Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6
M D Glew1, P F Markham, G F Browning
1Department of Veterinary Science, University of Melbourne, Parkville, Australia.
Abstract:
A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.
Insights
Mycoplasma gallisepticum expresses multiple pMGA genes, with pMGA1.1 being the most abundant. Transcriptional control appears to regulate pMGA gene selection in this avian pathogen.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- The avian pathogen Mycoplasma gallisepticum possesses a large pMGA gene family.
- Previously, only one pMGA gene member was known to be expressed in a single strain.
- This study investigates the expression of multiple pMGA genes across different strains.
Purpose of the Study:
- To analyze mRNA expression of the pMGA gene family in Mycoplasma gallisepticum strain 56.
- To understand the mechanisms of pMGA gene selection and regulation.
- To identify the predominant pMGA mRNA and its expression level.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect pMGA mRNA.
- Northern blot analysis quantified the relative abundance of different pMGA mRNAs.
- Magnesium concentration's effect on mRNA yield was assessed.
- Transcription start point and promoter for pMGA1.1 were determined.
Main Results:
- Four distinct pMGA mRNA molecules were detected, with pMGA1.1 mRNA being predominant.
- pMGA mRNA is 2.2 kb in size and monocistronic.
- Magnesium concentration significantly influenced the relative yields of pMGA mRNAs.
- pMGA1.1 mRNA was expressed at a level five times higher than the tuf gene.
Conclusions:
- Transcriptional control is a major factor in selecting pMGA gene expression in Mycoplasma gallisepticum.
- Multiple pMGA genes are transcribed, but expression levels vary significantly.
- The study provides insights into the regulation of virulence factor expression in M. gallisepticum.
