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Abstract:
This episode of "This Week in Microbiology" (TWIM 330) delves into two recent publications. The first segment discusses a Nature paper detailing the engineering of a genomically recoded E. coli strain ("ochre") that utilizes only a single stop codon (UAA). This feat involved replacing nearly 10,200 instances of the other two stop codons (UAG and UGA) genomically, modifying the RF2 release factor, and altering tryptophan tRNA to prevent unwanted recognition of the newly freed codons. While successfully demonstrating the incorporation of non-standard amino acids with high fidelity using the freed codons, the engineered strain suffers from a significant fitness defect (reduced growth rate), presenting a challenge for practical applications like novel protein synthesis or enhanced biocontainment.
The second segment explores an mBio paper investigating the molecular basis of co-aggregation between the oral bacteria Veillonella parvula and Streptococcus gordonii, key players in dental plaque formation. Using genetic tools on a naturally competent V. parvula strain, researchers identified specific Trimeric Autotransporter Adhesins (TAAs), particularly VTAA, VTAD, and VTAE, that mediate binding to different early colonizers (S. gordonii, S. oralis, Actinomyces oris). The study highlights the metabolic relationship where V. parvula consumes lactate produced by streptococci and demonstrates that disrupting these specific adhesions prevents proper mixed biofilm formation, leading to segregated structures. This research offers insights into the organized nature of dental plaque and potential targets for intervention. The episode also includes background on the first author of the second paper.
This Week in Microbiology (TWIM) 330: Recoding Genomes and Dental Biofilms
0:00:02 Introduction: Episode introduction, host check-ins, and brief weather updates from various locations.
0:02:07 Support Call: Request for listener support for the non-profit podcast production via microbe.tv/contribute.
0:02:28 Snippet 1 Intro: Engineering E. coli with One Stop Codon: Introduction to a Nature paper on creating a genomically recoded organism ("ochre") using only the UAA stop codon.
0:03:36 Genetic Code Background: Explanation of the degenerate genetic code, the three standard stop codons (UAA, UAG, UGA), and previous work eliminating the UAG codon in E. coli.
0:04:01 Recoding Goal: The aim was to eliminate the UGA stop codon as well, leaving only UAA, thereby "releasing" UAG and UGA for reassignment to incorporate non-standard amino acids.
0:06:07 Challenge 1: Mass Codon Replacement: Researchers had to replace nearly 10,200 TGA codons with TAA using Multiplex Automated Genome Engineering (MAGE), carefully excluding genes requiring UGA for selenocysteine incorporation (e.g., formate dehydrogenases).
0:08:27 Side Effect & Mitigation: The MAGE process introduced hundreds of unintended background mutations. Fitness (growth rate) was used as a selection criterion to carry forward faster-growing variants.
0:09:12 Challenge 2: Modifying Release Factor RF2: RF2 normally recognizes UGA. It needed modification to reduce UGA termination activity without losing its other essential functions. Specific mutations (e.g., R170K) were identified using structural modeling (AlphaFold) and assays.
0:11:25 Assay Technique: Fluorescence reporters were used to quantify stop codon read-through efficiency.
0:12:01 Challenge 3: Preventing tRNA Suppression: The tryptophan tRNA was found to read the UGA codon; the tRNA gene was mutated to abolish this recognition while still allowing tryptophan incorporation via its standard codon.
0:12:54 Challenge 4: Fitness Defect: The final "ochre" strain exhibited a significant fitness defect, with doubling time reduced by ~50%, largely attributed to the necessary RF2 modifications. Further "tuning" is required for practical use.
0:13:31 Successful Reassignment: Despite the fitness cost, the system allowed the incorporation of two different non-standard amino acids at the freed UAG and UGA codons with over 99% accuracy.
0:15:56 Potential Applications: Discussed uses include synthesizing proteins with novel properties and potentially enhancing biocontainment of genetically modified organisms.
0:20:37 Snippet 2 Intro: Dental Plaque Co-aggregation: Introduction to an mBio paper identifying adhesions mediating the interaction between Veillonella parvula and Streptococcus gordonii.
0:21:44 Dental Plaque Background: Explanation of plaque as a complex poly-microbial biofilm, its role in dental caries, gingivitis, and periodontitis, and the structural role of V. parvula as a "bridging species."
0:27:21V. parvula's Metabolic Role: Highlighted as a keystone species that consumes lactate (a byproduct of streptococci fermentation that causes enamel demineralization), thus potentially reducing cavities.
0:28:44 Research Challenge & Tool: Progress was previously limited by a lack of genetic tools for V. parvula. This study utilized a naturally competent strain (SKV38).
0:29:34 Adhesins of Interest: Focus on V. parvula's cell surface Trimeric Autotransporter Adhesins (TAAs), specifically VTAs and VMAs.
0:34:08 Experimental Approach: Co-aggregation assays were performed by mixing cultures of wild-type or specific V. parvula adhesion deletion mutants with various oral bacteria partners (S. gordonii, S. oralis, Actinomyces oris).
0:35:41 Key Findings: Specific Adhesions Identified: Different VTA proteins mediate binding to specific partners: VTAA involved with S. oralis; VTAA and VTAE with Actinomyces oris; VTAD and VTAE with S. gordonii. These adhesions do not seem to bind host cells.
0:39:38 Structural Importance: Preventing co-aggregation by deleting the relevant adhesions resulted in segregated biofilms, indicating these specific interactions are crucial for the integrated structure of the mixed biofilm.
0:41:08 Physiological Impact & Model: Transcriptomic analysis supported the findings. A model (Fig 7) summarizes the identified V. parvula adhesions (VTAA, D, E) and a new S. gordonii adhesion involved in co-aggregation, impacting partner physiology and biofilm structure.
0:41:45 Intercellular Communication: Adhesion brings cells closer, likely enhancing the efficiency of processes like quorum sensing and metabolite exchange (like lactate consumption).
0:44:15 Potential Applications & Discussion: Findings could inform strategies to disrupt plaque formation (e.g., mouthwash ingredients targeting specific adhesions). Discussion touches on the balance between needing lactate consumers (V. parvula) and the acid production by its partners.
0:46:12 Author Background: Details about the first author (Lewis Dorison) of the mBio paper, his academic journey, overcoming challenges (dysgraphia), mentors, collaborations, and reflections on the scientific process.
0:53:00 Closing Remarks: Podcast wrap-up and credits.