GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis
© The Author(s) 2016
Received: 17 February 2016
Accepted: 17 May 2016
Published: 17 June 2016
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10−3 cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.
KeywordsMycoplasma hyorhinis Plasmid GFP Expression
Mycoplasma hyorhinis (M. hyorhinis) is a commensal pathogen of swine that also causes lung lesions and inflammation (Razin et al. 1998), and is thought to contribute to the development of cell transformation in vitro (Namiki et al. 2009). These properties of M. hyorhinis have increased interest to the researchers.
Whole genome sequence of M. hyorhinis strain HUB-1 was determined (Liu et al. 2010), and expression of foreign antigens in M. hyorhinis might help to produce recombinant engineered strains. However, a method based on GFP expressing plasmids to evaluate the transformation and expression of foreign genes in M. hyorhinis has not been described. Several methods to monitor gene activity in cells are available such as the formation of fusion proteins with coding sequences for β-galactosidase, firefly luciferase, and bacterial luciferase (Stewart and Williams 1992). But, these methods are of limited use since they require exogenous substrates or cofactors. The green florescence proteins (GFP) of jellyfish Aequorea victoria is a unique tool to monitor gene transfer and expression (Cubitt et al. 1995). Using GFP might help to construct an efficient reporter system for M. hyorhinis. Here, we constructed a plasmid expressing GFP fluorescence and optimized conditions for transformation by electroporation.
M. hyorhinis strain HUB-1 (GenBank accession CP002170.1) was provided by Prof. Xiao Shaobo (Huazhong Agricultural University, China) and was grown at 37 °C in KM2 cell-free liquid medium (a modified Friis medium) containing 20 % (v/v) swine serum (Xiong et al. 2016). KM2-Agar was prepared by adding 0.7 % Agar (Biowest Agarose ®G-10; Gene Company Limited, Chi Wan, Hong Kong) to KM2 medium and was incubated at 37 °C to grow the visible colonies. Tetracycline hydrochloride (Sigma-Aldrich) was used at 0.01 μg/ml.
We previously constructed a plasmid pMD18-TOgfp encoding tetracycline resistance gene (tetM) controlled by the p97 gene promoter, GFP gene also controlled by the p97 gene promoter and oriC of M. hyopneumoniae attenuated strain (168L) (GenBank accession 507382422) (Ishag et al. 2016). The purpose of this plasmid was to express GFP in M. hyopneumoniae strain 168L. It is well known that, the p97 gene functions as an adhesion molecule for M. hyopneumoniae and the activity of this promoter was previously described in oriC-plasmids of M. hyopneumoniae (Maglennon et al. 2013). Here, we further evaluated the potential of this promoter in M. hyorhinis.
Primers used to amplify the oriC of M. hyorhinis to construct pMD18-O/MHRgfp plasmid
Oligonucleotides sequence (5′–3′)
Tetracycline-resistant mycoplasmas were analyzed for their plasmid content. Total genomic DNA was extracted using a TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) from either the pool of mycoplasma cultures containing 0.01 µg/ml tetracycline hydrochloride or from a single resistant clone sub-cultured in KM2 medium containing 0.01 µg/ml tetracycline hydrochloride. The presence of pMD18-O/MHRgfp was analyzed by the detection of GFP (750 bp) by PCR, and GFP could be detected from the total genomic DNA of the transformants, but not from untransformed mycoplasmas (Fig. 2b). One product amplified with GFP specific primers was sequenced and was indeed the expected GFP sequence (data not shown).
In the present report, the construction of a vector carrying the GFP gene was performed in order to develop a direct method for monitoring gene transfer and expression in M. hyorhinis in which the timing, as well the magnitude of gene expression, is being examined. This visual expression analysis system could also indicate that, the expression of the heterologous genes in M. hyorhinis is feasible.
HZAI designed, carried and drafted the manuscript. MJL assisted in the experiment design. QYX, ZXF, YRS and GQS, critically revised and approved the final manuscript. All authors read and approved the final manuscript.
The support from postdoctoral fund of Jiangsu Academy of Agricultural Sciences, China (6511318), National Natural Sciences Foundation of China (31550110211, 31300155) and the Natural Sciences Foundation of Jiangsu Province (BK2013070).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Bottin A, Larche L, Villalba F, Gaulin E, Esquerre-Tugaye MT, Rickauer M (1999) Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae. FEMS Microbiol Lett 176(1):51–56View ArticleGoogle Scholar
- Cordova CM, Lartigue C, Sirand-Pugnet P, Renaudin J, Cunha RA, Blanchard A (2002) Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids. J Bacteriol 184(19):5426–5435View ArticleGoogle Scholar
- Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20(11):448–455View ArticleGoogle Scholar
- Dybvig K, Alderete J (1988) Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures. Plasmid 20(1):33–41View ArticleGoogle Scholar
- Ishag HZA, Liu MJ, Yang RS, Xiong QY, Feng ZX, Shao GQ (2016) A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae. Genet Mol Res. doi:https://doi.org/10.4238/gmr.15027832
- Liu W, Fang L, Li S, Li Q, Zhou Z, Feng Z, Luo R, Shao G, Wang L, Chen H (2010) Complete genome sequence of Mycoplasma hyorhinis strain HUB-1. J Bacteriol 192(21):5844–5845View ArticleGoogle Scholar
- Maglennon GA, Cook BS, Matthews D, Deeney AS, Bossé JT, Langford PR, Maskell DJ, Tucker AW, Wren BW, Rycroft AN (2013) Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae. Vet Res 44(1):63View ArticleGoogle Scholar
- Namiki K, Goodison S, Porvasnik S, Allan RW, Iczkowski KA, Urbanek C, Reyes L, Sakamoto N, Rosser CJ, Tyson DR (2009) Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS ONE 4(9):e6872View ArticleGoogle Scholar
- Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62(4):1094–1156Google Scholar
- Stewart GS, Williams P (1992) Lux genes and the applications of bacterial bioluminescence. J Gen Microbiol 138(7):1289–1300View ArticleGoogle Scholar
- Xiong Q, Wang J, Ji Y, Ni B, Zhang B, Ma Q, Wei Y, Xiao S, Feng Z, Liu M, Shao G (2016) The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host cells. Vet Microbiol 186:82–89View ArticleGoogle Scholar