Mycoplasmal genetics.

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MYC()PLASMAL GENETICS Kevin Dybvig Departments of Com parative Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

KEY WORDS:

mycoplasmas, evolution, genetic transformation, genetic variation, extrachromosomal DNAs

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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PHyLOGENy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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GENE TRANSFER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Mechanisms of Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Restriction and Modification Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EXTRACHROMOSOMAL DNA.................................................................. Mycoplasma Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasm ids ..... . . ....... .... . ........ . . . . ........... . . . ....... . .... . ........ Transposable Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENETIC VARIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION The properties of mycoplasmas make them intriguing but difficult to study. These microorganisms are ubiquitous pathogens of man, animals, plants, and insects. Although they are phylogenetically related to gram-positive bacteria, mycoplasmas lack a cell wall. The mycoplasmal genome is thought to be the smallest of any free-living cell. The minimal coding capacity of their genomes indicates a limited biosynthetic capability, and indeed, mycoplasmas require a complex medium to support growth in vitro. For most species, defined media have not been developed and auxotrophic mutants are unavailable. Replica 81

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plating techniques have not been described as yet, probably because growth on agar results in microscopic colonies. Genetic studies are further com plicated by the fact that the mycoplasmal genetic code is different from that of other bacteria. Despite these inherent difficulties, promising approaches for the development of mycoplasmal genetic systems have recently become available. These genetic systems should help elucidate another newly emerg ing area of interest-the high rate of genetic variation that occurs in some mycoplasmal species. PHYLOGENY The phylogenetic relationship of mycoplasmas to other eubacteria is of central importance to our understanding of the basic biology and genetics of these organisms. There are six genera of wall-less prokaryotes collectively referred to in this article as mycoplasmas. These genera are Mycoplasma, Acholeplas ma, Spiroplasma, Ureaplasma, Anaeroplasma, and the newly recognized genus Asteroleplasma. Species of Mycoplasma, Acholeplasma, Ureaplasma, and Spiroplasma are facultative anaerobes. Obligate anaerobic mycoplasmas that require sterols for growth are classified as Anaeroplasma, and those that do not require sterols for growth are classified as Asteroleplasma (95). The mycoplasmas arose from the branch of gram-positive bacteria with DNAs having low guanine plus cytosine content (34, 62, 96). R ecently, Weisburg et al (11 8) have studied the phylogeny of the mycoplasmas based on the sequence of the 16S rRNA molecule from over 40 mycoplasmal species. They have divided the mycoplasmas into five phylogenetic groups designated as the pneumoniae group (six characterized species that include Mycoplasma muris, Mycoplasma pneumoniae, and Ureaplasma urealyticum), the hominis group (sixteen characterized species that include Mycoplasma arthritidis, Mycoplas ma !ermentans, Mycoplasma hominis, Mycoplasma hyopneumoniae, Myco plasma hyorhinis, and Mycoplasma pulmonis), the spiroplasma group (seven teen characterized species that include Mycoplasma capricolum, Mycoplasma mycoides, Spiroplasma apis, Spiroplasma citri, and Spiroplasma mirum), the anaeroplasma group (six characterized species that include Acholeplasma laidlawii, Anaeroplasma abactoclasticum, Anaeroplasma intermedium, and Anaeroplasma varium), and the asteroleplasma group (one characterized species,Asteroleplasma anaerobium). Some Mycoplasma species (e. g. M. capricolum and M. mycoides) are more closely related to Spiroplasma, and other Mycoplasma species (e. g. M. pneumoniae and M. muris) are more closely related to Ureaplasma. Although most of the mycoplasma phylogene tic groups are closely related to one another, Asteroleplasma is somewhat distant; it probably evolved from a gram-positive eubacterium distinct from the ancestor(s) of the other mycoplasmas.


MYCOPLASMAL GENETICS

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The genome size of Acholeplasma, Anaeroplasma, Asteroleplasma, and Spiroplasma species is about 1400-1800 kilobase pairs (kb), while Ureaplas ma and Mycoplasma species have smaller genomes of only 600-800 kb (4, 9, 82, 89, 95 , 120). According to estimates, the small genomes of Ureaplasma and Mycoplasma species have the coding capacity for a mere 500 genes (74). As one would predict from the position of the mycoplasmas on the phyloge netic tree of gram-positive bacteria, the mycoplasmal genome has a high A+T content. In fact, many mycoplasmas, such as M. capricolum, have an A+T content as high as 75%, the highest of all prokaryotes. In the case of M. capricolum, spacer regions between genes are about 80% A+T, protein genes are about 70% A+T, and rRNA and tRNA coding regions are about 50% A +T (74). These data suggest that the mycoplasmas are undergoing selective pressure to replace G+C base pairs with A+T base pairs. This pressure may have played a vital role in the evolution of the mycoplasmal genetic code. Some mycoplasmas use the codon UGA to code for tryptophan, whereas other eubacteria use UGG to code for tryptophan and UGA is used as a termination codon. The unusual mycoplasmal genetic code was first shown for M. capricolum (124), and this code has now been shown for numerous Mycoplasma and Spiroplasma species (e. g. 6, 19, 21, 46a, 86, 90, 91, 109). Recently, a DNA sequence thought to be the gene encoding the urease enzyme from U. urealyticum was cloned (7), and sequence data indicate that this organism also uses UGA to code for tryptophan (A. Blanchard, personal communication). Thus, it appears that most (probably all) Mycoplasma, Spiroplasma, and Ureaplasma species use UGA as a tryptophan codon; however, some mycoplasmas have the typical eubacterial genetic code. A. laidlawii apparently has a single tryptophan tRNA that recognizes UGG and not UGA (111). The DNA sequence of mycoplasma virus L2, a virus isolated from A. laidlawii, confirms that this organism uses UGA as a termination codon (J. Maniloff, personal communication). In terms of the phylogenetic groupings of Weisburg et aI, species within the pneumoniae, hominis, and spiroplasma groups apparently use UGA as a tryptophan codon, but at least one species of the anaeroplasma group (A. laidlawii) uses UGA as a termina tion codon. A proposed mechanism for the evolutionary change in the genetic code of mycoplasmas is that all UGA termination codons in the mycoplasma an cestor(s) may have been converted to UAA termination codons because of the bias toward A+T base pairs (74). This alteration would have permitted the use of UGA as a tryptophan codon, as is found with some mitochondrial DNAs (11). Mycoplasmas can also utilize UGG as a tryptophan codon, but because of the A+T bias, UGA is the preferred codon. The unusual genetic code is of practical importance because it results in the premature termination of mycoplasmal genes when expressed in other eubacteria, such as


