Biomedical subjects

K Dybvig

Publications and source records attributed to K Dybvig.

At least 55 records · Page 3Linked to original sources

PubMed1992

High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching.

Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high-frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.

Antigenic Variation↗

PubMed1992

Cloning and DNA sequence of a mycoplasmal recA gene.

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.

Acholeplasma laidlawii↗

PubMed1992

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.

Amino Acid Sequence↗

PubMed1991

Plasmid transformation of Mycoplasma mycoides subspecies mycoides is promoted by high concentrations of polyethylene glycol.

The recent isolation and characterization of two plasmids from Mycoplasma mycoides subspecies mycoides has opened up new possibilities for studying mycoplasmal genetics. In order to facilitate the development of a genetic system in M. mycoides subsp. mycoides, parameters of polyethylene glycol (PEG)-mediated transformation were examined, as existing protocols prove very inefficient in this organism. The effects of PEG concentration, DNA concentration, presence of Ca2+ ions, and choice of buffers on the transformation of the Tn916-containing plasmid pAM120 into M. mycoides subsp. mycoides were examined. The stability of Tn916 in the M. mycoides subsp. mycoides chromosome was also evaluated. The optimal PEG concentration (53-62% (w/v)) in the transformation mixture was substantially higher than the PEG concentration reported to be optimal for transformation of other mycoplasmas (36% (w/v)). The PEG concentrations used here were also higher than the concentration used to promote transformation or fusion of gram-positive bacterial protoplasts. A necessity for the presence of Ca2+ ions for optimal transformation was shown, as was the possible involvement of cell culture growth stage. Our results demonstrate the need for expanding current transformation techniques for mycoplasmas. Studies also indicate that once Tn916 inserts into the M. mycoides subsp. mycoides chromosome, it can transpose to other sites at a relatively high frequency.

Blotting, Southern↗

PubMed1990

Isolation of a second cryptic plasmid from Mycoplasma mycoides subsp. mycoides.

Plasmids have rarely been detected in organisms constituting the genus Mycoplasma. Recently, the isolation of a cryptic plasmid from Mycoplasma mycoides subsp. mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms.

Genetic Vectors↗

PubMed1990

Mycoplasmal genetics.

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Extrachromosomal Inheritance↗

PubMed1989

Transformation of Acholeplasma laidlawii with streptococcal plasmids pVA868 and pVA920.

One limitation with studying mycoplasma genetics is the lack of cloning vectors. Studies were undertaken to determine whether streptococcal plasmids could replicate in Acholeplasma laidlawii, for the purpose of identifying potential vectors. Plasmids pVA868 and pVA920 contain the same origin of replication and tetracycline resistance determinant. pVA920 also contains an erythromycin resistance determinant not present in pVA868. A. laidlawii was transformed with plasmids pVA868 (13.7 kb) and pVA920 (12.2 kb), and isolated from the transformants were deletion derivatives of the parent plasmids having sizes of 3.7 and 10.3 kb, respectively. The tetracycline and erythromycin resistance markers functioned in A. laidlawii, and the deletion derivatives may be useful for development of mycoplasma vectors. However, difficulties may arise due to plasmid instability.

Acholeplasma laidlawii↗

PubMed1989

Subunit structure of the variable V-1 antigen of Mycoplasma pulmonis.

It was previously shown that multiple structural variants of the V-1 antigen (variable antigen 1) of Mycoplasma pulmonis could be found within a single strain. This antigen is unusual in that it produces a ladder pattern after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The present study showed that some variants of V-1 could be extracted into the aqueous phase of a phenol-H2O system. Analysis with anti-V-1 monoclonal antibodies showed that the phenol-H2O-extracted V-1 had a regular spacing of 3.1 kilodaltons (kDa) between bands and trypsinization of this extracted V-1 resulted in the gradual symmetrical collapse (2.9-kDa increments) of the ladder into a single band, suggesting the presence of multiple identical subunits within the V-1 structure. The upper band from the phenol-H2O-extracted V-1 was isolated and analyzed by SDS-polyacrylamide gel electrophoresis immunoblotting, resulting in the regeneration of the original ladder pattern with 3.1-kDa spacing between bands. When V-1 was boiled for increasing times in the presence of SDS, the staining intensity of the upper band decreased with the concurrent appearance of additional lower-molecular-weight bands. Finally, by using whole cells, it was found that the lower-molecular-weight species of the ladder pattern selectively partitioned into the hydrophobic phase of a Triton X-114 phase partitioning system, and the higher-molecular-weight bands were found in the aqueous phase. These data indicate that the V-1 bands are composed of subunits which may aggregate via hydrophobic interactions and that these aggregates at least partially dissociate when exposed to harsh denaturing conditions, resulting in the characteristic ladder pattern of V-1.

Antigenic Variation↗

PubMed1989

High-frequency variation in Mycoplasma pulmonis colony size.

Heterogeneity in colony size of the murine pathogen Mycoplasma pulmonis was examined. Subcloning experiments showed that colony size variation resulted from high-frequency genetic changes. About 3% of the colonies from any given subclone were variants, with as much as a fourfold change in colony diameter. When the variants were propagated in liquid broth, their doubling times in logarithmic growth phase reflected the colony sizes obtained on agar. Colony size variation correlated with changes in the electrophoretic properties of the V-1 surface antigen.

Antigens, Bacterial↗

PubMed1988

Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures.

