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Near Saturation of Ribosomal L7/L12 Binding Sites with Ternary Complexes in Slowly Growing E. coli.

ABSTRACT: For Escherichia coli growing rapidly in rich medium at 37?°C, the doubling time can be as short as ~20?min and the average rate of translation (ktrl) can be as fast as ~20 amino acids/s. For slower growth arising from poor nutrient quality or from higher growth osmolality, ktrl decreases significantly. In earlier work from the Hwa lab, a simplified Michaelis-Menten model suggested that the decrease in ktrl arises from a shortage of ternary complexes (TCs) under nutrient limitation and from slower diffusion of TCs under high growth osmolality. Here we present a single-molecule tracking study of the diffusion of EF-Tu in E. coli growing with doubling times in the range 62-190?min at 37?°C due to nutrient limitation, high growth osmolality, or both. The diffusive properties of EF-Tu remain quantitatively indistinguishable across all growth conditions studied. Dissection of the total population into ribosome-bound and free sub-populations, combined with copy number estimates for EF-Tu and ribosomes, indicates that in all cases ~3.7 EF-Tu copies are bound on average to each translating 70S ribosome. Thus, the four L7/L12 binding sites adjacent to the ribosomal A-site in E. coli are essentially saturated with TCs in all conditions, facilitating rapid testing of aminoacyl-tRNAs for a codon match. Evidently, the average translation rate is not limited by either the supply of cognate TCs under nutrient limitation or by the diffusion of free TCs at high osmolality. Some other step or steps must be rate limiting for translation in slow growth.

SUBMITTER: Mustafi M

PROVIDER: S-EPMC6554028 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Near Saturation of Ribosomal L7/L12 Binding Sites with Ternary Complexes in Slowly Growing E. coli.

Mustafi Mainak M Weisshaar James C JC

Journal of molecular biology 20190430 12

For Escherichia coli growing rapidly in rich medium at 37 °C, the doubling time can be as short as ~20 min and the average rate of translation (ktrl) can be as fast as ~20 amino acids/s. For slower growth arising from poor nutrient quality or from higher growth osmolality, ktrl decreases significantly. In earlier work from the Hwa lab, a simplified Michaelis-Menten model suggested that the decrease in ktrl arises from a shortage of ternary complexes (TCs) under n ...[more]

PMID: 31051175

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Project description:BackgroundWe generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals.Methods and resultsFour recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine.ConclusionThus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Dataset Information

Near Saturation of Ribosomal L7/L12 Binding Sites with Ternary Complexes in Slowly Growing E. coli.

Publications

Near Saturation of Ribosomal L7/L12 Binding Sites with Ternary Complexes in Slowly Growing E. coli.

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