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Escherichia coli. Genes from other bacterial systems should for the most part be correctly translated in mycoplasmas, but readthrough of intended UGA termination codons by the mycoplasma should sometimes result in defective gene products. The unique properties of mycoplasmas led to their assignment into a taxonomic class, Mollicutes, separate from other bacteria. Historically, this classification was useful in that the distinction between mycoplasmas and bacterial L-forms was clear. However, there is much to be gained by emphasizing the si,milarities between mycoplasmas and other bacteria. For example, the proposed messenger RNA-binding sequence (101) at the 3' terminal region of the mycoplasmal 16S rRNA gene (35, 48, 90) resembles that of gram-positive bacteria. Differences in the mRNA-binding domain are thought to be one reason why many genes from gram-negative bacteria are poorly expressed in gram-positive bacteria (71), indicating that genes from gram-positive bacteria are more likely to be successfully expressed in myco plasmas than genes from gram-negative bacteria. Moreover, although data on mycoplasmal promoters and transcription termination signals are limited, these features for the most part resemble those of other eubacteria (e. g. 9, 37, 46, 74, 87, 90, 112, 113); thus, major surprises regarding expression of mycoplasmal genes may be limited to the genetic code. A logical strategy for development of systems for studying mycoplasmal genetics is to use gram positive bacteria as a potential source of genetic tools (e.g. antibiotic resis tance determinants, plasmid vectors, transposable elements, etc. ) and of information (e. g. methods for transformation and conjugation). This strategy has often been successful.

GENE TRANSFER Transformation Artificial transformation has been established for several species of mycoplasma. It is generally an inefficient process; most studies have used at least 5 fLg of plasmid or viral DNA per transformation, and have reported frequencies usually on the order of 10-4 to 10-8 transformants per recipient colony-forming unit (CFU). Most transformation studies have em ployed a polyethylene glycol- (PEG) mediated procedure similar to that used to transform protoplasts of gram-positive bacteria. Transformation of a myco plasma with PEG was first described for A. laidlawii in transfection studies using DNA from mycoplasma virus L2 (103). PEG-mediated transfection of A. laidlawii was also successful using mycoplasma viral L l (104) and L l72 (28) DNAs, but not with mycoplasma viral L3 DNA (55, 104). Perhaps PEG-mediated transfection with L3 DNA was not effective because of the large size of the DNA (39 kb) or the fact that this DNA is linear (Ll and L2 TRANSFECTION


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viruses have circular genomes of 4.5 kb and 11.6 kb, respectively). Using the PEG-mediated protocol developed for transfection of A. laidlawii, transfec tion of spiroplasmas with several viral DNAs has also been described (66, 80).


TRANSPOSABLE ELEMENTS

Transfection studies provided the basis for

establishing transformation of Acholeplasma and Spiroplasma species, but definitive transformation of Mycoplasma species accompanied the finding that gram-positive transposon Tn916 functions in these organisms (27). These initial studies describing transformation of Mycoplasma species utilized the Tn916-containing plasmid pAM120. pAMl 20 does not replicate in mycoplas mas, and transposition of Tn916 from the plasmid into the recipient chromo some is required for successful transformation. Transformants are selected using the transposon's tetracycline resistance determinant, tetM, as a marker. Tn916 has been useful for developing transformation methods for numerous microorganisms because of its very broad host range. Moreover, the tetM determinant usually results in a high level of tetracycline resistance, which allows for selection of transformants under conditions that have little if any background of spontaneous mutants. Using pAM120, researchers have es tablished PEG-mediated transformation for several mycoplasmal species in cluding M. pulmonis (25, 27, 60), M. hyorhinis (25), M. mycoides subsp. mycoides (121, 122), and M. gallisepticum (F. C. Minion, personal com munication). More recently, PEG-mediated transformation of M. pulmonis has been examined using another transposable element that functions in this organism,

staphylococcal transposon Tn4001 (61). In this case, the gentamicin resis tance determinant of Tn4001 served as the selectable marker. As with studies using Tn916, transformation with Tn400I-containing plasmids requires a transposition event. Transformation was successful with pISMlOOl, a 13.45 kb plasmid containing Tn400I, but not with pSK31, a 37.7 kb plasmid. As suggested from transfection studies with A. laidlawii, PEG-mediated transformation may be ineffective for large DNAs. Alternatively, pSK31 may be more susceptible to a host restriction and modification system than pISM1001 or pAM120. Other mycoplasmas (M. gallisepticum and A. laidla wii) have also been successfully transformed with pISMI001, indicating that Tn4001 may be useful as a genetic tool of mycoplasmas in general (F. C. Minion, personal communication). HOMOLOGOUS

RECOMBINATION

Mahairas & Minion (60) obtained

transformation of M. pulmonis in the absence of a transposable element or a plasmid capable of replication within the mycoplasma. These experiments relied upon homologous recombination for incorporation of antibiotic resis-


