Recombinant Vibrio vulnificus Error-prone DNA polymerase (dnaE2), partial

Introduction

Recombinant Vibrio vulnificus Error-prone DNA polymerase (dnaE2), partial, refers to a genetically engineered version of a specific DNA polymerase enzyme derived from the bacterium Vibrio vulnificus . DNA polymerases are essential enzymes involved in DNA replication and repair processes in all living organisms. Vibrio vulnificus is a bacterium known to cause severe infections in humans, particularly through the consumption of contaminated seafood or wound infections .

Characteristics of Recombinant Vibrio vulnificus Error-prone DNA Polymerase (dnaE2), partial

• Source Organism: Vibrio vulnificus (strain YJ016)

• Type: Recombinant Protein

• Purity: Greater than 85% as determined by SDS-PAGE

• Sequence: The partial amino acid sequence is:
  MFYAELFCQS NFSFLTGASH PEELIVQANF LGYHSLAITD ECSLAGIVRA
  HSAIKQHQLS VKQIVGSFFK LNTECQFVLL CPNREAYAEL CRIITNARRR
  CEKGQYQLSQ WDVMSLKHCL LIWLPTGQTS DEQWATWLSR
  YQQSRLWLGL QRHLHHDDEH YLHHCQTLAN QFDLPITACG GVLMHRPERL

• Storage and Shelf Life:

  • Liquid form: Stable for 6 months at -20°C to -80°C

  • Lyophilized form: Stable for 12 months at -20°C to -80°C

• Uniprot No.: Q7MLY4

Function and Significance

1. Error-prone DNA Polymerase: The dnaE2 polymerase is characterized as "error-prone," meaning it has a higher tendency to introduce mutations during DNA replication compared to high-fidelity polymerases . This characteristic is significant in bacterial evolution and adaptation, allowing for rapid genetic changes in response to environmental pressures .

2. Role in Bacterial Pathogenesis: Error-prone polymerases can contribute to antimicrobial resistance and virulence in bacteria. The imp genes, related to error-prone repair systems, have been found in various bacteria such as E. coli, Shigella flexneri, and Salmonella . These genes enhance mutagenesis and survival of repaired genomes, potentially leading to the development of antimicrobial resistance .

3. DNA Repair Mechanisms: The error-prone DNA polymerase is involved in translesion synthesis (TLS), a DNA repair mechanism that allows DNA replication to proceed across damaged DNA templates. This process is essential for bacterial survival under stress conditions but can also introduce mutations .

Applications

1. Real-Time PCR Analysis: Real-time polymerase chain reaction (PCR) is a sensitive method used for detecting and quantifying specific DNA sequences in biological samples . Studies have shown the effectiveness of real-time PCR in detecting Vibrio vulnificus DNA in clinical specimens, such as tissues and blood .

2. Biotechnological Applications: Viral DNA polymerases, including those with strand displacement activity, have numerous biotechnological applications . They are valuable in in vitro DNA amplification and isothermal reactions, which are crucial in various molecular biology techniques .

3. Detection of Vibrio vulnificus: Recombinant DNA polymerases are used in developing methods for rapid and accurate detection of Vibrio vulnificus in food and clinical samples . For example, real-time recombinase polymerase amplification (RPA) methods targeting genes like empV have been developed for V. vulnificus detection, showing comparable sensitivity to qPCR methods .

Research Findings

1. Increased DNA Copy Numbers in Tissues: Research indicates that Vibrio vulnificus DNA copy numbers are higher in tissues compared to blood samples in infected patients . This finding suggests that tissue specimens, such as skin lesions, are more useful for PCR analysis, especially in patients who have received antibiotics before admission .

2. Role of imp Genes in Error-Prone Repair: Studies on E. coli have shown that imp genes, which encode error-prone DNA polymerases, contribute to bacterial survival and mutagenesis under stress conditions, such as exposure to bile salts . Bile salts can induce the SOS response, leading to the upregulation of error-prone repair mechanisms .

3. Identification of Virulence Genes: Dual RNA-seq analysis has been used to identify key virulence genes in Vibrio harveyi during the early stages of infection . Such studies provide insights into the molecular mechanisms underlying bacterial pathogenesis and can aid in developing targeted interventions .