Analysis and Evaluation of Research Paper " The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB–dnaE2 gene cassette and generation of superior feather degrading bacteria"

 The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB–dnaE2 gene cassette and generation of superior feather degrading bacteria

Introduction

This era of the genome has brought in an unprecedented opportunity to engineer microorganisms for a multitude of diverse biotechnological applications. To accomplish this, the work and research requires rigorous scientific approaches rooted in methodical experimental design and execution. 

This paper explores the SOS pathway of TLS polymerase, mutagenesis and the generation of superior keratin breakdown pathways. Where polymerase I-III can stall at UV damage such as thymine dimmers in DNA strands, polymerase IV/V or TLS, Translesion polymerase, is able to move along and repair the damage, however by doing this it also induces mutation, a method of survival to continue the replication cycle of DNA. The team then continues to explore these genes by performing knockouts to deduce that the imuB and dnaE2 genes in the cassette are crucial to the production of TLS polymerase and the ability to therefore induce mutations with UV radiation. 

The team goes on to produce a mutation of D. ficus with increased keratinolytic activity. Feathers, containing mostly keratin, are waste products typically found in the poultry industry. They are difficult to degrade due to the rigid structures caused by disulfide bonds and cross-linkages, so having an efficient and effective way to decompose or bio-convert this material is beneficial to both the industry and the environment and by manipulating these natural methods of UV repair and mutagenesis a more effect way effective way of extracting these valuable proteins can be achieved. 

This also opens up other avenues of exploring methods and pathways of other proteins.

Methods & Materials Used

    Bacterial culture conditions and growth medium

• D. ficus and Grandis inoculated in NB (Himedia)
• Mutation frequency determined with concentration of 20ug/ml Rifampicin
• Transformation experiment of D. ficus used two-layer of NA plates. 
• Lower Layer contained 17g agar/8ug/ml ampicillin
• Upper Layer 7g without ampicillin
• Vector Transformation was cultivated with E. Coli (DH5a) in LB + 80ug/ml amp

    Phylogenetic Tree Construction

• To determine if ImuN protein exist in other Deinococcus species, they aligned ImuB sequence from D .Ficus to ImuY protein sequence from D. Deserti
• The Conserved ImuB as identified as F:HPGVPVAV and R:WLDPWAY
• 16S rDNA sequences between D. Ficus and D. Grandis have 96.1% similarity. 
• Determined if ImuB protein exist in D. Grandis or not
• CimuB Primers used to amplify ImuB section.
• Aligned using ClustalX
• Phylogenetic tree constructed with MEGA4, 10k replicates
• DnaE2 protein sequence of D. Ficus deposited in GenBank DB.         

    Construction of D. Ficus imuB and dnaE2 gene knockout strains-imuB- and dnaE2-

• imuB and dnaE2 genes were knocked out using insertion inactivation mutagenesis method
• recombinant vector constructed, (imuB - amp - dnaE2)
• Introduced to competent D. ficus cells to insert amp resistance gene into imuB or dnaE2 gene by homologous recombination.
• Confirmed by PCR
• (Shen and Young 2005) method used for DNA isolation
• Double Recombination then preformed by PCR
• imuB and dnaE2 gene disruption were then performed using similar methods. 
• Reactions were then amplified via PCR and ran on gel for confirmation of knockout

    Determination of UV survival and mutation. 

• For survival rate analysis
• D. ficus wild-type (CC-FR2-10) and mutant strains (imuB- and dnaE2-) were grown to pre-stationary phase (approximately 5 × 108 CFU/ml)
• 5ml of culture was UV-irradiated @ 999 mJ/cm2 in classic petri dish for 5 and 10 minutes. 
• Aliquots were removed before and after irradiation, diluted and plated on NA. 
• Colonies were then counted after 48h @ incubation of 30C
• For mutation rate analysis 
• WT, imuB- and dnaE2- were grown to pre-stationary phase
• 5ml of each culture was irradiated with UV for 10 min.
• Before and after 1ml of culture was added to 5ml of NB and incubated @ 30C, 150rpm for 48 hours. 
• 0.1ml spread on NA containing rifampicin, incubated at 30C till colonies formed. 

    Determination of Keratinolytic Activity 

• 10-minute UV exposure
• Viable cells isolated and inoculated in liquid feather medium
• Soluble keratin prepared from black chicken feather using method (Wawrzkiewicz 1987)
• Activity was determined by method (Gradisar 2005)

    Evaluation of UV-induced mutagenesis in D. ficus mutant strain

• Determined using the Bio-Rad Assay
• Compared zymogram profiles of extracellular proteins in WT, CC-ZG207 and CC-ZG227 strains of ficus. 

