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.

The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis.

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.

Sequence analysis of the Mycoplasma arthritidis bacteriophage MAV1 genome identifies the putative virulence factor.

The bacteriophage MAV1 is required for the development of arthritis in rats after infection with its host Mycoplasma arthritidis. To identify the phage-encoded virulence factor for this arthritis, the complete nucleotide sequence of MAV1 was determined. The linear double-stranded genome of MAV1 is 15644bp and contains 15 ORFs. Putative protein products from these ORFs were identified by comparison of the deduced amino acid sequences to known proteins and comprise DNA replication, restriction-modification, structural, regulatory, and integration/excision proteins. Eight putative promoters were identified; four of these would produce polycistronic transcripts. Translation of each ORF appears to be initiated independently, with each having its own RBS. A single ORF, vir, was identified on the minus strand of the phage genome. The putative protein product of vir contains a classic prokaryotic lipoprotein signal sequence and is a strong candidate for the MAV1-encoded virulence determinant.

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.

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.

Association of a major protein antigen of Mycoplasma arthritidis with virulence.

Mycoplasma arthritidis causes acute polyarthritis in rats and chronic proliferative arthritis in mice. M. arthritidis-induced arthritis serves as a model for arthritis caused by infectious agents and as a model for examining the role of the superantigen MAM (M. arthritidis T-cell mitogen) in the development of autoimmunity. M. arthritidis strain 158-1 is a spontaneous mutant of strain 158 that has a drastic reduction in virulence. We show that the mutant is missing a major antigen of 47 kDa (P47) and has acquired a protein of 67 kDa (P67). P47 and P67 partitioned into the detergent phase by extraction with Triton X-114. Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels show that P67 is produced in abundance. Analysis of gel-purified P67 by mass spectrometry led to its identification as a lipoprotein (the open reading frame [ORF] 619 gene product) predicted from the genome sequence of M. arthritidis. PCR analysis of genomic DNA from 158 and 158-1 indicates that P47 and P67 are encoded by the same ORF 619 gene and differ only in the number of repeats in a tandem repeat region. By two-dimensional polyacrylamide gel analysis, no protein differences were detectable between 158 and 158-1 other than P47 and P67. Collectively, the data suggest that the tandem repeat region of P47 and P67 influences disease outcome.

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.

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.

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.

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.

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.

Integration and lysogeny by an enveloped mycoplasma virus.

The 11.8 kilobase pair (7.8 X 10(6) mol. wt.) genome of mycoplasma virus L2 in lysogenic Acholeplasma laidlawii cells was examined. For this study, DNAs were analysed by agarose gel electrophoresis and viral DNA sequences identified by DNA-DNA hybridization. L2 DNA was found to be integrated into the lysogenic host cell chromosome at a unique site in both viral and cellular DNA. The viral DNA site was roughly mapped and the approximate time of integration during the L2 non-cytocidal infectious cycle was determined.

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.

The hsd loci of Mycoplasma pulmonis: organization, rearrangements and expression of genes.

Two paralogous, site-specific invertible loci, designated hsd1 and hsd2 (host specificity determinant), have been identified in the Mycoplasma pulmonis genome. They encode putative type I restriction and modification (R-M) systems with maximum sequence homology to the type IC family, which includes EcoR124II and EcoDXXI. Each locus encodes an endonuclease subunit (HsdR), a methylase subunit (HsdM) and two DNA specificity subunits (HsdS). The gene organization at each locus is such that hsdR and hsdM are flanked by two hsdS genes. Within each locus, one of the hsdS genes, hsdR and hsdM, is encoded in tandem by the same DNA strand, while the second hsdS gene is encoded by the complementary strand but without overlap with the other three hsd genes. The hsdR and hsdM sequences of one locus are almost identical to their counterparts in the other. The four hsdS genes (two per locus) are highly homologous at their 5' ends and also share sequence similarities in the 3' ends of their corresponding coding regions. Owing to the disposition of and sequence similarities among the hsdS genes, they form inverted repeats at each locus. Analysis by polymerase chain reaction (PCR) has shown that both loci behave as site-specific DNA invertible elements with multiple inversion sites, termed 'vipareetus', occurring within the hsdS genes. The inversions lead to a reassortment of hsdS sequences, generating an array of recombinant genes that probably encode S subunits possessing alternative DNA-binding specificities. Sequence information obtained from the analysis of hsd2 transcripts by 5' RACE (rapid amplification of cDNA ends) indicates that inversion induces the transcription of alternative hsdS genes by the relocation of coding sequences downstream of a promoter and ribosome-binding site (RBS) situated at one end of each locus.

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.

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.

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.

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.