RESUMEN

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Asunto(s)

Enterobacteriaceae/genética , Plásmidos/genética , beta-Lactamasas/genética , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Conjugación Genética/genética , Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/enzimología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Orden Génico , Genes Bacterianos/genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Plásmidos/química , Polonia , Análisis de Secuencia de ADN , Resistencia betalactámica/genética

RESUMEN

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

Asunto(s)

Klebsiella pneumoniae/genética , Plásmidos/genética , Resistencia betalactámica/genética , beta-Lactamasas/biosíntesis , ADN Bacteriano/análisis , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genética

RESUMEN

The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.

Asunto(s)

Plásmidos/metabolismo , Plásmidos/fisiología , Bacteriófagos/química , Modelos Genéticos , Oligonucleótidos Antisentido/química , Operón , Plásmidos/química , Recombinación Genética

RESUMEN

Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded omega product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), delta (Pdelta), and omega (Pomega) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of omega protein to the repeats on the Pcop1, Pcop2, Pdelta, and Pomega promoters represses transcription initiation by a mechanism that does not exclude sigma(A)RNAP from the promoters. These results indicate that omega protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

Asunto(s)

Replicación del ADN , Regulación Bacteriana de la Expresión Génica , Plásmidos/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica

RESUMEN

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.

Asunto(s)

Bacillus/enzimología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN-Citosina Metilasas/química , ADN-Citosina Metilasas/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus/genética , Secuencia de Bases , Sitios de Unión , Secuencia Conservada/genética , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN-Citosina Metilasas/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Escherichia coli/genética , Células Eucariotas/enzimología , Evolución Molecular , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia

RESUMEN

The hemolysin (HlyA) secretion system was used to achieve the sec-independent secretion of streptokinase (Skc) originating from Streptococcus equisimilis into the medium by Escherichia coli cells. The in-frame fusions of the skc gene, either possessing or lacking a region encoding the signal peptide (SP) with the 3'-end of the hlyA gene of various lengths were analysed. All hybrids retained Skc activity. Hybrid proteins devoided of the N-terminal SP, regardless of length of the hlyA secretion signal (62 vs. 194 amino acids), were secreted into the medium by the E. coli HlyA transporter at similar levels. Considerable amounts of hybrid proteins were still, however, associated with E. coli cells, mainly in the degraded form.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estreptoquinasa/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/metabolismo , Proteínas Portadoras/biosíntesis , Electroforesis en Gel de Poliacrilamida , Fibrinolisina/metabolismo , Genes Bacterianos , Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/aislamiento & purificación , Plasminógeno/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Streptococcus/enzimología , Estreptoquinasa/biosíntesis , Estreptoquinasa/aislamiento & purificación , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/aislamiento & purificación , beta-Galactosidasa/metabolismo

RESUMEN

The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR). (i) It fully complements the fol "null" mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes. (iii) Overexpression of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity. In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.

Asunto(s)

Genes Bacterianos , Lactococcus lactis/enzimología , Lactococcus lactis/genética , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Expresión Génica , Lactococcus lactis/efectos de los fármacos , Datos de Secuencia Molecular , Fenotipo , Homología de Secuencia de Aminoácido , Transcripción Genética , Trimetoprim/farmacología

RESUMEN

The gene organization of the broad-host-range low-copy-number pSM19035-derived plasmid pDB101 is presented. Analysis of the 19,202-bp sequence revealed thirteen different open reading frames (orfs). Nine of these orfs (repS-orf-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1 have been previously identified [Ceglowski et al., Gene 136 (1993) 1-12]. The extraordinarily long inverted repeated sequence, which includes orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta, comprises 76% of the pDB101 molecule. The gene order in pDB101 is repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1-orf zeta-orf epsilon-orf delta-orf gamma-orf beta-orf alpha-orf eta-orf theta-orf1-copS. The organization of genes of the orf eta-orf gamma region resembles the organization of genes in the orfA-orfI region of pAM beta 1. Except for Orf1, bands of radioactive proteins corresponding to the molecular mass of the deduced reading frames (26.7, 14.3 and 10.3 kDa) were detected using the T7 promoter-expression system. The orf1 encoded a product (deduced molecular mass 28.3 kDa) which shows anomalous electrophoretical mobility corresponding to 60 kDa. The copS- and orf1-encoded proteins share homology to plasmid copy number control systems and Gram+ cocci surface proteins, respectively. The orf eta and orf theta encode proteins with unknown activity.

