Identification and characterization of the omaA gene encoding the major outer membrane protein of Azospirillum
S Burdman1, R De Mot, J Vanderleyden
1Department of Plant Pathology and Microbiology, and The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel.
Abstract:
The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligonucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilense in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.
Insights
Researchers purified the major outer membrane protein (MOMP) from Azospirillum brasilense and sequenced its gene. The Azospirillum brasilense MOMP sequence shows similarity to bacterial porins.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Chemistry
Background:
- Azospirillum brasilense is a nitrogen-fixing bacterium crucial for plant growth.
- The major outer membrane protein (MOMP) plays a vital role in bacterial structure and function.
- Understanding MOMP's structure can reveal insights into bacterial physiology.
Purpose of the Study:
- To isolate and characterize the gene encoding the major outer membrane protein (MOMP) of Azospirillum brasilense.
- To determine the amino acid sequence and structural properties of A. brasilense MOMP.
- To investigate the evolutionary relationship of A. brasilense MOMP with other bacterial proteins.
Main Methods:
- Purification of Azospirillum brasilense major outer membrane protein (MOMP).
- Oligonucleotide primer design based on amino acid sequences.
- Polymerase Chain Reaction (PCR) amplification and Southern hybridization.
- Gene cloning, sequencing, and bioinformatic analysis.
Main Results:
- A 766-bp PCR fragment was generated and used for screening a genomic library.
- A clone containing a 20-kb EcoRI insert was identified, from which a 3.5-kb fragment was subcloned.
- The sequenced gene encodes a mature protein of 367 amino acids with a molecular mass of 38,753 Da.
- The deduced amino acid sequence revealed a putative signal peptide and similarity to bacterial porins.
Conclusions:
- The gene for Azospirillum brasilense MOMP has been successfully isolated and sequenced.
- The deduced protein sequence suggests a porin-like structure and function.
- This study provides a foundation for further research into the role of MOMP in Azospirillum brasilense.
