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F L Macrina

Publications and source records attributed to F L Macrina.

At least 73 records · Page 4Linked to original sources
PubMed1983

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning.

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cmr, Tcr, and Emr determinants that are expressed in E. coli. Only the Emr determinant is expressed in S. sanguis. Both the Cmr and the Tcr of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras into S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345-353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.

Base Sequence↗
PubMed1983

Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives.

The streptococcal resistance plasmid pIP501 (30 kilobase pairs [kb]) encodes resistance to chloramphenicol (Cmr) and erythromycin (Emr) and is capable of conjugative transfer among numerous streptococcal species. By using a streptococcal host-vector recombinant DNA system, the Cmr and Emr determinants of pIP501 were localized to 6.3-kb HindIII and 2.1-kb HindIII-AvaI fragments, respectively. pIP501 was lost at a frequency of 22% in Streptococcus sanguis cells grown at 42 degrees C but was stable in cells grown at 37 degrees C (less than 1% frequency of loss). Sequences from a cryptic multicopy plasmid, pVA380-1, were substituted for the pIP501 Emr determinant in vitro, and the resulting recombinant plasmid, designated pVA797, was recovered in transformed S. sanguis cells. The replication of pVA797 was governed by the pVA380-1 sequences based on temperature-stable replication and incompatibility with pVA380-1-derived replicons. The self-ligation of partially cleaved HindIII pIP501 DNA fragments allowed the localization of a pIP501 region involved in autonomous plasmid replication. A small pIP501 derivative (pVA798) obtained from this experiment had a greatly increased copy number but was unstably inherited. Our data indicate that the sequences encoding the resistance determinants and some of the plasmid replication machinery are relatively clustered on the pIP501 molecule. The properties of pVA797 and pVA798 indicate that these molecules will enhance current streptococcal genetic systems from the standpoint of conjugative mobilization (pVA797) and gene amplification (pVA798).

Base Sequence↗
PubMed1982

A competence specific inducible protein promotes in vivo recombination in Streptococcus sanguis.

We describe the first example of a recombination-specific protein induced during the development of competence for transformation in Streptococcus sanguis. Elaborated in response to stimulation by competence-protein, the 51,000 Molecular Weight (MW) polypeptide is one of at least 10 new polypeptides transiently induced during the competence phase. Biochemical and genetic analyses of the parental, cipA+ (competence specific inducible polypeptide A), and mutant, cipA, strains have shown that the 51,000 MW polypeptide has two roles: its low level constitutive synthesis is required for repair of damage to DNA due to UV light and methylmethane sulfonate; its induced synthesis (3--6 x 10(4) copies/cell) during the competence phase is essential for promoting recombination between donor single-standard DNA and the recipient chromosome. Also, ccc plasmid donor DNA transformation, which occurs as a decreasing probability of the increasing donor plasmid MW, requires the inducible function specified by the 51,000 MW polypeptide. The MW independent low level transformation with ccc plasmids, the inheritance of plasmids by conjugation, and the stable maintenance of plasmids introduced by transformation and conjugation, respectively, are independent of the function specified by the 51,000 MW polypeptide.

Conjugation, Genetic↗
PubMed1982

A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis.

A plasmid that is able to replicate in both Escherichia coli and Streptococcus sanguis has been constructed by the in vitro joining of the pACYC184 (Cmr Tcr) and pVA749 (Emr) replicons. This plasmid, designated pVA838, is 9.2 kb in size and expresses Emr in both E. coli and S. sanguis. Its Cmr marker is expressed only in E. coli and may be inactivated by addition of DNA inserts at its internal EcoRI or PvuII sites. The pVA838 molecule also contains unique SalI, SphI, BamHI, NruI and XbaI cleavage sites suitable for molecular cloning. pVA838 may be amplified in E. coli but not in S. sanguis. We have used the pVA838 plasmid as a shuttle vector to clone streptococcal plasmid fragments in E. coli. Such chimeras isolated from E. coli were readily introduced into S. sanguis by transformation.

Base Sequence↗
PubMed1982

Novel antibiotic resistance transfer in Bacteroides.