Asunto(s)

Genes Bacterianos , Plásmidos/genética , Streptococcus pyogenes/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Péptidos/genética , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido

RESUMEN

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (orf) [alpha and beta]. Inactivation or deletion of orf beta results in SegA- plasmids. Better than random segregation requires an active segB region. The segB region contains two orfs (orf epsilon and orf zeta). Inactivation of either of the orfs does not lead to an increase in cell death, but orf zeta- plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.

Asunto(s)

Bacillus subtilis/genética , Replicación del ADN , Genes Bacterianos , Plásmidos , Streptococcus pyogenes/genética , Análisis Mutacional de ADN , ADN Bacteriano/genética , Sistemas de Lectura Abierta , Replicón , Mapeo Restrictivo , Eliminación de Secuencia , Especificidad de la Especie

RESUMEN

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Bacillus subtilis/genética , Plásmidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta , Homología de Secuencia de Aminoácido , Transcripción Genética , Transposasas

RESUMEN

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.

Asunto(s)

Bacillus subtilis/genética , Plásmidos , Replicón , Secuencia de Bases , Replicación del ADN , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , Datos de Secuencia Molecular

RESUMEN

A partial library of BclI-generated chromosomal DNA fragments from Streptococcus equisimilis H64A (Lancefield Group C) was constructed in Escherichia coli. Clones displaying either streptokinase or deoxyribonuclease (streptodornase; SDC) activities were isolated. The gene (sdc) expressing the SDC activity was allocated on the 1.1-kb AccI DNA subfragment. Sequence analysis of this DNA fragment revealed the presence of one open reading frame, which could encode a protein of 36.8 kDa. The N-terminal portion of the deduced protein exhibited features characteristic of prokaryotic signal peptides. The sdc gene was expressed in E. coli, Bacillus subtilis and Lactococcus lactis. As observed for S. equisimilis, in the heterologous Gram + hosts, at least part of the SDC protein was secreted into the medium.

Asunto(s)

Bacillus subtilis/genética , Desoxirribonucleasas/genética , Escherichia coli/genética , Lactococcus lactis/genética , Streptococcus/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Desoxirribonucleasas/biosíntesis , Desoxirribonucleasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Lactococcus lactis/enzimología , Datos de Secuencia Molecular , Mapeo Restrictivo , Streptococcus/enzimología , Estreptoquinasa/biosíntesis , Estreptoquinasa/metabolismo

RESUMEN

A recE mutant (recE6) of Bacillus subtilis was constructed by insertion of a selectable marker into the recE coding region. The insertional inactivation of the recE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. The recE6 allele was then introduced into a set of DNA repair-deficient strains of B. subtilis. The removal of DNA damage by the recF, addA addB, recH, recL and recP gene products is strictly dependent on an active recE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in the uvrA42 recE6 and polA5 recE6 strains suggests that such lethal lesions may be removed either by the recE-dependent or by the recE-independent pathway.

Asunto(s)

Bacillus subtilis/genética , Reparación del ADN , ADN Bacteriano/genética , Proteínas de Escherichia coli , Exodesoxirribonucleasas/genética , 4-Nitroquinolina-1-Óxido/farmacología , Alelos , Clonación Molecular , Daño del ADN , Elementos Transponibles de ADN , Exones , Genes Bacterianos , Marcadores Genéticos , Mutación , Recombinación Genética , Transducción Genética , Transfección , Transformación Bacteriana

RESUMEN

The mechanism of bactericidal activity of lactostrepcin 5 (Las 5), a bacteriocin produced by Streptococcus cremoris 202, was investigated. Las 5 did not kill protoplasts of sensitive cells, and its activity was decreased about 10-fold after pretreatment of the cells with trypsin, suggesting the involvement of the cell wall in the activity of this bacteriocin. In susceptible cells, the bacteriocin slowed down and then stopped synthesis of DNA, RNA, and protein, although this did not appear to be the primary effect of Las 5 action. Las 5 also inhibited uridine transport in susceptible cells and induced leakage of K+ ions and ATP. Survival of cells treated with Las 5 in phosphate buffer was higher in the presence of K+, CA2+, or Mg2+ ions.

Asunto(s)

Bacteriocinas/toxicidad , Streptococcus/fisiología , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/biosíntesis , Cationes/farmacología , Pared Celular/efectos de los fármacos , ADN Bacteriano/biosíntesis , Concentración de Iones de Hidrógeno , Lactococcus lactis/efectos de los fármacos , ARN Bacteriano/biosíntesis , Streptococcus/efectos de los fármacos

RESUMEN

Hybrid plasmids were constructed in which the transcription of regions of inserted DNA was defined. Cells containing these plasmids were transformed with monomeric forms of a different hybrid plasmid, which contained, however, the same inserted DNA as the resident plasmid. The transformation frequencies observed indicated that transcription of homologous DNA in resident plasmids and also tertiary DNA structure interfered with transformation.

