Dataset Information

Development of oriC-plasmids for use in Mycoplasma hyorhinis.

ABSTRACT: Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5?×?10-4 and 2.0?×?10-5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P?

SUBMITTER: Ishag HZA

PROVIDER: S-EPMC5587638 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Development of oriC-plasmids for use in Mycoplasma hyorhinis.

Ishag Hassan Z A HZA Xiong Qiyan Q Liu Maojun M Feng Zhixin Z Shao Guoqing G

Scientific reports 20170906 1

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were succ ...[more]

PMID: 28878274

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Project description:The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

Dataset Information

Development of oriC-plasmids for use in Mycoplasma hyorhinis.

Publications

Development of oriC-plasmids for use in Mycoplasma hyorhinis.

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