Mechanisms of Genetic Instability in Mollicutes (Mycoplasmas)

Russian Journal of Genetics, Vol. 37, No. 9, 2001, pp. 979–992. Translated from Genetika, Vol. 37, No. 9, 2001, pp. 1173–1187. Original Russian Text Copyright © 2001 by Momynaliev, Govorun. THEORETICAL PAPERS AND REVIEWS Mechanisms of Genetic Instability in Mollicutes (Mycoplasmas) K. T. Momynaliev and V. M. Govorun Research Institute of Physicochemical Medicine, Moscow, 118128 Russia; e-mail: [email protected] Received December 28, 2000; in final form, March 28, 2001 Abstract—Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses. INTRODUCTION Mollicutes (mycoplasmas) are the smallest known microorganisms capable of independent division [1–5]. They lack a rigid cell wall and have only a surrounding membrane. Mycoplasmas were first described about 100 years ago; at present, more than 180 mycoplasma species are known [6, 7]. The characteristic feature of mollicutes is their wide distribution as parasites of multicellular organisms and the species specificity with respect to a host macroorganism in which many mycoplasma species persist in vivo [1]. As mycoplasmas are “perfect” parasites, they can induce drastic pathologic changes in the macroorganism in some states, such as inflammations accompanied by long-term persistence of infection (and often with complications expressed as autoimmune disorders) [8, 9]. Outbreaks of epidemic inflammatory infectious diseases provoked by mycoplasmas were described in farm animals, birds, plants, and in humans [1, 8–12]. In addition, when mycoplasmas contaminate cell lines, they cause great commercial losses in modern fields of modern biotechnology. Despite the fact that the molecular organization of mycoplasmas was studied extensively, main mechanisms of pathogenesis causing mycoplasmoses and responsible for the formation of resistance of mollicutes to antibiotics and for the ability to bypass the host immunity control are still unrevealed. Nevertheless, considerable experimental material clearly shows a high rate of intraspecific genetic divergence and high metabolic plasticity of mollicutes determined by genetic chromosomal rearrangements, mobile ele- ments, and viruses. In this review, we attempted to consider main mechanisms responsible for genetic instability of mollicutes, assuming that ample information available may promote research of geneticists, immunologists, and clinical physicians designed to establish main features of mollicute persistence in a host organism. GENETIC HETEROGENEITY OF MOLLICUTES The genome of one of the class members, Mycoplasma genitalium, is only several times greater than that of large viruses, amounting to 580 070 bp [13]. To date, complete nucleotide sequences in genomes of four representatives of mollicutes—M. genitalium (strain G37), M. pneumoniae (strain M129), M. pulmonis UAB CTIP, and Ureaplasma urealiticum (serotype 3)— have been determined, and genomic sequences of M. capricolum (214 000 bp) and M. hypopneumoniae (891 267 bp) have been partially analyzed [13–18]. The M. genitalium chromosome contains 479 open reading frames (ORF); 30% of them are related to genes encoding membrane proteins (Table 1). In M. pneumoniae, the genome constitutes 816 394 bp and contains 677 ORF. The genome of U. urealiticum contains 751 719 bp and 695 putative ORF. Research activities of the group headed by R. Herrmann in 1996 resulted in complete sequence analysis of the M. pneumoniae genome [14]. To conduct the functional analysis of M. pneumoniae, a combination of two methods was used: transcriptional analysis (transcription profile) [19] of all putative 677 ORF [20] (transcriptome) with protein analysis for the identification of all proteins 1022-7954/01/3709-0979$25.00 © 2001 MAIK “Nauka /Interperiodica” 980 MOMYNALIEV, GOVORUN UU rnpA rpmH 113 48 dnaA 457 dnaN 379 160 bp MG rnpA rpmH 128 48 94 kb MP AL MC rnpA rpmH 118 48 7620 bp 196 bp BS dnaN 364 735 bp dnaA 450 gyrB 650 gyrA 836 dnaA 440 gyrB 650 gyrA 839 1020 bp dnaN 380 2299 bp 1035 bp rpmH dnaA dnaN gyrB ? 