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Molecular Biology of Mycoplasmas: from the Minimum Cell Concept to the Artifi Cial Cell

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Molecular Biology of Mycoplasmas: from the Minimum Cell Concept to the Artifi Cial Cell Anais da Academia Brasileira de Ciências (2016) 88(1 Suppl.): 599-607 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201620150164 www.scielo.br/aabc Molecular biology of mycoplasmas: from the minimum cell concept to the artifi cial cell CAIO M.M. CORDOVA1, DANIELA L. HOELTGEBAUM1, LAÍS D.P.N. MACHADO2 and LARISSA DOS SANTOS3 1Departamento de Ciências Farmacêuticas, Universidade de Blumenau, Rua São Paulo, 2171, Campus III, 89030-000 Blumenau, SC, Brasil 2Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina, Rua Delfi no Conti, s/n, 88040-900 Florianópolis, SC, Brasil 3Programa de Pós-Graduação em Química, Universidade de Blumenau, Rua Antônio da Veiga, 140, Campus I, 89012-900 Blumenau, SC, Brasil Manuscript received on May 13, 2015; accepted for publication on June 2, 2015 ABSTRACT Mycoplasmas are a large group of bacteria, sorted into different genera in the Mollicutes class, whose main characteristic in common, besides the small genome, is the absence of cell wall. They are considered cellular and molecular biology study models. We present an updated review of the molecular biology of these model microorganisms and the development of replicative vectors for the transformation of mycoplasmas. Synthetic biology studies inspired by these pioneering works became possible and won the attention of the mainstream media. For the fi rst time, an artifi cial genome was synthesized (a minimal genome produced from consensus sequences obtained from mycoplasmas). For the fi rst time, a functional artifi cial cell has been constructed by introducing a genome completely synthesized within a cell envelope of a mycoplasma obtained by transformation techniques. Therefore, this article offers an updated insight to the state of the art of these peculiar organisms’ molecular biology. Key words: mycoplasma, molecular biology, microbial genetics, synthetic biology, gene transfer techniques. INTRODUCTION the term mollicutes has come into use as a generic name for all microorganisms in the class (Razin et It is believed that mycoplasmas originated through al. 1998). The criteria for defi ning the taxonomy degenerative evolution from Gram-positive of mollicutes are established by the Subcommittee ancestors, with the loss of non-essential genes on the Taxonomy of Mollicutes, linked to the (Dybvig and Voelker 1996). The term mycoplasma International Committee on Systematics of is often used to refer to all microorganisms in the Prokaryotes, and can be found at its website: Mollicutes class, regardless of which genus they <http://icsp.org/subcommittee/mollicutes/>. belong to. In order to prevent confusion with MOLECULAR BIOLOGY OF MOLLICUTES microorganisms specifi c to the Mycoplasma genus, Correspondence to: Caio Mauricio Mendes de Cordova Aside from the fundamental differences between E-mail: [email protected] these and other prokaryotes, the molecular biology An Acad Bras Cienc (2016) 88 (1 Suppl.) 600 CAIO M.M. CORDOVA et al. of mollicutes is very typical in some respects. with the sequencing of the complete genome of Having evolved from Gram-positive bacteria, gene various species. Currently, the complete genome expression and other genetic characteristics of sequences of 46 species and strains have been mollicutes are similar to those of their ancestors. published. For a detailed analysis, we suggest On the other hand, because of the limited capacity visiting MolliGen.org. MolliGen is a database of their genome, mollicutes do not have many dedicated to the comparative genomics of bacteria of the enzymatic pathways characteristics of the belonging to the Mollicutes class. It offers sequence majority of bacteria (Dybvig and Voelker 1996). and analysis of data collected on 54 genomes These characteristics make mollicutes particularly belonging to 37 species. The MolliGen database interesting in molecular and cellular biology provides pages with genome and genetic elements, studies, as, even with these limitations, they are homology relationships defined by various capable of independent replication and parasitism, methods, precompiled statistical data, metabolic causing diseases that lead to losses in agriculture as pathways, and a large collection of tools to explore well as in human health. and compare genomes (Barré et al. 2004). Currently there is an effort, sponsored by the International GENOME STRUCTURE AND ORGANIZATION Organization for Mycoplasmology (IOM), to Mollicutes have a chromosome made up of circular encourage genome sequencing of all species of double stranded DNA, which can vary between mollicutes. 