Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Plasmids are crucial extrachromosomal DNA elements in bacteria.
• Understanding plasmid biology is key to genetic engineering and biotechnology.
• Acidiphilium multivorum harbors unique plasmids relevant to extreme environments.

Purpose of the Study:

• To fully sequence and analyze the cryptic plasmid pAM5 from Acidiphilium multivorum JCM-8867.
• To characterize the functional elements of pAM5, including replication and mobilization modules.
• To develop a novel cloning shuttle vector for acidophilic bacteria and E. coli.

Main Methods:

• Complete DNA sequencing of plasmid pAM5 using HindIII-PstI fragment cloning and primer walking.
• Bioinformatic analysis to predict open reading frames (ORFs) and functional protein domains.
• Construction and functional testing of derivative plasmids (pSK1, pSK2) in various bacterial hosts.

Main Results:

• Plasmid pAM5 is 5161bp with five putative ORFs, including mobilization (Mob) and replication initiation (Rep) proteins.
• The oriT region and MOB(Q) family characteristics were identified, enabling mobilization to E. coli.
• A derivative plasmid, pSK2, containing the pAM5 replication module, replicates in Acidiphilium, Acidocella, and E. coli, serving as a shuttle vector.
• Replication of pSK2 in acidophiles is dependent on the pAM5 Rep protein.

Conclusions:

• Plasmid pAM5 is a cryptic theta-replicating plasmid with functional replication and mobilization modules.
• The pAM5 replication module is essential for replication in acidophiles.
• The derivative plasmid pSK2 is a versatile cloning shuttle vector for Gram-negative bacteria, including acidophiles and E. coli.