Suicide Vectors for allelic exchange in Cellulophaga Lytica

By Mehul Puri

Introduction 

C. Lytica 

The gram-negative marine-based bacteria C. lytica has a genome of 3,765,936 base pairs, including 3,303 protein-coding genes and 55 RNA genes (Pati et al., 2011). It can grow in a wide range of temperatures between 4 °C and 40 °C in an 8% NaCl concentration with optimal growth between 22 °C to 30 °C (Pati et al., 2011). In the absence of flagella and pili, C. lytica cells depend on gliding motility to transport themselves. This translocation mechanism is also used to form biofilm colonies, which consist of colonies of C. lytica cells that can grow on non- biological surfaces such as rocks and metals. This allows them to survive in hostile environments and colonize new environments with ease. (Hall-Stoodley et al., 2004). 

Additionally, biofilm colonies serve as a foundation for larvae growth due to chemical and physical cues (Unabia et al., 1999). The formation of biofilm colonies by C. lytica also produces iridescence or coloration created by light reflection on intricately organized cells resembling crystals, as shown in Figure 1 (DeSimone, 2021). Though this iridescence has not been observed in natural environments, it has been observed in colonies grown in lab environments, and the significance of this iridescence in nature has not been elucidated to date. It is one of the identifiers or markers of biofilm colonies for C. lytica cells (Kientz et al., 2016). 

Figure 1. Colonies of C. lytica grown on Black ink plates (Adapted from M. DeSimone’s thesis, DeSimone 2021) 

Goal 

This study hypothesizes that deletion of the GldB gene in the bacteria Cellulophaga Lytica is responsible for gliding motility and can disrupt the formation of colonies. The ability to disrupt the formation of uniform colonies of C. lytica can impact the biofilm formation and allow us to control the iridescence of the bacteria. 

Approach/Methodology 

Designing the Vector 

In this project, the PYT313 suicide vector (Donated by a collaborator’s lab, Dr. Yontao Zhu, Minnesota State University Mankato) was used as it works with F. johnsoniae related to C. lytica. The suicide vector, as shown in Figure 2, contains sacB and the promoter of F. johnsoniae, ompA, which is used to construct chromosomal gene deletions specific to gliding (Zhu, 2017). Additionally, PYT313 is resistant to the antibiotic ampicillin due to the presence of AmpR. 

Figure 2. The plasmid map of PYT313 donated by Dr. Yongtao Zhu indicating the presence of the sacB, erythromycin resistance (ermF), and the promoter of F. johnsoniae, ompA (Zhu et al., 2017). 

Four primers are designed to isolate the GldB (gliding motility) gene within the C. Lytica DNA and are then used to create a new suicide vector using PYT313. As shown in figure 3, primers a and d contain restriction enzyme sites on their 3’ and 5’ sites, respectively. These sites correspond to specified restriction enzyme sites on the PYT313 vector. Primers c and d are homologous 1 kb upstream and downstream of the GldB gene from the start and stop codons, respectively. Through three polymerase chain reactions (PCR), the AB fragment and CD fragment are used to create the AD fragment which contains the GldB gene with restriction enzyme sites upstream and downstream of the DNA (Francis et al.). 

 

Figure 3. Four primers are designed for Overlap PCR. Through two PCR rounds, the gene is removed from the bacteria C. Lytica and ligated onto the PYT313 suicide vector. Figure from (Francis et al.). 

Then, through double digestion, the PYT313 vector is digested at the two specified restriction enzyme sites. After running the gel purification through electrophoresis, the larger digested PYT313 DNA is extracted and ligated with the AD fragment containing the GldB gene. This creates a new vector specifically designed to replace the GldB gene within C. Lytica with an inactive copy of the gene through transformation and conjugation processes (Francis et al.). 

Transformation and Conjugation 

Bacterial transformation is the process of environmental DNA uptake by competent cells. In this project, chemically competent E. Coli S17 λ Pir cells are used to uptake the GldB gene-inclusive PYT313 suicide vector. S17 cells allow for better DNA transfer during conjugation, which is why DNA uptake during transformation is crucial for GldB gene deletion. (Chen et al.) Then, the transformed E. Coli S17 cells are conjugated with C. Lytica cells for a direct transfer of DNA. 

Bacterial conjugation directly transfers genetic material from the E. Coli S17 λ Pir cells to the C. Lytica. During the conjugation, the mutant GldB gene is introduced to the recipient C. Lytica. As shown in figure 4, a two-step homologous recombinant event occurs: first and second crossover. 

