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K Dybvig

Publications and source records attributed to K Dybvig.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular biology of mycoplasmas.

Although mycoplasmas lack cell walls, they are in many respects similar to the gram-positive bacteria with which they share a common ancestor. The molecular biology of mycoplasmas is intriguing because the chromosome is uniquely small (< 600 kb in some species) and extremely A-T rich (as high as 75 mol% in some species). Perhaps to accommodate DNA with a lower G + C content, most mycoplasmas do not have the "universal" genetic code. In these species, TGA is not a stop codon; instead it encodes tryptophan at a frequency 10 times greater than TGG, the usual codon for this amino acid. Because of the presence of TGA codons, the translation of mycoplasmal proteins terminates prematurely when cloned genes are expressed in other eubacteria, such as Escherichia coli. Many mycoplasmas possess strikingly dynamic chromosomes in which high-frequency changes result from errors in DNA repair or replication and from highly active recombination systems. Often, high-frequency changes in the mycoplasmal chromosome are associated with antigenic and phase variation, which regulate the production of factors critical to disease pathogenesis.

Chromosomes, Bacterial↗
PubMed1995

Mycoplasma virus P1 has a linear, double-stranded DNA genome with inverted terminal repeats.

Mycoplasma virus P1 is a tailed, polyhedral virus isolated from Mycoplasma pulmonis. To characterize the P1 genome, stocks of virus were prepared free of host cell nucleic acids. A single DNA species of 11.3 kb that was shown by plaque hybridization to be of P1 origin was extracted from the virus. Efficient isolation of P1 DNA required digestion with proteolytic enzymes prior to phenol extraction. Although P1 DNA was double-stranded and linear following such treatment, it was resistant to digestion with the 5'-specific lambda exonuclease. Electron microscopic analysis indicated that globular material is complexed to the ends of P1 DNA. The globular material was not observed on protease-treated P1 DNA molecules, suggesting that it is composed of protein. Removal of the putative terminal protein by chemical treatment allowed the cloning of the P1 DNA ends, and nucleotide sequence analysis revealed that these ends contain a 350-bp inverted terminal repeat.

Bacteriophages↗
PubMed1995

Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site-specific DNA inversions.

The chromosome of the murine pathogen Mycoplasma pulmonis undergoes rearrangements at a high frequency. We show that some of these rearrangements regulate the phase-variable expression of a cluster of genes (the vsa locus) that encode the variable V-1 surface antigens. Only one vsa gene is associated with an expression site; the other vsa genes are transcriptionally silent. The silent genes lack the 5' end region (promoter and ribosome-binding site) that is present in the expressed gene, and DNA rearrangements regulate gene expression by reassorting the 5' end region from an expressed gene with the 3' end region from a previously silent gene. All vsa rearrangements identified so far are site-specific DNA inversions that occur between copies of a specific 34 bp sequence that is conserved in each vsa gene. Interestingly, DNA inversions within the vsa locus apparently occur in concert with inversion of the hsd1 element, which regulates restriction and modification activity in M. pulmonis.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of the recA genes from Mycoplasma pulmonis and M. mycoides subsp. mycoides.

The RecA protein has a central role in DNA repair and is essential for homologous recombination in most eubacteria. Little is known about these critical processes in mycoplasmas. By using standard and inverse polymerase chain reactions (PCR) coupled with conventional cloning techniques, a series of overlapping fragments comprising the entire recA genes of Mycoplasma mycoides subsp. mycoides (Mm) and Mycoplasma pulmonis (Mp) were generated. Each gene was sequenced in its entirety. The recA genes of Mm and Mp would encode proteins of 345 amino acids (aa) and 339 aa, respectively. The mycoplasmal RecA proteins revealed strong conservation when compared with RecA sequences from other bacterial species.

Amino Acid Sequence↗
PubMed1994

Mycoplasmal cloning vectors derived from plasmid pKMK1.

Only two plasmids have been isolated and characterized from the entire genus Mycoplasma, which includes over 90 recognized species. Both of these plasmids were obtained from the same species, Mycoplasma mycoides subsp. mycoides. We have previously characterized one of these plasmids, pKMK1, as a preliminary step in developing mycoplasmal cloning vectors. In the present study, we have separately combined pKMK1 with two different Escherichia coli replicons and a tetracycline resistance (tetM) gene. One of the constructs, plasmid p2D4, was shuttled from E. coli to M. mycoides subsp. mycoides and back to E. coli with no deletions or rearrangements occurring in the plasmid. In the second construct, the E. coli replicon was deleted when the plasmid was transformed into M. mycoides subsp. mycoides. This derivative, designated plasmid pIK delta, is noteworthy in that it could be transformed into M. mycoides subsp. mycoides at a much higher frequency than the parental plasmid. A gram-positive bacterial erythromycin resistance determinant (erm) was cloned into both p2D4 and pIK delta. Resistance to erythromycin was stably maintained using both constructs, even in the absence of erythromycin selection, indicating that these plasmids will be useful mycoplasmal cloning vectors.

Blotting, Southern↗
PubMed1994

Transformation of Mycoplasma capricolum and examination of DNA restriction modification in M. capricolum and Mycoplasma mycoides subsp. mycoides.