445 325 615 338 bp 1645 bp 1497 bp rnpA rpmH 111 25 gyrA 840 368 bp dnaA 437 7239 bp gyrB 650 dnaN 210 gyrB 146 gyrA 824 gyrA 37 165 bp rnpA rpmH dnaA dnaN 116 44 446 378 626 bp 191 bp gyrB 638 1791 bp gyrA 821 211 bp Fig. 1. Genetic organization of the dnaA–gyrA region of mycoplasmas: UU, U. urealiticum (serotype 3); MG, M. genitalium G37; MP, M. pneumoniae M129; AL, A. laidlawii PG-8B; MC, M. capricolum. For comparison, the similar region of Bacillus subtilis (BS) is presented. Numerals indicate amino acid sizes of corresponding proteins and a distance between them. Arrows indicate the direction of ORF transcription for dnaA, dnaN, gyrB, gyrA, rnpA, and rpmH. excreted from the bacterial cell (proteome) [21, 22]. R. Herrmann et al. [23] detected 450 polypeptides after staining gels with silver. Meanwhile, only 225 polypeptides were visualized when using Kumassy blue, and 90% of them were identified. Thus, a comparison of results obtained in transcriptome and proteome analyses showed that the number of transcripts in M. pneumoniae does not correspond to the number of polypeptides and the reverse. A comparison of published data on nucleotide sequences of M. genitalium and M. pneumoniae genomes showed high homology. The genome of M. pneumoniae includes all ORF of the M. genitalium genome and additional 209 ORF absent in M. genitalium [13, 14, 24]. Only 110 of all these ORF were specific for M. pneumoniae and had no homology with M. genitalium genes. Of the remaining 99 ORF, 23 encode from 1 to 3 copies of repetitive sequences, which have a low homology with sequences of the M. genitalium genome. Some ORF (76) are amplified gene copies, represented by single copies in M. genitalium. In contrast, a large set of ORF [15], which did not have orthologs in the genomes of M. genitalium and M. pneumoniae was detected in the U. urealiticum genome (Table 2). Moreover, M. genitalium and M. pneumoniae have the similar disposition of genes (with the exception of several operons: S10, spc, and α), whereas no such gene order was observed in U. urealiticum. The organization of genes in the region of the replication origin (oriC) in the genome of mollicutes should be the focus of attention, because we know that the order of eubacterial genes contained in oriC (rnpA– rpmH–dnaA–dnaN–recF–gyrB) is highly conserved. This order is partially retained in mycoplasmas (Fig. 1). The absence of the recF gene is typical for them, except for A. laidlawii [13, 25–28]. A and B subunits of DNA gyrase (gyrA and gyrB) in Gram-positive bacteria are located close to oriC, although in Gram-negative bacteria, gyrA is usually located at the other chromosomal locus. Interestingly, tandem-bound subunits of DNA gyrase were found in oriC of M. genitalium [25], M. pneumoniae [26], M. gallisepticum [29], and Spiroplasma citri [28], but not in M. capricolum or M. hominis where the gyrB and gyrA genes are located separately, outside this region [30, 31]. Considering the data on the structural organization of M. genitalium and M. pneumoniae genomes, Hilbert et al. [26] concluded that genes located in the dnaA region of these mycoplasmas are unique, because their analogs have not been found in the oriC region of other bacteria, while the gyrB, dnaA and dnaN genes are arranged in a different order or orientation relative to each other (Fig. 1). Specific nucleotide sequences TTATCCACA required for binding the DnaA protein responsible for initiation of DNA replication were detected in the dnaA region of mycoplasmas [32]. In M. pneumoniae, no TTATCCACA sequences adjacent to the dnaA region were found [26]. Nucleotide sequences responsible for binding the DnaA protein are currently assumed to differ in various mycoplasma species [33]. Thus, the function- RUSSIAN JOURNAL OF GENETICS Vol. 37 No. 9 2001