active as a single subunit. To a large extent, mutagenesis appears to be the result of (dis)functions of the DNA replication machinery. It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events.
Asunto(s)
ADN Polimerasa I/fisiología , Escherichia coli/enzimología , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Escherichia coli/genética , Mutagénesis/fisiología , Recombinación Genética/fisiología , Respuesta SOS en Genética/fisiologíaRESUMEN
In this work, the previously described inversion in the lactose region of the Escherichia coli K-12 chromosome (D. J. Savic, J. Bacteriol. 140:311-319, 1979) is analyzed in greater detail. The results presented indicate that the inversion most likely occurred by a homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements.
Asunto(s)
Inversión Cromosómica , Cromosomas Bacterianos , Escherichia coli/genética , Operón Lac , Mapeo Cromosómico , Enzimas de Restricción del ADN , ADN Bacteriano/análisisRESUMEN
We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Genes Bacterianos , Histidina/biosíntesis , Mutación , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , OperónRESUMEN
A spontaneous mutant of Escherichia coli K-12, strain SY99, with an inversion in the lactose region was isolated and partially characterized. The inversion was detected due to inverse chromosomal conjugational transfer after introduction of an F42 (F'lac) episome. The termini of the inversion are between proAB and lac on one side and lac and proC on the other. The inverse conjugational transfer in SY99 did not appear to be absolute but was always accompanied by a residual "normal" counterclockwise mobilization. This residual transfer was further shown to be caused by the intrinsic instability of this region (at least in the line W3110). The possible involvement of IS3 elements flanking the lactose operon is discussed.
Asunto(s)
Cromosomas Bacterianos/fisiología , Escherichia coli/genética , Operón Lac , Recombinación Genética , Conjugación Genética , Factor F , Marcadores GenéticosRESUMEN
An unlinked regulatory mutation hisT1504, causes an approximate 11-fold derepression of the histidine (his) operon and a linked constitutive mutation hisO1242 causes an approximate 15-fold derepression. In this study we demonstrate that hisT1504 provokes a significant increase in the UV-induced reversion frequency of his ochre and frameshift mutations. Analysis of revertants derived from frameshift mutants show that this increment in derepressed strains compared to the repressed strains is due to better growth of suppressed revertants by weak frameshift suppressors. The frequency of revertants suppressed by strong frameshift suppressors appears to be the same in repressed and derepressed strains. In contrast, intragenic revertants appear at two-fold decreased frequency in derepressed strains carrying either of the histidine constitutive mutations, hisT1504 or hisO1242. A possible competition is indicated between frequently transcribing RNA polymerase and error-promoting recombinational repair within the histidine operon.