Asunto(s)

Bacillus subtilis/genética , Transformación Genética , ADN Bacteriano/genética , Plásmidos

RESUMEN

A new Streptococcus sanguis strain Wicky endonuclease was isolated, purified and partially characterized. This nuclease acts preferentially on thermally denatured DNA, is not inhibited by RNA and is activated 3-5 times by trypsin. This activation is accompanied by the reduction of molecular weight of the enzyme. These features distinguish the new S, sanguis nuclease from the 3 previously described S. sanguis endonucleases. With covalently closed circular plasmid DNA, the enzyme causes first the appearance of a single stranded nick, then the second nick on the opposite DNA strand, resulting in plasmid DNA linearization. This nuclease most likely is located at the cell surface. The possible relationship of the described nuclease with ability of S. sanguis cells to take up DNA in genetic transformation is discussed.

Asunto(s)

Endonucleasas/aislamiento & purificación , Streptococcus sanguis/enzimología , Membrana Celular/enzimología , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Peso Molecular , Plásmidos , Streptococcus sanguis/genética , Streptococcus sanguis/ultraestructura , Transformación Genética , Tripsina/farmacología

RESUMEN

Two HindIII generated DNA fragments of 3.0 and 2.3 Kb derived from rRNA genes of B. subtilis were cloned in E. coli with pBR322. The 3.0 Kb fragment could be subcloned in B. subtilis using pC194. However, only the multimeric, but not the monomeric derivatives of this hybrid plasmid were active in transformation of B. subtilis cells. The 2.3 Kb fragment could neither be subcloned in pC194 nor in pPL603, using both cell and protoplast transformation. We attribute the nonclonability of the 2.3 Kb fragment in B. subtilis to the presence of strong promoter activity in this fragment. Direct proof for the presence of a strong promoter, which is apparently responsible for the transcription of the rRNA gene, was obtained in experiments with B. subtilis and E. coli promoter search plasmids.

Asunto(s)

Bacillus subtilis/genética , Plásmidos , ARN Ribosómico/genética , Transformación Genética , Clonación Molecular , ADN/genética , ADN Bacteriano/genética , ADN Ribosómico , Escherichia coli/genética , Genes Bacterianos , Operón

RESUMEN

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.

Asunto(s)

Bacillus subtilis/genética , Escherichia coli/genética , Genes Bacterianos , Factores R/efectos de los fármacos , Antibacterianos/farmacología , Clonación Molecular , Hibridación Genética , Modelos Genéticos

RESUMEN

In a previous report we demonstrated the presence of a factor binding deoxyribonucleic acid (DNA) in vitro (BF) in cell leakage fluids from transformable Streptococcus sanguis strains Wicky, Challis, and Blackburn. BF originating from strain Wicky was purified to homogeneity, and its properties are described. In this work, it was found that BF occurs at the surface of Wicky cells in two forms, loosely bound (LB-BF) and strongly bound to the cell envelope. It was demonstrated that LB-BF formed fast-sedimenting complexes with exogenous DNA at the surface of Wicky cells. About 10-fold-more DNA became associated as a fast-sedimenting complex in competent than in incompetent cells. Thus, LB-BF is a cell receptor for exogenous DNA. However, the comparison of the effects of some agents on the transformation yield and the formation of LB-BF-DNA complexes, showed that the influence of these agents on both observed phenomena is not parallel and may be even opposite. These results are interpreted to mean that the LB-BF-DNA complexes do not take part in transformation. The problem of participation of BF strongly bound with the cell membrane fraction remains to be elucidated.

Asunto(s)

Proteínas Portadoras/genética , ADN/genética , Streptococcus sanguis/genética , Transformación Bacteriana , Proteínas Portadoras/aislamiento & purificación , Membrana Celular , Centrifugación por Gradiente de Densidad , ADN/aislamiento & purificación , ADN Bacteriano/genética , Proteínas de Unión al ADN

RESUMEN

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S(1) action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.

Asunto(s)

Proteínas Bacterianas/aislamiento & purificación , ADN Helicasas/aislamiento & purificación , Streptococcus sanguis/análisis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , ADN Helicasas/análisis , ADN Helicasas/metabolismo , ADN Bacteriano/metabolismo , Desoxirribonucleasas/farmacología , Hidrolasas/farmacología , Punto Isoeléctrico , Peso Molecular , Streptococcus sanguis/genética , Transformación Bacteriana