580 and 1,800 kb (Razin et al. 1998). The 580 kb DNA REPLICATION AND REPAIR chromosome of the human pathogen M. genitalium is currently the smallest known genome (Lucier Studies in this area have long maintained the idea et al. 1994). The GC content of the majority of that mollicutes were very limited with respect to mollicutes’ chromosomes is around 35%, with their DNA repair mechanisms. However, at least 25% being the smallest (Glass et al. 2000). Even in M. pulmonis, transposon induced mutagenesis though they have such small genomes, some in the recA, ruvA, ruvB, and ruvC genes produced mollicutes have repeated sequences of DNA in defi cient phenotypes (French et al. 2008). Through their chromosomes. The repeated elements found in silico analysis of sequenced genomes, the only in mollicutes are homologous to members of the complete repair mechanism found in mycoplasmas IS3 Family Insertion Sequence (Westberg et al. is that of repair by nucleotide excision repair 2002, Pilo et al. 2003, Ditty et al. 2003, Thomas et (NER), probably complemented by enzymes from al. 2005, Lysnyansky et al. 2009). the base excision repair (BER) mechanism and the With the exception of spiroplasmas, recombination mechanism (Carvalho et al. 2005). extrachromosomal DNA is rarely found in The NER mechanism was also identified in M. mollicutes. Currently, only a few plasmids have genitalium, in which the RecA protein has an active been isolated from M. mycoïdes subsp. mycoïdes role in the antigenic variation mechanism but not and from S. citri (Bergemann et al. 1989). On in DNA repair (Burgos et al. 2012). A histone-like the other hand, numerous replicative forms of protein that recognizes nucleotide incorporation viral genomes (phages) have been isolated from errors in DNA was identifi ed in M. gallisepticum. Acholeplasma and other species of Mycoplasma However, this does not appear to be a rule among and Spiroplasma (Maniloff 1992). mycoplasmas, which, during their evolution, lost In recent years, the greatest advances in the the DNA mismatch repair mechanism (MMR) understanding of mollicute biology were obtained (Kamashev et al. 2011). Nevertheless, despite their An Acad Bras Cienc (2016) 88 (1 Suppl.) MOLECULAR BIOLOGY OF MYCOPLASMAS 601 limitations, it is evident that mycoplasmas possess Nevertheless, the efficiency of DNA transfer in an adequate set of genes and gene products, even these cases was low. if they have not yet been completely identifi ed, VECTORS to promote DNA repair in a way that makes their development possible, causing signifi cant diseases Transfection experiments of S. citri with the SpV1 in a wide range of hosts (Gorbachev et al. 2013). virus containing an inserted gene that codes the M. GENETIC RECOMBINATION pulmonis P1 adhesin resulted in an expression of this spiroplasma fragment. However, this system Until recently, there was no evidence that homol- is inefficient, since there is a rapid loss of the ogous recombination could occur in mollicutes’ cloned DNA fragment. Transposons have also genomes. In M. agalactiae, the necessary genes been used in the transformation of A. laidlawii for homologous recombination appear to be and M. pulmonis in antibiotic-resistant mollicutes reduced, showing only the components involved using the pAM120 plasmid, containing the Tn916 in the formation of the presynaptic complex (recD, streptococcus transposon, which confers resistance recO, and recR), strand exchange (recA), branch to tetracycline through the expression of the tetM migration (ruvA and ruvB), and the Holliday gene (Dybvig and Cassell 1987). Other transposons junction resolution (recU and yqgF); however, it have also been used with various species of has already been demonstrated in other bacteria mollicutes since then, such as Tn1545 and Tn4001 that, with the absence of complete sets of genes, (Cao et al. 1994, de Barbeyrac et al. 1996, Foissac homologous recombination can occur (Rocha et al. 1997, Kapke et al. 1994). The transposons et al. 2005). Gene disruption by homologous undergo excision from the plasmid that contains recombination has already been proven in M. them, and integrate with the mollicute chromosome genitalium (Dhandayuthapani et al. 1999) and in in random positions (Foissac et al. 1997). S. citri (Duret et al. 1999), and in Mycoplasma Mutagenesis by transposon is done by inserting it mycoïdes subsp. capri, inclusion of recA from E. into genes, causing the inactivation of transcription coli in a suicide plasmid favored the occurrence or the formation of truncated products. The genes of mutants (Allam et al. 2010). Apparently there containing the transposon are then mapped and are various mechanisms that can be explored to identifi ed, and the defi nitive proof of its function is increase the effectiveness of this approach, which given by the complementation of the mutant with may be species-dependent. the
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