Search PubMed⌕ Search

Biomedical subjects

W R Guild

Publications and source records attributed to W R Guild.

At least 19 recordsLinked to original sources
PubMed1988

Two genes for chloramphenicol resistance common to staphylococci and streptococci.

Southern blot hybridization and pneumococcal transformation were used to study the epidemiology at a molecular level of the genes for chloramphenicol resistance (cat) in streptococci and staphylococci. The cat gene of staphylococcal plasmid pC194 showed homology to the cat genes of the chromosomal elements of 5 different strains of Streptococcus pneumoniae and of Streptococcus agalactiae B109. DNA sequence homology was also detected between the cat gene of staphylococcal plasmid pC221 and the cat gene of broad host range conjugative plasmid pIP501, originally isolated from S. agalactiae. Two different cat genes appear to be present in clinical isolates of both streptococci and staphylococci.

Chloramphenicol Resistance↗
PubMed1987

Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA).

PspA is a cell surface protein of Streptococcus pneumoniae that is present on a number of clinical isolates as well as the nonencapsulated laboratory strain Rx1. In a previous report we have shown that mAbs directed against PspA can protect mice from at least some of the pneumococcal strains bearing this protein. In our present report we have produced insertional inactivation mutants that lack PspA and have used these mutants to demonstrate that PspA can play a role in pneumococcal virulence and that anti-PspA immunity can lead to protection against pneumococcal infection. PspA- mutants were obtained using derivatives of plasmid pVA891 carrying chromosomal fragments from Rx1. From one of the mutants, we cloned a 550 bp fragment of the pneumococcal gene into pVA891 and transferred this chimeric plasmid, designated pKSD300, into Escherichia coli. After transformation of pKSD300 into Rx1, PspA production is not detected. In colony hybridization experiments, the 550 bp fragment hybridizes specifically to pneumococcal isolates in a pattern consistent with the hypothesis that the fragment is a portion of the pspA structural gene that is different from the portions coding for the antigenic determinants detected by mAbs Xi64 or Xi126. When X-linked immunodeficient (xid) CBA/N mice were immunized with wild-type Rx1, they were resistant to challenge with type 3 strain WU2. However, when these mice were immunized with a PspA- mutant of Rx1, they failed to survive the subsequent challenge, indicating that immunity to PspA can contribute to the resistance to pneumococcal infection. Using pKSD300 we insertionally inactivated pspA in D39, a virulent strain of S. pneumoniae. When injected intravenously there was a 10-fold greater reduction of the mutant pneumococci in the blood, as compared to the wild-type D39.

Animals↗
PubMed1986

Cloning and physical characterization of chromosomal conjugative elements in streptococci.

We used a directed insertion method to introduce a nonreplicating vector plasmid into the large conjugative cat-tet element found in the chromosome of Streptococcus pneumoniae BM6001 and derivatives. To direct insertion preferentially to the conjugative element, we transferred it by conjugation to Streptococcus faecalis and then used DNA from this strain as a source of restriction nuclease fragments for ligation to digests of the vector pVA891, which can replicate in Escherichia coli but not in streptococci. This ligation mix was used to transform pneumococcal cells carrying the cat-tet element, with selection for the erythromycin resistance carried by pVA891. Eight such isolates were found, and transformation and conjugation tests showed that in each case the vector had inserted into the conjugative element, as expected. DNA from these pneumococcal strains generated a variety of E. coli plasmids which provide tools for obtaining a detailed restriction map and for defining other structural features of the streptococcal conjugative element.

Bacterial Proteins↗
PubMed1986

Structure of a conjugative element in Streptococcus pneumoniae.