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tance markers into recipient chromosomes. Several plasmids that replicate in E. coli were constructed containing either the gentamicin resistance determi nant from Tn4001 or the tetM gene,combined with fragments of chromosom al DNA from M. pulmonis. Transformation resulted in insertion of the plasmid marker into the recipient chromosome at a frequency generally ranging from 10-4 to 10-6 transformants per CPU. Transformation frequen cies were apparently affected by the specific chromosomal insert present on the plasmid. These results suggest the presence of high levels of homologous recombination, and they should provide a basis for introducing a variety of genes into M. pulmonis. Of particular importance is the capability of introduc ing cloned genes with specific mutations into M. pulmonis in order to study the effect of mutations in vivo. Electroporation is another transformation method that will probably prove to be effective for numerous mycoplasmas. For example, electroporation was successful for transfection of A. laidlawii with L3 viral DNA, while PEG was ineffective (55). However, electroporation was less effective than PEG for transfection with L1 viral DNA (55). Therefore,the method of choice, electroporation or the PEG procedure,may depend on the cell strain and the particular DNA in question. Electroporation has also been successfully used for transfection of S. citri with SVTS2 DNA (65) and for transformation of Mycoplasma mycoides subsp. mycoides with pAM120 (122). In the S. citri transfection study, electroporation was about as efficient as PEG-mediated transformation. One would anticipate that electroporation will become increasingly effective as advances occur in this relatively new technique.

ELECTROPORAnON

TRANSFORMATION STUDIES Some reports have described transformation in the absence of PEG (or electroporation), but these results have been difficult to evaluate. Transformation of Mycoplasma hominis cells made competent by treatment with divalent cations has been reported (14, 36), but this approach was not successful in another laboratory (93). Transfec tion of A. laidlawii with viral L1 DNA has been reported in the absence of PEG or other treatments involving divalent cations (53, 54), but subsequent reports of transfection of these cells have required PEG (or electroporation) (e.g. 55,104). Transformation of A. laidlawii using DNA from a neomycin resistant strain of A. laidlawii in the absence of PEG or divalent ion treatment has also been reported (41). In a more recent study, the addition of PEG did not improve the transformation frequency of untreated S. citri in serum-free medium (102). These studies suggest the possibility of natural transformation of some mycoplasmas. Even if natural transformation can occur,however,it would probably be inefficient and difficult to reproduce. OTHER


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Other Mechanisms of Gene Transfer CONJUGAL TRANSFER OF TN916 Conjugal transposon Tn91 6 has been transferred from Enterococcus faecalis to numerous gram-positive bacteria. Roberts & Kenny (93) using M. hominis first described transfer of Tn916 into a mycoplasma, and conjugal transfer using E. faecalis as the donor will clearly be an effective mechanism for introducing Tn916 into a variety of mycoplasmas [e. g. M. pulmonis (24)]. Transfer of a conjugative DNA ele ment is a likely mechanism for how clinical isolates of M. hominis and U. urealyticum acquired the tetM gene (92, 94). Conjugal transfer is usually more convenient than transformation for the introduction of Tn916 into mycoplasmas because fewer manipulations are required and the need to isolate plasmid DNA is eliminated. Moreover,Tn91 6-mediated conjugation in bacilli has recently been shown to promote cotransfer of plasmids not harboring the transposon (75). Therefore,T091 6 may prove to be useful for transferring a variety of plasmids from gram-positive bacteria into mycoplas mas. To date, conjugal transfer of T091 6 using a mycoplasma as a donor has not been described.

Transfer of chromosomal DNA between spiro plasmas using resistance to arsenic acid and vanadium oxide as markers has recently bel:.:n described (3). Gene transfer occurred in growth medium in the absence of PEG,and control evidence strongly argues that the mechanism of gene transfer was neither transformation nor transduction. Conjugation and cell fusion are possible mechanisms. This is an important finding,especially because similar results have been obtained with M. pUlmonis using the tetM gene from Tn916 and the gentamicin resistance determinant from Tn4001 as markers (G. G. Mahairas & F. C. Minion, personal communication). Mahairas & Minion constructed several strains carrying these antibiotic resis tance markers at a variety of chromosomal sites. These constructs were obtained by transforming cells with plasmids that inserted into the chromo some by homologous recombination, as described above. Genetic exchange in M. pulmonis occurred on agar surfaces at a frequency ranging from 10-4 to 10-8 transformants per CFU, depending on the location and nature of the marker. These studies establish new techniques for experimental genetic manipulation of mycoplasmas, and they suggest that genetic exchange be tween mycoplasmas may be an important natural phenomenon.

MYCOPLASMAL MATING

Although transduction has not been described for the mycoplasmas, the prevalence of viruses isolated from these organisms in dicates thalt transduction is feasible and that it may occur in nature. Until

TRANSDUCTION

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recently, demonstration of transduction in the laboratory has been hampered by the lack of markers. The markers presently available (e.g. the tetM gene) should facilitate development of a transducing system.