During the first crossover, C. Lytica acquires the plasmid from the S17 cells, including the ampicillin antibiotic resistance. The conjugated bacteria is isolated using antibiotics, and a second crossover event occurs using the SacB sucrose counter-selection gene. During this event, the remaining part of the vector is removed from the C. Lytica, including the ampicillin resistance, leaving behind the mutant GldB gene or a wild-type GldB gene. Colony PCR is then conducted to differentiate between the two outcomes. C. Lytica cells with mutant GldB gene are grown and tested for results (Old Reliable: Two-Step Allelic Exchange by Bitesize Bio). 

Figure 4. Two Step Homologous Recombinant Event occurs, creating two types of bacteria. First conjugated C. Lytica has a wild-type allele, and second has the desired mutant allele. Adapted from Old Reliable: Two-Step Allelic Exchange by Bitesize Bio. 

Projected Outcome 

To test if the combination of DNA transfer is successful, the conjugated C. Lytica cells are introduced to antibiotic ampicillin. If the cells survive, the conjugation was successful, otherwise the cells would deteriorate. Additionally, successful conjugation will result in the C. Lytica cells losing their ability of iridescence. 

Results 

Spring 2024 

During the Spring 2024 semester, getting results on the transformation and conjugation was emphasized rather than altering the PYT313 to create a new vector. C. Lytica and PYT313 were grown in BB2 Agar and LB Agar plates respectively, as shown in Figure 5. Afterwards, a 50 ml culture was made using the colonies from both plates. Additionally, a 50 ml culture of S17 λ pir cells was grown for transformation. Transformation protocol was conducted using the S17 cells and PYT313. Transforming the S17 λ pir cells using the PYT313 was successful as it resulted in colony growth on LB Agar plates. Colony growth on multiple plates with antibiotic present is shown in Figure 6.

Figure 5. Growth of C. Lytica and PYT313 in BB2 Agar and LB Agar plates. LB Agar plate has ampicillin antibiotic added to it which demonstrates PYT313 ampicillin resistance. 

Figure 6. LB Agar plates with PYT313 suicide vector transformed S17 λ pir cells. Growth shows successful transformation due to the presence of antibiotic ampicillin. 

However, conjugation was unsuccessful, as no growth was present in the Conjugation Plates after a week of incubating. This could be due to multiple factors such as too many antibiotics or less cell density of C. Lytica or transformed S17 cells before conjugation. Additionally, due to time constraints, multiple trials of transformation and conjugation could not be completed. 

Summer 2024 

During Summer 2024, our aim is to establish which primers to use for the PYT313 for the two- step allelic exchange and complete successful transformation and conjugation with the new vector. Because we know transformation is possible, and conjugation can also be achieved with multiple trials, we hope to achieve complete deletion/replacement of the GldB by the end of summer. Additionally, the OUR grants have greatly enhanced this project by providing funds for resources and have made my research goals possible.

References 

ChenInês, et al. “The Ins and Outs of DNA Transfer in Bacteria.” Science, vol. 310, no. 5753, 2 Dec. 2005, pp. 1456–1460, www.ncbi.nlm.nih.gov/pmc/articles/PMC3919525/, https://doi.org/10.1126/science.1114021. 

DeSimone, Mark, Development of Genetic Engineering Tools for the Iridescent Bacteria Cellulophaga lytica, A Thesis (2021) 

 Francis, Matthew S, et al. “Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components.” Methods in Molecular Biology, 12 Nov. 2016, pp. 11–31, pubmed.ncbi.nlm.nih.gov/27837478/, https://doi.org/10.1007/978-1-4939-6649-3_2. 

Accessed 13 June 2024. 

Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Nature Reviews Microbiology, 2(2), 95–108. https://doi.org/10.1038/nrmicro821 

Kientz, B. et al. A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms. Sci. Rep. 6, 19906; doi: 10.1038/srep19906 (2016). 

McBride, M. J., & Zhu, Y. (2013). Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes. Journal of Bacteriology, 195(2), 270–278. https://doi.org/10.1128/jb.01962-12 

“Old Reliable: Two-Step Allelic Exchange.” Bitesize Bio, 17 July 2018, bitesizebio.com/41461/old-reliable-two-step-allelic- exchange/#:~:text=The%20idea%20behind%20suicide%20vectors,understandably%2C%20can’t %20replicate. 

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Unabia, C., Hadfield, M. Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans. Marine Biology 133, 55–64 (1999). https://doi.org/10.1007/s002270050442