General Analysis and Take Away

    Deinococcus as a genus has been observed to be almost famously effective at DNA repair of damage caused by ionizing radiation and desiccation. Unlike D. ficus, both D. radiodurans and D. geothermalis were not mutable by UV, and accurately repaired DNA damaged caused by this in the absence of TLS Polymerase genes in their genome. Through knockout the team found and confirmed that imuB and dnaE2 are essential at the production of TLS polymerase. This function is then exploited to induce mutations in D. ficus and grown in a liquid chicken feather broth to simulate the high keratin environment to produce a more efficient strains at breaking down keratin. 

Future Plans

Regarding the future of D. ficus in our lab over the summer and into the fall we plan on transforming D. ficus with pRAD1 from E. coli as a starting point. Exploring the SOS mutagenesis genes and it's resistance to UV and ability to repair in general.  

By using the methods of inducing UV lesions and letting TLS polymerase create mutations we can attempt to replicate the methods performed on other genes of interest. 

The past hasn't presented too fruitful in terms of much progress with D. Ficus. We have prepared competent cells for transformation, where we have also started to prep the e. Coli plasmid for uptake to the competent D. ficus cells. So far, we have run the gel to confirm the presence of pRAD1 plasmid, which has provided some interesting results of a 4th band that appears to be a multimer, but more research is needed to confirm. 

Glossary of Terms

Gene Cassette - A group of linked genes, co-transcribed and functionally related. Often involved in the same process or pathway. 

lexA2-imuA-imuB-dnaE2 - A specific gene cassette found in certain bacterial species. It encodes specifically encodes proteins involved in SOS response to DNA damage and translesion DNA synthesis.

    lexA2 - regulator gene

    imuA & imuB  - mutagenesis proteins

    dnaE2 - encodes error-prone DNA polymerase. 

TLS (Translesion Synthesis) Polymerase - Specialized DNA polymerase that can replicate across DNA lesions or damage that would normally stall the more higher-fidelity polymerase. TLS typically has lower fidelity and can introduce mutations along the way

Keratinolytic - The ability to degrade or breakdown keratin. 

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) - Analytical technique used to separate proteins based on molecular weight. 

Zymogram Analysis - Technique that combines electrophoretic separation of proteins via SDS-PAGE, and an in-gel enzyme activity assay. 

Mutagenesis - Process of inducing genetic mutations, either spontaneously or through the use of mutagens like radiation or chemicals. Increases genetic diversity within a population.

Phylogenetic Tree - A branching diagram that represents the inferred evolutionary relationship and ancestry among biological entities based on similarities and differences in their genetic characteristics.

ClustalX - tool used to perform multi-sequence alignment and analysis of protein or nucleic acid sequences. It requires providing one or more sequence files (e.g. fasta format) and will perform an alignment of the sequences, bringing into alignment the conserved or identical amino acid or nucleotide positions. 

MEGA4 - analysis program that allows vector-based sequence annotation, alignment viewing, and phylogenetic tree construction.

Rifampicin - an antibiotic used in microbiology research and experiments. Frequently used for selection of bacterial mutants that are resistant to the antibiotic. 

AI NOTES

D. ficus naturally produces keratinase enzymes that can degrade feather keratin.

Microbial keratinolytic enzymes have potential industrial applications in processes like:

• Conversion of feather waste into animal feed/fertilizers
• Dehairing in the leather industry
• Prion decontamination

This gene cassette might give indication to the flexibility of UV-inducible mutation in D. ficus

TLS polymerases have more open and flexible active sites that can accommodate distorted DNA structures caused by lesions. They are able to insert nucleotides across from the lesion, allowing the replication fork to continue rather than stalling

The discovery and characterization of a gene cassette involved in TLS polymerase activity and mutagenesis in the radiation-resistant bacterium Deinococcus ficus, with implications for generating mutants with improved feather-degrading capabilities.

While regular DNA polymerases stall at lesions to maintain replication accuracy, TLS polymerases sacrifice accuracy to allow lesion bypass and continued replication, introducing mutations in the process. This TLS mechanism allows cells to survive acute DNA damage at the risk of increased mutagenesis.

While regular polymerases prioritize replication accuracy and fidelity, TLS polymerases sacrifice accuracy to allow lesion bypass and permit replication on damaged templates, introducing mutations but preventing replication blockage and cell death after DNA damage.