A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum

Abstract

BackgroundMycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited.ResultsIn this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14 C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis.ConclusionThis is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.

Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum

“…M. gallisepticum strain S6 was grown in mycoplasma broth or on mycoplasma agar (Panicker et al, 2012) at 37 C, with 16 µg gentamicin ml À1 (Invitrogen) included in the medium to select for transformants. E. coli DH5a cells were used as the host for genetic manipulation and cloning of plasmids.…”

“…The pTAP plasmid has been described previously (Panicker et al, 2012). Briefly the promoter region of the gene for elongation factor Tu (ltuf) of M. gallisepticum, the leader sequence and acylation sequence of the vlhA1.1 gene from M. gallisepticum strain S6, and the E. coli phoA gene, which codes for alkaline phosphatase, were ligated into transposon Tn4001 in pISM2062.2, generating the pISM2062.2ltufacyphoA plasmid (pTAP), which thus contained a PhoA lipoprotein gene in which cysteine was the first amino acid of the mature PhoA lipoprotein.…”

“…Briefly the promoter region of the gene for elongation factor Tu (ltuf) of M. gallisepticum, the leader sequence and acylation sequence of the vlhA1.1 gene from M. gallisepticum strain S6, and the E. coli phoA gene, which codes for alkaline phosphatase, were ligated into transposon Tn4001 in pISM2062.2, generating the pISM2062.2ltufacyphoA plasmid (pTAP), which thus contained a PhoA lipoprotein gene in which cysteine was the first amino acid of the mature PhoA lipoprotein. The PhoA lipoprotein has been shown to be exported through the membrane and exposed as acylated lipoprotein on the external surface of the cytoplasmic membrane (Panicker et al, 2012).…”

“…The transformants containing the desired mutations were selected and plasmid DNA isolated using QIA-GEN Plasmid Midi kit (Qiagen) as described previously. The DNA sequences of the pTAP +2 plasmids were confirmed using BigDye Terminator v3.1 cycle sequencing (Perkin Elmer Applied Biosystems) and the plasmids then used to transform M. gallisepticum cells by electroporation and the colonies obtained picked and grown in mycoplasma broth containing gentamicin, as described previously (Panicker et al, 2012). The presence of the gentamicin resistance gene was confirmed by PCR using the oligonucleotide primers GmF and GmR (Table 1).…”

Disruption of the Membrane Nuclease Gene (MBOVPG45_0215) of Mycoplasma bovis Greatly Reduces Cellular Nuclease Activity

“…Intact M. bovis cells were treated with trypsin to partially digest cell surface proteins, as described previously (12,21). M. bovis cells were cultured, and the cell pellet was washed in 50 mM Tris, 0.145 M NaCl, pH 7.4 (Tris-salt [TS] buffer).…”