Resistance to tetracycline and lincosamide antibiotics was transferred en bloc from a strain of Bacteroides fragilis (V503) to a plasmidless strain of Bacteroides uniformis (V528) during in vitro filter matings. Resistance transfer was detected at frequencies of 10(-5) to 10(-6) drug-resistant progeny per input donor cell and was dependent on cell-to-cell contact of donors and recipients. Transfer was insensitive to DNase and was not mediated by chloroform- or filter-sterilized donor broth cultures. A determinant for resistance to cefoxitin in V503 was not transferred in this system. V503 contained a 3.7 x 10(6)-dalton plasmid (pVA503). Drug-resistant progeny of V503 x V528 matings usually contained pVA503, but up to 20% of the total progeny of such crosses were plasmid free. Filter blot DNA hybridization studies (Southern method) confirmed that pVA503 was not integrated into the host chromosome of the plasmidless progeny. Drug-resistant progeny from V503 x V528 matings (with or without pVA503) conjugally transferred clindamycin resistance an tetracycline resistance to a suitable recipient strain. None of the drug resistance determinants of V503 were affected by treatment with standard plasmid curing regimens, and methods designed to detect very large plasmid molecules failed to suggest the involvement of extrachromosomal DNA in this resistance transfer system. The well-characterized Bacteroides R plasmid, pBF4 (conferring clindamycin resistance), was found to share hybridizing sequences with bulk cellular V503 DNA when examined by filter blot hybridization. Similarly sized sequences were found in drug-resistant progeny recovered from matings. Neither of the two pBF4 derivatives carrying deletions that abolished clindamycin resistance hybridized with V503 DNA.

Anti-Bacterial Agents↗
PubMed1982

Two independent conjugal transfer systems operating in Bacteroides fragilis V479-1.

Bacteroides fragilis V479-1 (also designated strain 92) has previously been shown to contain a conjugative plasmid, pBF4, that specifies resistance to clindamycin (Cc). A report of inducible tetracycline (Tc) resistance in this strain suggested that this phenotype was also plasmid associated (G. Privitera et al., Nature [London] 278:657-659, 1979) and prompted further investigation. Mating experiments with V469-1 and a Cc-sensitive derivative of V479-1, V598, showed that Tc resistance transfer occurred by a conjugation-like event which was insensitive to DNase, was not mediated by donor culture cell-free filtrates, and required cell-to-cell contact. Results from transfer experiments with V479-1 indicated that Tc and Cc resistance determinants were not linked and segregated independently in matings. Progeny recovered from matings with the V479-1 or V598 donor strain were able to transfer the Tc resistance marker in secondary crosses. Tc resistance transfer from V479-1 or V598 was greatly stimulated by pregrowth in the presence of Tc but not Cc. pBF4-mediated Cc resistance transfer was not affected by pregrowth in the presence of Cc or Tc. Filter blot DNA hybridization studies revealed that pBF4 sequences were not involved in either the Tc resistant phenotype or its associated conjugal transfer properties. The Tc resistance transfer element was not associated with pBF4 or any other extrachromosomal DNA element.

Bacteroides↗
PubMed1982

Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

Chimera↗
PubMed1982

Hybridization studies reveal homologies between pBF4 and pBFTM10, Two clindamycin-erythromycin resistance transfer plasmids of Bacteroides fragilis.

Two clindamycin-erythromycin resistance transfer factors of Bacteroides fragilis, pBF4 and pBFTM10, were analyzed for regions of DNA homology. Although the plasmids were derived from different clinical isolates of B. fragilis and have different sizes, they showed homology in the clindamycin-erythromycin resistance region; no homology could be detected outside of this region.

Bacteroides fragilis↗
PubMed1981

Transferable tetracycline resistance in Clostridium difficile.

The transfer of tetracycline resistance among strains of Clostridium difficile is described. Transfer occurred by a conjugation-like event that was insensitive to deoxyribonuclease, could not be mediated by donor culture filtrates or chloroform-treated donor cultures, and required cell-to-cell contact. Tetracycline-resistant progeny recovered from matings displayed a resistance phenotype identical to that of the donor in level of resistance, constitutive expression, and transmissibility. Although the original tetracycline-resistant donor contained 5 x 10(6)- and 22 x 10(6)-dalton plasmids, standard physical analyses of antibiotic-resistant transconjugants revealed no plasmid deoxyribonucleic acid molecules in common with the donor strain. Furthermore, tetracycline-susceptible derivatives of the original donor always possessed a plasmid complement identical to that of the resistant parental strain as determined by restriction endonuclease digestion analysis. The results indicate that the tetracycline resistance determinant(s) was not encoded by readily detectable plasmid deoxyribonucleic acid and may be chromosomally located.

Clostridium↗
PubMed1981

Physical characterization of Bacteroides fragilis R plasmid pBF4.