Restriction and Modification Systems

Ideally, designs of gene transfer experiments would take into account host restriction and modification systems. For most mycoplasmas, little is known about these systems. Historically, these systems have been identified in bacteria by comparing relative titers of bacteriophage assayed on different host strains. A similar approach has been useful for examining restriction and modification systems in mycoplasmas. Probably the best-studied mycoplas mal restriction system is that of A. laidlawii strains JAI and 1305. Strain 1305 modifies the cytosine nucleotide in the sequence GATC to 5 methylcytosine (31). Mycoplasma viruses L l and L2 propagated o n strain JAI have unmethylated GATC sequences that are restricted (cleaved) during viral infection of strain 1305. Strain JAI has an unusual restriction system. DNA containing 5-methylcytosine is cleaved by strain JAI with little or no sequence specificity ( lOS). Because Ll and L2 viruses propagated on strain 1305 contain 5 -methylcytosine, they are restricted by strain JAI. Strain JAI modifies these genomes only in the sense that progeny viral DNA is un methylated. An important mutant of strain JAI has been isolated that lacks the restriction system described above (105). This strain, designated strain 8195, is valuable because it is thought to lack any restriction system that would be a barrier to gene-transfer studies. Restriction and modification systems are present in many mycoplasmas. U. urealyticum strain 960 has a restriction endonuclease that recognizes the sequence GCNGC (18). A restriction endonuclease from M. Jermentans with specificity for the sequence CAATTG has been described (43). A restriction endonuclease isolated from S. citri strain ASP2 is an isoschizomer of HhaI, recognizing the sequence GCGC (107). Many other mycoplasmal species have an uncharacterized endonuclease activity (83). For M. pulmonis, en donuclease activity has been shown to be membrane bound, suggesting that it may affect transformation studies (70). As expected if we assume mycoplas mal restriction systems are prevalent, many mycoplasmas contain methylated bases (88). For example, Spiroplasma sp. strain MQ- l apparently methylates the dinucleotide CG (79). The adenine nucleotide in the sequence GATC is methylated in the DNA of M. hyopneumoniae (15) and M. mycoides subsp. mycoides (K. Dybvig, unpublished data), but other Mycoplasma species [e.g. Mycoplasmaflocculare (15) and M. pulmonis (K. Dybvig, unpublished data)] lack this methylation. Whether the function of adenine methylation within the GATC sequences of some mycoplasmal DNAs is in any way related to that of DAM methylation in E. coli [see Messer & Noyer-Weidner (68) for a review] is unknown.


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EXTRACHROMOSOMAL DNA Extrachromosomal DNAs that can either replicate autonomously or move by transposition are important resources for development of genetic systems. These DNAs are also clinically important if they affect virulence of pathogen ic organisms. In this section, extrachromosomal DNAs that function in mycoplasmas are reviewed, with emphasis on utilizing these DNAs as genetic tools.


Mycoplasma Viruses

Information on viruses isolated from mycoplasmas is too extensive to be reviewed here, and the reader is referred to a recent review of these viruses by Maniloff (63) for a more detailed discussion. Little is known about most viruses isolated from organisms within the genus Mycoplasma; the character ized mycoplasma viruses have been isolated from organisms within the Spiroplasma and Acholeplasma genera. The utility of these viruses as genetic tools will probably be restricted to the host range of the virus, which in most cases is apparently limited to certain strains of a single species. For most mycoplasma viruses, too little information is available to speculate about their potential as genetic tools. One exception is the L2 virus isolated from A. laidlawii. L2 is a lysogenic virus with potential both as a transducing agent and as a cloning vehicle. L2 virions have an unusual morphology in that they have a lipid envelope but no apparent protein capsid. The L2 genome consists of an 11. 6 kb double-stranded, circular DNA molecule (76). At least 3 distinct L2 virions are produced during infection, which results in heterogeneity in both size and density of the virus particles (81). One of these virus types is thought to contain at least 2 copies of the L2 genome per virion, suggesting flexibility in the amount of DNA the virus can package. Further evidence for packaging flexibility came from the isolation of L2 miniviruses and insertion variants (30). The smallest L2 minivirus contains a 3. 3 kb genome. Miniviruses with multimeric genomes of 6. 6 and 9.9 kb are also observed, and higher multi meric forms are probably present as well. The insertion variants have genome sizes of 14.9 kb and apparently consist of the 3. 3 kb miniviral genome inserted into wild-type L2 DNA. Minivirus genomes are packaged into noninfectious, defective particles, which potentially could be developed into cloning vectors. The flexibility in DNA packaging of this system indicates that minivirus genomes would probably accommodate inserts as large as 10 kb. Plasmids

Several cryptic plasmids have been isolated from mycoplasmas, mostly from Spiroplasma species (9). Extrachromosomal

MYCOPLASMAL PLASMIDS

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DNAs are ubiquitous in spiroplasmas, and differentiating between true plas mids and viral genomes is not a simple matter. Although the development of


vectors from spiroplasma plasmids may be fruitful,plasmid instability may pose a problem in some cases. In addition to potential problems with plasmid segregation, some plasmids may integrate into the spiroplasma chromosome. An example of an unstable extrachromosomal molecule isolated from S. citri was originally thought to be a plasmid and designated as pRAI (8 kb). Fragments of pRAl are incorporated into the chromosome during passaging (77). Insertion of pRAl sequences generates interspersed, repetitive DNA elements approaching 30--40 copies per genome (78). Recent data indicate

that pRAI is actually a replicative form of viral DNA (8, 85), but spiroplasma plasmids not of viral origin may exhibit similar properties. For example, sequences homologous to plasmid pMHI (7 kb) are associated with the chromosomal DNA of some spiroplasmas (72). Simoneau & Labarere (102) recently described one attempt to construct a

vector from pMHl . They isolated a segment of plasmid pBR328 containing the chloramphenicol acetyl transferase (CAT) gene without it s promoter. This DNA was cloned into plasmid pUC13 (generating a plasmid denoted as pCAT) , and a promoter derived from S. citri was placed upstream from th e CAT gene. This construct containing a CAT gene driven by a spiroplasma promoter was combined with pMHl and transformed into S. citri, selecting for chloramphenicol resistance. Transformants were obtained, and plasmids isolated from them had homology to pMH l and to pCAT. However, the plasmids from the transformants had lost the ability to replicate in E. coli, and their mobility on agarose gels suggested that deletions had occurred. With passaging, these plasmids displayed segregational instability and additional

