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.
Highlights
Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry
Construction of plasmid ltufacyphoA The elongation factor Tu promoter region of 277 bp (GenBank accession: X16462) and the leader sequence of the vlhA1.1 gene (GenBank accession: U90714) from M. gallisepticum were originally amplified by PCR from the genomic DNA of M. gallisepticum strain S6 and ligated into the pISM2062.2lac [14] vector to produce the ltufsiglac construct [20]
The ltuf promoter region was amplified from M. gallisepticum genomic DNA by PCR using the LNF and TSR oligonucleotide primers (Table 1), and the vlhA export signal sequence of 51 bp was amplified from M. gallisepticum genomic DNA using the TSF and LBR primers
Summary
Mycoplasma 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. Mycoplasmas cause disease in a number of animal species and are able to survive and persist in the face of host defences, even though they possess a relatively small genome and are bounded by a single protective plasma membrane. The proteins within the single limiting membrane of mycoplasmas fulfill many of the critical functions related to morphology, nutrient transport, environmental adaptation and colonisation of the host [4]. Mycoplasmas possess an unusually large number of lipoproteins, which are anchored to the cell membrane by a lipid moiety, with the polypeptide moiety exposed on the cell’s outer surface [8]. The significance of mycoplasma lipoproteins in interactions with the host emphasises the need to better understand how they are processed, and the mechanisms controlling their expression [4]
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