We have cloned and mapped a 69-kilobase (kb) region of the chromosome of Streptococcus pneumoniae DP1322, which carries the conjugative omega (cat-tet) insertion from S. pneumoniae BM6001. This element proved to be 65.5 kb in size. Location of the junctions was facilitated by cloning a preferred target region from the wild-type strain Rx1 recipient genome. This target site was preferred by both the BM6001 element and the cat-erm-tet element from Streptococcus agalactiae B109. Within the BM6001 element cat and tet were separated by 30 kb, and cat was flanked by two copies of a sequence that was also present in the recipient strain Rx1 DNA. Another sequence at least 2.4 kb in size was found inside the BM6001 element and at two places in the Rx1 genome. Its role is unknown. The ends of the BM6001 element appear to be the same as those of the B109 element, both as seen after transfer to S. pneumoniae and as mapped by others in pDP5 after transposition in Streptococcus faecalis. We see no homology between the ends of the BM6001 element and find no evidence suggesting that it ever circularizes.

Cloning, Molecular↗
PubMed1985

Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae.

We compared the efficiencies of two different processes that can direct integration of plasmids into chromosomes of recipient cells during transformation. A donor-recipient system was constructed to allow a single donor plasmid to use either flanking homology, involving an apparent double crossover, or the insertion duplication process that has been described as due to a "Campbell-like" single crossover between the chromosome and a circular duplex. The latter process gave 600-fold fewer insertions that did the former, confirming expectations from prior work showing that insertion of heterologous DNA by use of flanking homology is highly efficient. It has some advantages for cloning and mapping purposes and can be exploited once it is recognized.

Chromosomes, Bacterial↗
PubMed1983

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination.

Transformation-deficient mutants of Streptococcus pneumoniae were isolated after nitrosoguanidine mutagenesis. Seventeen mutants developed normal peaks of competence, as tested by their ability to degrade one strand of donor DNA, but they yielded transformants for chromosomal point markers at efficiencies from less than 0.001 to 0.04 that of the wild type. Some of the mutants were defective in DNA uptake and are described as entry defective (Ent-). Others took up DNA in normal quantities, but they failed to give stable transformants and are described as recombination defective (Rec-). In two of the Rec- mutants, normal levels of transformation by plasmid DNA occurred; in the others, it was reduced as much as chromosomal transformation. Conjugative transfers of a chromosomal omega (cat tet) element and of the plasmid pIP501 occurred at normal levels both to and from Rec- mutants. Transfer of chloramphenicol resistance by transformation with omega (cat tet) donor DNA, however, was blocked in Rec- mutants to about the same extent as was transformation for point markers.

Conjugation, Genetic↗
PubMed1982

Enhancement of pneumococcal transfection by protamine sulfate.

Protamine sulfate enhanced transfection of Streptococcus pneumoniae by DNA of omega 3 phage by factors as large as 10(5)-fold, provided it was present at the time the cells were added to the DNA. For DNA concentrations well below 1 microgram/ml, the optimum amount of protamine sulfate was near 1 microgram/ml of cells. Higher DNA concentrations required more protamine for maximum effect, and in all cases transfection fell when protamine was in excess. Transformation was not enhanced by low protamine levels and was inhibited by higher levels. A recipient strain with low but finite endonuclease activity and normal transformability showed higher transfection than did the wild type at low DNA concentrations but less than did the wild type at high DNA concentrations. Protamine sulfate enhanced its transfection at low, but not high, DNA concentrations. The behavior of this strain and the enhancement of transfection by protamine sulfate of wild-type cells were each consistent with less cutting of the donor DNA at the cell surface, which is part of the normal entry process in naturally competent gram-positive bacteria. Less cutting would lead to entry of fewer but longer strands that would be more efficient in reconstruction of the 33-megadalton phage replicon. We suggest that in this system protamine enhances transfection by inhibition of the surface nuclease action that is part of the normal entry process.

Bacteriophages↗
PubMed1981

Monomer plasmid DNA transforms Streptococcus pneumoniae.

The covalently closed (CC) monomer form of plasmid pMV158 was found to transform pneumococcus (Streptococcus pneumoniae) and to do so with two-hit kinetics. The evidence came from analysis of the behavior of the transforming activity in fractions from preparative gel electrophoresis. Activity in the first major peak to elute (i) co-eluted with monomer CC as detected on analytical gels, (ii) banded as CC in dye-buoyancy gradients, (iii) sedimented with the velocity expected for monomer CC, and (iv) gave two-hit kinetics as functions of both concentration and time of exposure of the cells to DNA. A second major peak of activity behaved physically as though mostly due to dimer CC forms and gave single-hit response curves. Because almost no dimer was detectable optically on analytical gels of starting preparations, its specific activity was high relative to that of the monomers.