Bacteroides fragilis V479-1 has previously been shown to harbor a self-transmissible 27 X 10(6)-dalton plasmid (pBF4) which confers lincosamide-macrolide resistance. The present study has focused on the physical properties of pBF4. The plasmid was found to be present in 1 to 2 copies per chromosomal equivalent. pBF4 was genetically stable, although spontaneously occurring plasmidless segregants could be detected at low frequency (approximately 1%). This frequency was unaffected by growth of cells in ethidium bromide. About one-third of all spontaneously occurring macrolide-lincosamide-sensitive clones of strain V479-1 were found to contain pBF4 molecules that carried deletions. Ten independently obtained deletion derivatives of pBF4 from lincosamide-macrolide-sensitive strains were compared with the parental pBF4 by restriction endonuclease cleavage analysis. A restriction site map of pBF4 was constructed, and the location of the deletions was approximated. Self-annealed pBF4 molecules, examined by electron microscopy, revealed the presence of two pairs of inverted repeat (IR) sequences on the plasmid. IR-1 was about 400 base pairs in length, and its two component members were separated by an intervening sequence of about 15 kilobases. IR-2 was about 75 base pairs in length, and its component members were separated by 4.2 kilobases. Each of the deletions of pBF4 studied had a terminus at or near the same IR-2 sequence.

Bacteroides fragilis↗
PubMed1981

Transformation of Streptococcus sanguis with monomeric pVA736 plasmid deoxyribonucleic acid.

Monomeric and oligomeric forms of a 5.0 x 10(6)-dalton plasmid (conferring erythromycin resistance) were able to genetically transform naturally competent Streptococcus sanguis. Transformation with electrophoretically purified monomer was a second-order process, whereas transformation with a dye-buoyant density gradient-purified plasmid preparation followed one-hit kinetics.

Centrifugation, Density Gradient↗
PubMed1980

Sequential outbreaks of infection due to Klebsiella pneumoniae in a neonatal intensive care unit: implication of a conjugative R plasmid.

Sequential outbreaks of infection in a neonatal intensive care unit were due to multiple antibiotic-resistant strains of Klebsiella pneumoniae of different serotypes. In investigations of these outbreaks, the transfer of resistance to gentamicin, ampicillin, cephalothin, carbenicillin, and kanamycin from gentamicin-resistant organisms to standard laboratory recipients and between recipients was observed. Purified plasmid DNA, isolated from all multiple antibiotic-resistant strains, was analyzed by agarose gel electrophoresis, which revealed a common, large plasmid component with a molecular size of 71 megadaltons. Analysis of drug-resistant progeny suggested this plasmid encoded resistance to antibiotics and the information needed for its transmission. The identity of the plasmid from three different sources was established by the use of restriction-enzyme fingerprinting. The dissemination and persistence of this plasmid in environmental and fecal organisms, despite the disappearance of multiple antibiotic-resistant K. pneumoniae, provided a potential source for spread to other bacteria.

DNA↗
PubMed1980

Simple method for demonstrating small plasmid deoxyribonucleic acid molecules in oral streptococci.

A simple procedure for rapidly demonstrating small plasmids (less than 10 megadaltons) in oral streptococci is described. Logarithmic-phase, glycine-treated cells from 1.5-ml broth cultures were converted to osmotically fragile forms and lysed with sodium dodecyl sulfate. After the hydrodynamic shearing of host chromosomal deoxyribonucleic acid, such lysates were analyzed by low-voltage agarose gel electrophoresis. Small plasmids, migrating significantly faster than chromosomal deoxyribonucleic acid under such conditions, were readily visualized by ethidium bromide staining.

DNA, Bacterial↗
PubMed1980

Genetic transformation of Streptococcus sanguis (Challis) with cryptic plasmids from Streptococcus ferus.

By using the basic methodology initially published by Kretschmer et al. (J. Bacteriol. 124:225-231, 1975), we have been able to introduce phenotypically cryptic plasmids from Streptococcus ferus (formerly Streptococcus mutans subsp. ferus) into Streptococcus sanguis by genetic transformation. In this system, the entry of the cryptic plasmids is selected indirectly. This is effected with transforming deoxyribonucleic acid mixtures in which the cryptic plasmid deoxyribonucleic acid is present in an approximate 10-fold molar excess with respect to a plasmid (pVA1) known to confer erythromycin resistance. Under such conditions, 5 to 10% of the pVA1-containing erythromycin-resistant transformants were cotransformed with cryptic plasmid deoxyribonucleic acid. pVA1 may be selectively eliminated by growth of its S. sanguis host strain at 42 degrees C, enabling the construction of isogenic strains with and without S. ferus cryptic plasmids. Comparative physiological studies of such strains have failed to reveal any plasmid-conferred phenotypes in S. sanguis. With this procedure, we have been able to physically separate two small cryptic plasmids (2.4 x 10(6) and 2.8 x 10(6) daltons) of S. ferus. Although these plasmids were found naturally to exist in a single S. ferus host, they were able to replicate independently of one another in S. sanguis. Restriction enzyme fingerprinting indicated that these plasmids did not share a common ancestry.

Bacteriocins↗