deletions were also observed. Similar plasmid instability has been observed when A. laidlawii was transformed with streptococcal plasmids (see below). There have been numerous reports of plasmids in mycoplasmal genera other than Spiroplasma (e.g. 22,45, 49, 125). Some of these reports may be valid, but confirmation has been difficult. One bona fide plasmid has recently been isolated from M. mycoides subsp. mycoides (5). This plasmid,designat

ed pADB201, has a sequence only 1,717 base pairs long (6). The sequence data indicate homology between an open reading frame in pADB201 and the replication proteins of staphylococcal plasmid pEl94 and streptococcal plas mid pLS 1. Replication of pE194 and pLS 1 is thought to involve intermediates containing single-stranded DNA [reviewed by Gruss & Ehrlich (42)], and pADB201 presumably has a similar mode of replication. In fact, by compar ing the sequence of pADB201 with that of plasmids that replicate by a

single-stranded mode, Gruss & Ehrlich (42) have identified the origin of replication (ori) for the plus strand of pADB201. Because plasmids that replicate via single-stranded intermediates tend to undergo deletion



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events at high frequency (2, 32), a vector developed from pADB201 would likely have some inherent problems with instability. Cryptic plasmids may be more prevalent in organisms within the genus Mycoplasma than currently realized. A cryptic plasmid has been isolated from other strains of M. mycoides subsp. mycoides. An examination of eight strains obtained from A. J. DaMassa revealed that two strains, strain GMl2 and strain GM928C, harbored a small plasmid of about 1 . 85 kb (K. Dybvig, unpublished data). The restriction maps of the 1.85 kb plasmids were in distinguishable from one another, indicating that the plasmids were closely related if not identical. The plasmid isolated from strain GMI2, designated plasmid pKMK l , was compared to pADB20 l using Southern blot techniques. Although their restriction maps were dissimilar, a limited region of homology existed between pADB201 and pKMKI as shown in Figure 1. BACTERIAL PLASMIDS Many plasmids isolated from gram-positive bacteria have extremely broad host ranges that include not only many genera of gram-positive bacteria but, in some cases, some gram negative bacteria. Some of these plasmids will probably replicate in myco plasmas. Surprisingly, the first example of a gram-positive bacterial plasmid that replicates in mycoplasmas comes from plasmids that had not been previously shown to replicate in organisms other than streptococci. Plasmid pVA380-1 is a 4. 2 kb cryptic plasmid originally isolated from Streptococcus ferus (58 ). Several streptococcal cloning vehicles have been constructed by combining the ori from this plasmid with various antibiotic resistance mark ers. The ability of the ori from pVA380-1 to replicate in mycoplasmas was examined by transforming A. Laidlawii strain 8195 with plasmids pVA868 and pVA920 (23). These plasmids contain the tetM determinant, and tetracy cline resistant-transformants of A. laidlawii were obtained at low frequency. Plasmid DNAs isolated from A. laidlawii transformed with pVA868 or pVA920 were deletion derivatives of the parent plasmids. The deletion derivative of pVA868 (13.7 kb) was a 3. 7 kb plasmid designated pKJI (Figure 2A). pKl1 was stable in A. laidlawii in the sense that additional deletions in the plasmid were not detected, and transformation of A. laidlawii with pKJl occurred at a relatively high frequency of 10-6 transformants per CFU. The deletion derivative of pVA920 (12.2 kb) was a 10. 3 kb plasmid designated pKJ3 (Figure 2B). pVA920 contains an erythromycin resistance determinant in addition to the tetM determinant. The erythromycin resistance determinant is still present in pKB, and it renders A. laidlawii cells resistant to this antibiotic. Although transformation of A. laidlawii with pKJ3 occurred at a frequency comparable to that of pKJ1, transformants receiving pKB had further deletions in the plasmid. These deletion events resulted in transfor mants that were resistant to tetracycline or erythromycin, depending on which GRAM-POSITIVE

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A Alu I


pADB201 1717bp

Hindm Hindm

B

Alu I

Sau 3A

Figure 1 Restriction maps of cryptic plasmids isolated from M. mycoides subsp. mycoides. (A) Map of pADB20 l compiled from the sequence data of Bergemann et al (6). (B) Map of pKMKI. Striped areas show regions of homology between the two plasmids. Dark areas are open reading frames (ORF). The large open reading frame in A is thought to code for a replication (rep) protein. The region of homology in A extends into the rep region only to the Spe I site. The shaded area shows the origin of replication deduced from sequence data (42). pKMKI does not have recognition sites for EcoRV, HindIII, Clal, NdeI, DdeI, Seal, HaeIII, BgnI, and BamHI. The Sau3A sites in pKMKI were also recognition sites for DpnI but not MboI, indicating that the adenine nucleotide within the GATe sequences is methylated .

antibiotic was used for selection, but not resistant to the other antibiotic. Plasmids pKJ1 and pKJ3 also exhibited a high degree of segregational instability. The plasmids were lost from most cells after a few passages in medium without antibiotic selection. Although pKJ3 is unstable in A. laidlawii. pKll may be useful as a vector. It is apparently stable in A. laidlawii (except for segregational instability) with a copy number of at least 10 molecules. per cell (K. Dybvig, unpublished


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A

B

HincII

Figure 2 Restriction maps of plasmids pKJI (A) and pKJ3 (8). Dark areas are coding regions specifying resistance to tetracycline (tet) or erythromycin (erm) as indicated. The origin of replication in 8 is within the shaded area. Additional information on the relationships between pKJI and pKB and their parent streptococcal plasmids is available (23).

data). It has an unique Hindlll site, which should be available for cloning because it is located within sequences that are not involved with plasmid replication or antibiotic resistance. pKJl potentially could be used as a streptococcus-mycoplasma shuttle vector; both pKJl and pKJ3 have retained the capabililty of replicating in streptococci. Unfortunately, pKll may not be useful as a vector in Mycoplasma; attempts to transform M. pulmonis with pKJl and pKJ3 have not been successful. Transposable Elements