A procedure for transformation of the murine pathogen Mycoplasma pulmonis with plasmid pAM120 was developed. This plasmid replicates in Escherichia coli and contains the gram-positive transposon Tn916. The transformation protocol also proved effective for the swine pathogen Mycoplasma hyorhinis. The tetracycline resistance determinant of Tn916 was expressed in transformed myocoplasma cells, and Tn916 was found inserted into numerous sites in the recipient chromosomes of M. pulmonis and M. hyorhinis, indicating that transposition had occurred. Interestingly, some transformants of M. pulmonis and M. hyorhinis contained cointegrate structures which apparently had a complete copy of the entire donor plasmid (pAM120) inserted into the recipient chromosome. Subsequent transposition of inserted Tn916 was observed in passaged clones of transformed M. pulmonis.

Blotting, Southern↗

PubMed1988

Adsorption of mycoplasma virus P1 to host cells.

The adsorption of mycoplasma virus P1, a virus which infects some strains of Mycoplasma pulmonis, to host cells was examined. Mutants of M. pulmonis to which P1 virus did not adsorb were isolated. Proteins from the mutants and from wild-type cells were compared by two-dimensional polyacrylamide gel electrophoresis, and the only observed difference was in the surface antigen V-1. The electrophoretic properties of V-1 also correlated with the host range of the virus. These data strongly suggest that the V-1 antigen affects adsorption of P1 virus to host cells.

Adsorption↗

PubMed1987

Transposition of gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis.

Mycoplasma genetics has been limited by a lack of genetic tools such as selectable markers, methods to transfer DNA, and suitable vectors for cloning. Studies were undertaken to examine the potential of using the streptococcal transposon Tn916 as a mycoplasma genetic tool. The Escherichia coli plasmid pAM120, which contains Tn916, was transformed into Acholeplasma laidlawii and Mycoplasma pulmonis. Transposition of Tn916 into the mycoplasma chromosome apparently occurred by an excision-insertion mechanism. This example shows that newly introduced DNA from other bacteria can be successfully expressed in mycoplasma and that Tn916 should serve as a powerful genetic tool for the study of mycoplasmas.

Acholeplasma laidlawii↗

PubMed1987

Protein variability among strains of Mycoplasma pulmonis.

The proteins of Mycoplasma pulmonis were examined by two-dimensional polyacrylamide gel electrophoresis and immunoblotting. Comparison of profiles from silver-stained two-dimensional polyacrylamide gel electrophoresis of 18 strains of M. pulmonis allowed identification of five proteins that were strain variable. These variable proteins were not dependent on the medium used to grow the organisms and were shown by reaction with serum samples from naturally infected rats and mice to be produced in vivo and not to be medium components. Identification of strain-variable proteins may lead to an explanation of the differences in properties found among M. pulmonis strains.

Bacterial Proteins↗

PubMed1987

Isolation of a virus from Mycoplasma pulmonis.

A virus designated mycoplasma virus P1 has been isolated from Mycoplasma pulmonis. The virus infects M. pulmonis strain UAB 6510, and a plaque-forming unit assay has been developed. P1 has a tailed, polyhedral morphology with a head diameter of about 28 nm. Nucleic acid isolated from crude preparations of P1 virus contains double-stranded RNA, suggesting that P1 may be the first example of an RNA-containing mycoplasma virus.

Bacteriophages↗

PubMed1987

Mycoplasma pulmonis proteins common to murine mycoplasmas.

The proteins of Mycoplasma pulmonis were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunoblots using the following sera: antimedium, anti-M. arthritidis, anti-M. collis, anti-M. neurolyticum, and anti-M. muris. Fifteen proteins identified as genus-common were shared by M. pulmonis and at least one other mycoplasmal species. These proteins were shown not to be medium components, based on their reaction with antimedium serum.

Animals↗

PubMed1986

Isolation of mycoplasma virus L2 insertion variants and miniviruses.

We isolated two spontaneous variants of mycoplasma virus L2. Both variants, designated L2ins1 and L2ins2, contained a 3.1-kilobase-pair (kbp) insertion in the 11.8-kbp wild-type L2 genome. The insert DNA was shown to be derived from two noncontiguous regions of the L2 genome, and L2ins1 and L2ins2 differed only in the location of the 3.1-kbp insertion. We also isolated L2 miniviruses from serial passages of L2, L2ins1, and L2ins2 viruses. Miniviruses contained circular DNA molecules of 3.1 kbp or multimers of 3.1 kbp. Minivirus 3.1-kbp DNAs had the same sequences as the 3.1-kbp insert DNAs found in L2ins1 and L2ins2 viruses. Miniviruses were not infectious and interfered with the growth of L2, L2ins1, and L2ins2 viruses; hence, L2 miniviruses appeared to be defective interfering particles.

Acholeplasma laidlawii↗

PubMed1985

Identification of an enveloped phage, mycoplasma virus L172, that contains a 14-kilobase single-stranded DNA genome.

We have found that mycoplasma virus L172 is an enveloped globular virion containing circular, single-stranded DNA of 14.0 kilobases. L172 has been reported by other workers to have a double-stranded DNA genome of 13 to 17 kilobase pairs and has been classified as a plasmavirus, a group for which mycoplasma virus L2 is the type member. Mycoplasma viruses L172 and L2 differ in genome size and structure, DNA base composition, and protein composition, and they have no detectable DNA homology. As the only reported enveloped virion containing single-stranded DNA, L172 represents a new group of viruses.

Acholeplasma laidlawii↗

PubMed1983

A chicken repetitive DNA sequence that is highly sensitive to single-strand specific endonucleases.

A DNA sequence consisting of the 5-mer AGAGG repeated tandemly 32 times has been detected in a chicken genomic clone and found to be present in about 2000 copies per chicken genome. This sequence was highly susceptible to single-strand specific endonucleases isolated from Aspergillus oryzae (S1) and mung bean, but cleavage by a single-strand specific endonuclease isolated from Neurospora crassa occurred only at a pH below 5.5. Endonucleolytic cutting of the AGAGG sequence by the single-strand specific enzymes required a supercoiled substrate and was independent of ionic strength.

Animals↗