Plasmids pIK delta and pIK delta-erm have recently been developed as mycoplasmal cloning vectors. In this report, we demonstrate that these plasmids can replicate in Mycoplasma capricolum, a mycoplasmal species for which transformation had not previously been characterized. Both plasmids are stably maintained at a higher copy number than in their parental species, Mycoplasma mycoides subsp. mycoides. We have also examined the possibility of one or more restriction-modification systems affecting transformation frequencies in both species.

Cloning, Molecular↗
PubMed1994

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

Amino Acid Sequence↗
PubMed1994

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis.

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.

Amino Acid Sequence↗
PubMed1993

Mycoplasma corogypsi sp. nov., a new species from the footpad abscess of a black vulture, Coragyps atratus.

Strain BV1 was isolated from the exudate of the footpad abscess of a black vulture (Coragyps atratus). The colonies had a "fried-egg" appearance consistent with that of mycoplasmal species. Electron microscopic examination of the cells revealed irregular elongated or elliptical forms and smaller circular budding processes. Profuse growth was observed in Frey medium supplemented with 20% swine serum at 37 degrees C in a humidified atmosphere of 10% CO2 and air. Typical of mycoplasma, strain BV1 required sterol for growth and catabolized glucose but did not hydrolyze arginine or urea. The guanine-plus-cytosine content of the DNA was 28 mol%. The organism demonstrated the ability to hemolyze, absorb onto, and agglutinate the erythrocytes from several animal species. Strain BV1 was serologically unrelated by the growth inhibition test to previously established Mycoplasma, Acholeplasma, Entomoplasma, and Mesoplasma species, as well as to strains belonging to these genera but not identified to species level. Moreover, BV1 had a 16S rRNA gene with a nucleotide sequence distinct from reported sequences of other mycoplasmas. This organism represents a new species for which the name Mycoplasma corogypsi is proposed. Strain BV1 (ATCC 51148T) is the type strain of Mycoplasma corogypsi sp. nov.

Abscess↗
PubMed1993

DNA rearrangements and phenotypic switching in prokaryotes.

Microorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high-frequency DNA rearrangements which are the subject of this review.

Chromosome Inversion↗
PubMed1993

Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family.

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined. IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M. pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species.

Amino Acid Sequence↗
PubMed1993

Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence.

Cilia-associated respiratory (CAR) bacillus, an unclassified gliding bacterium associated with respiratory disease in rats, mice, and rabbits, has previously been cultivated only in embryonated chicken eggs, cell culture, or cell culture medium supplemented with conditioned medium from cultured tracheas. A reference strain of CAR bacillus, originally isolated in eggs, grew in cell culture flasks as adherent individual bacilli and ropy, whorled fascicles in cell culture media supplemented only with fetal calf serum. Using Dulbecco's minimal essential medium, we isolated CAR bacillus from naturally infected rats and a naturally infected rabbit and from experimentally inoculated mice and rats. Isolates were maintained for up to 20 passages. Isolates from rats were similar in morphology to the reference strain, but most were more actively motile and formed pincushion-like aggregates. The rabbit bacilli were smaller and formed fewer aggregates. DNAs of rat isolates differed only slightly in restriction fragment patterns from that of the reference strain, whereas that of the rabbit isolate was distinctly different. Cultures of CAR bacilli of all strains from rats contained Mycoplasma fermentans, Mycoplasma pulmonis, or both, and cultures of the CAR bacillus from the rabbit contained an unidentified arginine-utilizing mycoplasma. The sequence of the 16S rRNA gene of the reference strain was determined by amplification by polymerase chain reaction, cloning of the product, and sequencing by the dideoxynucleotide chain termination method. Comparison of the sequence with sequences in the GenBank data base indicated that CAR bacillus is a unique organism most closely related to Flavobacterium ferrugineum and Flexibacter sancti.

3T3 Cells↗
PubMed1993

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction.

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.

Base Sequence↗
PubMed1992

Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline.

Tetracycline resistance in Mycoplasma hominis and Ureaplasma urealyticum has been associated with the tetM determinant and has recently been increasing in incidence. We report here a rapid method for detection of the tetM determinant based on the use of the polymerase chain reaction (PCR) to amplify a 397-bp DNA fragment from the tetM gene and verification of specificity using the restriction enzyme TaqI. Analysis of 42 U. urealyticum and 49 M. hominis isolates indicates that the PCR method may be clinically useful for determination of tetracycline sensitivity, as tetM is presently the only known determinant associated with tetracycline resistance in these two organisms. All of the tetM-positive M. hominis isolates were sensitive to doxycycline, indicating that tetM does not necessarily confer resistance to this antibiotic.

Base Sequence↗
PubMed1992

Nucleotide sequence of Mycoplasma mycoides subspecies Mycoides plasmid pKMK1.

To facilitate the development of mycoplasmal cloning vectors, we have determined the nucleotide sequence of pKMK1, a cryptic plasmid isolated from Mycoplasma mycoides subsp. mycoides. It is 1875 bp in length and contains two open reading frames (ORFs) that share homology with ORFs from members of a large family of gram-positive bacterial plasmids which replicate via a single-stranded DNA intermediate. Putative origins of replication and candidate cloning sites have been identified.

Amino Acid Sequence↗
PubMed1992

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment.

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The phenotype of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.

Acholeplasma laidlawii↗