Tn916 (16 kb) [reviewed by Clewell & Gawron-Burke (16)] is a fascinating transposon because of its conjugative properties and its structure. It may


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transpose via circular intermediates by a process involving excision events that are frequently precise (17, 38, 98). Tn916 has been shown to be useful as an insertional mutagen in several systems (e. g. 12, 38, 47,50,84,97),and its properties in mycoplasmas suggest that it will also be useful as a mutagen in this system. As described above, Tn916 can be introduced into mycoplasmas by transformation or by conjugal transfer from E. faecalis. Although several Tn916-containing plasmids are available, mycoplasmal transformation studies involving Tn916 have been limited to plasmid pAM120 (25, 27). pAM120 (26.5 kb) consists of the transposon cloned into plasmid pGLlOI, a plasmid that replicates in E. coli (39). As mentioned above, pAM120 does not replicate in mycoplasmas and successful transformation requires a transposition event. If transposition following transformation were a rare event, transformation with pAM120 would occur at a relatively low frequency. This is not the case. Transformation of A. laidlawii with pAM120 occurs at a frequency of about 10-6 transformants per CFU (27). Transformation with plasmids pKl1 and pKJ3, which replicate in A. laidlawii, occur at the same frequency. This suggests that Tn916 trans poses into the recipient chromosome in nearly every cell that is transformed. These results are in accord with data from gram-positive bacterial systems that indicate that transposition is induced when Tn916 is first introduced into a cell ( 1 6). Studies with M. puimonis indicate that once Tn9/6 is inserted into the chromosome, it can undergo additional transpositions to other chromosomal sites (25). Similar results regarding transposition of Tn916 have been obtained with M. mycoides subsp. mycoides (K. W. King & K. Dybvig, unpublished data). Therefore, Tn916 functions as a transposable element in mycoplasmas. Transposition events in M. pulmonis apparently involve exci sion of Tn916 from the donor site as has been shown for transposition of Tn916 in streptococci. The transposition frequency in M. pulmonis is about 10-3 transpositions per CFU per generation; this is considerably higher than the reported transposition frequency for Tn916 in E. faecalis (16). However, the reported transposition frequency in M. puimonis may be misleading because it was based on only one clone, and Tn916 transposition frequencies are thought to vary depending on the site of the insertion (38). An unusual feature of transformation of mycoplasmas with pAM120 is that cointegrate structures are sometimes produced (25). Similar structures are commonly associated with some transposable elements, but cointegrate struc tures involving Tn916 have not been described in other bacteria. These structures seem to consist of a complete copy of pAM120 inserted into the mycoplasmal chromosome, usually (but not always) with a second copy of Tn916 present as well. With other types of transposons, structures such as these are thought to be generated by transposition events involving replicative intermediates. Because Tn916 transposition is not thought to involve these



95

types of intennediates, some other mechanism must be responsible for cointe grate fonnation. One possibility is that the large amount of plasmid used for PEG-mediated transfonnation (usually 10-20 jLg) can result in a single cell being simultaneously transfonned with two copies of pAM120. Tn916 could then transpose from one copy of pAMl20 into the mycoplasmal chromosome as a simple insertion. The other copy of pAM120 could then insert into the chromosome by homologous recombination, generating the cointegrate struc ture. This model is supported by the experiments of Mahairas & Minion (60) demonstrating homologous recombination in these cells. The ability to use Tn916 (or Tn4001) as an insertional mutagen should facilitate mycoplasmal genetic research by enabling investigators to identify genes important to the basic biology and disease pathogenesis of these organisms. Although Tn916 might have hotspots for insertion that could limit its usefulne$s as a mutagen, indications are that this is not a significant problem. Independent Tn916-containing transfonnants (or transconjugants) of A. laidlawii. M. pulmonis. M. hyorhinis. M. hominis, and M. mycoides subsp. mycoides have been shown to have Tn916 inserted at different sites (25, 27, 93, 122). The target site for Tn916 insertion is thought to be an A + T-rich sequence (17). The fact that most mycoplasmal genomes are extremely A + T rich suggests that Tn916 target sites will be prevalent. Spacer regions between genes will possibly be preferred target sites because these regions generally are more A + T rich than coding regions; however, the difference illl A + T content between coding regions and spacer regions is probably not great enough to present a major problem. Unfortunately, myco plasmal mutants generated by Tn916 insertion have not yet been described, and no data exist concerning the frequency or Tn916 insertions into coding regions. Tn4001 is a 4. 7 kb transposon isolated from Staphylococcus aureus (56). It encodes a bifunctional peptide specifying resistance to kanamycin and genta micin, and it is flanked by a 1. 3 kb insertion element, IS256 (57). Like Tn916. Tn4001 may be capable of precise excision events (59). Transfonna tion of M. pulmonis with the Tn4001-containing plasmid pISMlOO l resulted in insertion of the transposon into the recipient chromosome at several sites (61). Southern blot analysis indicates that the IS256 insertion element can sometimes transpose independently of the intact transposon. As with Tn916, the utility of Tn4001 as an insertional mutagen of mycoplasmas needs further investigation. Its prospects are promising in that Tn4001 has been shown to be useful as a mutagen of S. aureus (59). GENETIC VARIATION Relatively little work has been done with mutagenesis of mycoplasmas, and data on spontaneous mutation rates are limited. However, sequence analysis


