Mikoplazmų tyrimus apsunkina genetinių įrankių trūkumas. Ne išimtis ir Mycoplasma gallinarum – platų šeimininkų ratą turinti silpno patogeniškumo bakterija. Šiame darbe aprašomas pirmųjų M. gallinarum skirtų vektorių, į kurių sudėtį įeina chromosominė replikacijos pradžios sritis (oriC), kūrimas. Be to, šio tyrimo metu pirmą kartą pademonstruota sėkminga šios mikoplazmų rūšies transformacija. Iš šešių sukonstruotų vektorių penki replikavosi M. gallinarum ląstelėse, o šeštasis nebuvo funkcionalus dėl AT turtingoje srityje pasroviui nuo antrosios DnaA dėžutės atsiradusios mutacijos. Kai kurie vektoriai, kultivuojant transformantus in vitro, integravosi į chromosomą. Homologinės rekombinacijos dažnis dar labiau padidėjo po transformacijos ilgą laiką gaivinant ląsteles augimo terpėje be antibiotiko. Sukurti oriC vektoriai galėtų būti naudojami genetiniams M. gallinarum tyrimams, ypač genams inaktyvuoti ir ekspresuoti, bei suteiktų daugiau žinių apie molekulines šios bakterijos savybes. / Genetic studies of mycoplasmas are limited by the lack of a replicable vector system. Mycoplasma gallinarum, an opportunistic pathogen having a wide range of hosts, is not the exception. This study describes the identification and cloning of the M. gallinarum origin of replication (oriC) in order to construct the first vectors for this species. Additionally, this report provides the first evidence of the successful transformation of M. gallinarum. Five out of six developed vectors have been functional. The replication of the sixth vector has not been supported due to the mutation in the AT rich region downstream the second DnaA box. During in vitro passages, some vectors have been integrated into the chromosome. The rate of chromosomal integration has been even further increased by the lack of antimicrobial selection pressure during cultivation. Hence developed oriC plasmids are useful for genetic studies, including inactivation or expression of selected genes, in M. gallinarum, and lead to a better understanding of its molecular biology.

Unlike most other host-specific mycoplasmas, Mycoplasma gallinarum exists as a commensal with a host range including most poultry as well as some mammals. This property of M. gallinarum may reflect unique mechanisms for its colonization and persistence in hosts. Whereas M. gallinarum shows leucine and arginine aminopeptidase activity, the genes encoding the enzymes had not been cloned and characterized. We identified an aminopeptidase gene (APN) by oligonucleotide hybridization to a genomic library of M. gallinarum in lambda ZAPII bacteriophage. Nucleotide sequence analysis of overlapping phage clones identified a 1,362 bp open reading frame (ORF) encoding a putative leucine aminopeptidase gene. Database searches indicate that this ORF has 68% nucleotide identity and 51% amino acid identity with the M. salivarium leucine aminopeptidase gene. The active sites of the leucine aminopeptidases in other eukaryotes and prokaryotes were conserved in the cloned aminopeptidase gene. Northern-blot hybridization analysis showed that this ORF is expressed as a 1.5 kb transcript. Southern-blot hybridization analysis demonstrated this gene was present as a single copy in M. gallinarum. Characterization of the leucine aminopeptidase demonstrated that it is a metallo-aminopeptidase (EC 3.4.11.1) and is located in the cytoplasm with a weak interaction with the cell membrane. The subcellular location was further confirmed by immunoblotting with polyclonal anti-recombinant APN serum and M. gallinarum Triton-114 partitions. Immunoblotting results with sera from three chickens experimentally infected with M. gallinarum showed that there were very few proteins in M. gallinarum exposed to the host immune responses and that leucine aminopeptidase was not able to stimulate production of specific humoral antibody. Our results suggest that this leucine aminopeptidase play a role in nutrition supply for the host adaptation of M. gallinarum and that the enzyme was not strongly immunogenic.