Centrifugation, Density Gradient↗
PubMed1981

Pathway of plasmid transformation in Pneumococcus: open circular and linear molecules are active.

We have extended the analysis of plasmid transformation in Streptococcus pneumoniae by finding that monomeric and dimeric open circular and linear forms of pMV158 were active in transformation. Their efficiencies were at least 35-fold lower than those of the corresponding closed circular forms. The evidence came largely from analysis of S1 nuclease-digested plasmid deoxyribonucleic acid by combinations of dye-buoyancy, gel electrophoresis, and sedimentation velocity methods. As with closed circular forms, monomer open circular forms gave second-order kinetics and dimer forms gave first-order kinetics. Unique linear products of digestion by either of two restriction enzymes were inactive, but a mixture of the two digests was active, as was the mixture of linear monomer deoxyribonucleic acids produced by S1 nuclease. Absolute efficiencies of transformation were low even for closed circular donors. All of the results, including the low efficiencies, were consistent with the interpretation that plasmid replicons were assembled in the recipient cell by pairing of fragments of single strands that had entered the cell separately from duplex donors that had been cut on the cell surface.

DNA Restriction Enzymes↗
PubMed1981

Homology among tet determinants in conjugative elements of streptococci.

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes.

Base Sequence↗
PubMed1980

Properties and transforming activities of two plasmids in Streptococcus pneumoniae.

Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The major of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.

Centrifugation, Density Gradient↗
PubMed1980

Improved method for conjugative transfer by filter mating of Streptococcus pneumoniae.

The frequency of conjugation during filter mating of pneumococcus was increased 10- to 100-fold when the filter was embedded in agar during incubation instead of being on the surface. The major effect was not due to protection from oxygen. The factor of increase was similar for transfer of plasmids and of chromosomal insertions of drug resistance elements.

Agar↗
PubMed1979

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.

Bacteriophages↗
PubMed1979

A plasmid in Streptococcus pneumoniae.

Plasmid deoxyribonucleic acid has been detected in three related laboratory strains of Streptococcus pneumoniae. Strains D39S, R36, and R36NC each contain a minimum of two copies per cell of a 2.0-megadalton plasmid (pDP1). A plasmid twice as large as this smaller one is also present in much lower quantity in these strains, but neither plasmid is present in four strains related to these or in a drug-resistant clinical isolate from Paris. The plasmid yield was not amplified in the presence of chloramphenicol. No phenotype has been correlated with the presence of pDP1, which has existed in strains carried for many years in laboratory collections.

Anti-Bacterial Agents↗
PubMed1979

Organization and transfer of heterologous chloramphenicol and tetracycline resistance genes in pneumococcus.

The cat and tet genes of chloramphenicol- and tetracycline-resistant clinical isolates of Streptococcus pneumoniae from Paris and Japan were shown to be contained in adjacent heterologous insertions into the chromosome. The two insertions transformed laboratory strains at frequencies that were low, unequal, and, for tet, very sensitive to the length of the donor deoxyribonucleic acid strand. In contrast, the transforming activity of cat was relatively stable. There was an unusual asymmetric cotransfer, in that a majority of the tet transformants also acquired cat, whereas only a few of the cat transformants also acquired tet. The evidence for chromosomal insertion came from genetic data showing linkage of cat to a chromosomal gene and from cosedimentation of cat with chromosomal markers in both velocity and dye-buoyancy experiments. Genes on a known plasmid introduced into pneumococcus from Streptococcus faecalis showed very different physical behavior. Most of the transformation properties of these genes can be readily accounted for by analogy to transformation of deletions of normal genes. Whether transposition contributes any of the transfers remains to be determined. The presence of one of the genes in the recipient promoted the integration of the other, demonstrating enhanced accumulation of heterologous genes by a process that did not involve plasmids in the species of concern.

Chloramphenicol↗