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of mycoplasmal rRNAs indicate that these organisms are in a rapid state of evolution. A striking characteristic of 16S rRNA sequences is that positions that tend to be invariant in most bacteria show significant variation in myco plasmas, indicating a high rate of mutation. Woese (123) has suggested that mycoplasmas may be able to withstand a high mutation rate because of their small genome size, and some mycoplasmas may be expected to have high mutation rates because their DNA polymerase reportedly lacks a 3' to 5' exonuclease activity ( 1 0, 69). In contrast to earlier studies, Maurel et al (64) have recently reported that a 3' to 5' exonuclease activity is associated with the purified DNA polymerase(s) isolated from several mycoplasmal species; however, this study did not incorporate controls necessary to determine the specificity of the nuclease activity. An intriguing picture suggests that some mycoplasmal phenotypic traits can vary at high frequency. These variations have a genetic basis. Mutants of M. pneumoniae that fail to hemadsorb were first isolated following treatment with nitrosoguanidine (44), but subsequent studies described the isolation of spontaneous mutants at a high frequency of about 7 x 10-3 mutants per CPU (52). Several classes of nonhemadsorbing mutants were obtained. Most mutants, class I, were deficient in four high-molecular-weight proteins. Hemadsorption-positive revertants of these mutants regained all of these proteins (5 1 ) . Similarly, another class (class III) of nonhemadsorbing mutants lacked proteins designated A, B, and C. Hemadsorbing revertants of these mutants also regained all three proteins (5 1). These data suggest that expres sion of proteins involved with hemadsorption may be coordinately regulated. The high frequency of mutant isolation indicates that a phenomenon may be occurring that is similar to phase variation in Neisseria gonorrhoeae ( 1 06). In M. pulmonis, genetic variation occurs at an even higher frequency than does variation in M. pneumoniae hemadsorption. Watson et al (117) per formed analysis of the proteins from M. pulmonis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed that individual subclones may have any of numerous (hundreds), structurally distinct forms of a surface antigen designated as V-I. In any given subclone, variation in V-I occurs at a rate of about 2 x 10-3 variants per cell per generation (1 1 7). It takes about 30 generations for a single cell of M. pulmonis to grow first into a colony on agar, and then,after being picked as an agar plug, to grow into a 1 ml culture containing about 109 CPU. Such a culture is the most homogeneous stock of 109 M. pulmonis CFU that is obtainable, but already 6% (2 x 10-3 cells/generation x 30 generations) of the cells have a variant form of V-I ( 1 1 7). Assuming the growth rate of M. pulmonis is independent of V-I, the percentage of cells that are V-I variants after additional passages in broth can be calculated. After two additional passages in broth (each passage being a 1 to 1,000 dilution into fresh medium



97

and requiring about 10 generations to regain high titer), 10% of the cells would be V-I variants. After 22 passages in broth, approximately 50% of the cells would be V-I variants, and essentially every cell would have a form of V-I different from the parent after about 46 passages. Although the assump tion that cell growth is independent of V-I is probably not valid (see below), these calculations serve to emphasize the high rate of variability in this antigen. Variation in V-I has been shown to be associated with variation in other phenotypic traits of M. pulmonis. The size of M. pulmonis colonies is a highly variable trait that correlates with variation in V-I (29). Subclones with varied colony size (and V-I antigens) have different growth rates in broth, suggest ing that V··1 can affect growth of the organism on agar and in broth. M. mycoides subsp. mycoides also exhibits genetic variation affecting colony size ( 1 16), and other species (M. hyorhinis and M. hominis) exhibit significant variation in colony size that may be due to a related phenomenon (29). Whether colony size variation in each of these systems is associated with a V-l -like molecule remains to be determined. Another variation that occurs in M. pulmonis at a rate of 1 0-3 mutants per cell per generation results in spontaneous mutants that are resistant to mycoplasma virus PI (26). In this study, a minority of the PI virus-resistant mutants had altered forms of V-I antigen; PI virus failed to adsorb to these mutants, suggesting that V-I plays a role in virus adsorption. However, the majority of PI virus-resistant mutants had no obvious alteration in V-I. The virus readily adsorbed to these mutants, and the reason for the resistance phenotype is unknown. High-frequency variation in some molecule(s) other than V-I may account for these virus resistant mutants. Many mycoplasmal species, perhaps all, have an antigen similar to V-I. V-I antigen is distinctive in that it forms a characteristic ladder pattern observed on immunoblots of SDS-PAGE gels. Ladder patterns similar to that of V -1 have been observed on immunoblots of proteins from mycoplasmas as diverse as U. urealyticum (1 3), A. laidlaw;;, M. arthritidis, and M. muris (H. L. Watson" personal communication). The antigen giving rise to the ladder pattern from U. urealyticum may be one of the primary determinants of serotype specificity of this organism (1 3). As in the case of V-I, the antigen from ureaplasma undergoes a high rate of variation when passaged in broth (H. L. Watson, personal communication). In conclusion, the surface proper ties of mycoplasmas constantly change as a result of genetic variations affecting V-I-like antigens. The potential significance of these surface varia tions in regard to pathogenesis of mycoplasmal diseases is enormous. Mechanisms for high frequency, genetic variation in mycoplasmas are unknown, but they may be similar to those proposed for phenotypic variation in other bacteria (73, 100). One possible mechanism is gene conversion (67,


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99, 110). The finding that mycoplasmal genomes contain repetitive sequences argues for this possibility. Sequences with homology to part of the gene coding for the M. pneumoniae adhesin protein PI have been identified at several sites within the chromosome (108, 119). A different repetitive se quence is also dispersed along the M. pneumoniae chromosome within se quences unrelated to the PI gene (119). The finding that some mycoplasmas (M. pulmonis) apparently have high levels of homologous recombination supports the hypothesis that genetic variation in mycoplasmas may result from recombination involving repetitive sequences. However, these ideas are spec ulative. High frequency genetic variation within the PI gene or other known repetitive elements within mycoplasmal genomes has not been documented, and the gene(s) involved with known examples of high frequency genetic variation of mycoplasmas have not been analyzed. Mycoplasmas may have indigenous transposable elements that contribute to genetic variation. A 1.5 kb repetitive element found in porcine mycoplas mal species contains 28 base pair inverted repeats (33, 114, 115). About 16 copies of this element have been found in all strains of M. hyorhinis that have been examined, and it has also been found in fewer copies in M. hyopneumo niae and M. flocculare. The structural similarities between this element and prokaryotic insertion sequences and the possibility of horizontal transmission of the element among porcine mycoplasmas suggests that it may be a transposable element. The insertion of spiroplasma "plasmid" pRAI (dis cussed above) into chromosomal sequences also suggests a transposable element. Elements such as these may contribute to phenotypic variation by inserting in or near antigen coding regions.

CONCLUDING REMARKS Despite the inherent difficulties in working with fastidious organisms with an unusual genetic code, significant progress is being made toward development of gene transfer systems. Elucidation of the evolution of mycoplasmas has given impetus to the productive idea of using gram-positive bacteria as a source of genetic tools. Initial successes along these lines have shown that some gram-positive bacterial transposons function in mycoplasmas. Many gram-positive bacterial plasmids may be capable of replication in mycoplas mas, and development of some of these plasmids into useful cloning vectors should be possible. In addition to the exciting approach of using gram positive bacterial DNA elements as tools to study mycoplasmas, DNA ele ments of mycoplasmal origin are becoming available. Numerous mycoplasma viruses have been isolated, and cryptic plasmids are now being found as well. Exploitation of these genetic elements should greatly facilitate research on mycoplasmal molecular biology.


MYCOPLAS MAL GENETICS

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One of the most puzzling aspects of mycoplasmas is their high rate of genetic variation. This rate of variation may be partially explained by a deficiency in mycoplasmal DNA repair ( 1 , 20, 40, 8 1 ) coupled with a high level of DNA recombination. Little data support the notion that natural transformation systems contribute to genetic variation, but it appears that conjugation-like gene transfer systems may be present. Also, the role myco plasma viruses may play as transducing agents is unknown. Because of the high rate of genetic variation, cultures of mycoplasmas must be viewed as dynamic in that subpopulations are always present. Matters are further complicated by the fact that some subpopulations may have different growth properties than others (29). Because much of the documented ex amples of high frequency genetic variation affect mycoplasmal surface prop erties, nearly all studies in mycoplasmal research, especially genetics, may be affected. For example, some subpopulations may be preferentially trans formed, resulting in situations where most transformants would not be repre sentative of the culture as a whole. Similarly, some subpopulations may be more proficient at conjugation-like gene transfer or more susceptible to viral infection (as is apparently the case for mycoplasma virus PI). Moreover, mutagenesis experiments may be adversely affected by instability of the desired phenotype. These possibilities suggest that the emerging field of mycoplasmal genetics promises to be a complex and fascinating area of study. ACKNOWLEDGMENTS I acknowledge support from the National Institutes of Health (Grants A125640 and RR00959). I thank my many colleagues who provided me with helpful discussions and unpublished information. Literature Cited I . Aoki,. S Ito, S Watanabe, T. 1979. UY survival of human mycoplasmas: evidence of dark reactivation in Myco plasma buccale. Microbiol. lmmunol. 23 : 1 47-58 2. Ballester, S . , Lopez, P Espinosa, M . , Alonso, J . C . , Lacks, S . A . 1989. Plas mid structural instability associated with pC I 94 replication functions. J. Bacteri01. 1 7 1 :2271-77 3. Barroso, G . , Labarere , J. 1988. Chro mosomal gene transfer in Spiroplasma citri. Science 24 1 :959-61 4. Bautsch, W. 1 988. Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional field inver sion gel electrophoresis techniques. Nucleic Acids Res. 1 6 : 1 146 1-67 5. Bergemann, A. D . , Finch, L. R. 1 988. Isolation and restriction endonuclease . •

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Mycoplasrrul pneumoniae. Infect. Im mun. 57: 1059-65 Das, J. , Maniloff, J . , Bhattacharjee, S . B . 1972. Dark and light repair i n Ul traviolet-irradiated Acholeplasma laidla wii. Biochim. Biophys. Acta 159: 1 8997 Dudler, R. , Schmidhauser, C . , Parish, R. W . , Wettenhall, R. E. H . , Schmidt, T. 1988. A mycoplasma high-affinity transport system and the in vitro in vasiveness of mouse sarcoma celJs. EMBO J. 7:3963-70 Dugle, D. L . , Dugle, J. R. 1 97 1 . Pres ence of two DNA populations in Myco plasma laidlawii. Can. J. Microbiol. 17:433-34 Dybvig, K. 1989. Transformation of Acholeplasma laidlawii with streptococ cal plasmids pVA868 and pVA920. Plasmid 2 1 : 1 55-60 Dybvig, K. 1990. Genetic manipulation of mycoplasmas. Zentralbl. Bakteriol. Mikrobiol. Hyg. In press Dybvig, K. , A lderete, J . 1988. Transformation of Mycoplasma pulmo nis and Mycoplasrrul hyorhinis: transposition of Tn916 and formation of cointegrate structures. Plasmid 20:3341 Dybvig, K. , Alderete, J. , Watson, H. L . , Cassell, G. H. 1 988. Adsorption of mycoplasma virus PI to host cells. J. Bacteriol. 1 70:4373-75 Dybvig, K. , Cassell, G. H. 1987. Transposition of gram-positive trans poson Tn916 in Acholeplasma laidlawii and Mycoplasrrul pulmonis. Science 235: 1 392-94 Dybvig, K. , Nowak, J. A . , Sladek, T. L . , Maniloff, J. 1985 . Identification of an enveloped phage, mycoplasma virus

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