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Escherichia Coli Ribosomal Proteins Involved in Autogenous Regulation of Translation
Published in James F. Kane, Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
S4 is known to play a central role in organizing ribosome structure.48 S4, like S7 or S8, is required for the binding of several other r-proteins during the reconstitution of 30S subunits.40,49 It is likely that S4 interacts with one or several other r-proteins as well as with rRNA. S4, in fact, is buried in the 30S ribosomes by the presence of S5 and S12, at least to the extent that it is inaccessible to anti-S4 immunoglobulin.50 Several experimental results suggest that: (1) the N-terminal portion of the molecule is required for binding several other r-proteins, but is not required for binding to 16S rRNA (and hence, presumably to mRNA target sites); (2) the C-terminal portion of the molecule is essential for binding to 16S rRNA and also to mRNA target sites. First, removal of N-terminal 46 amino acids from the 203 residue protein by mild trypsin digestion did not abolish 16S rRNA binding activity, but the use of this altered S4 in ribosome reconstitution resulted in the formation of particles deficient in several r-proteins (SI, S2, S10, S18 and S21).51 Second, S4 molecules with up to 20% of the protein missing from the C-terminal end have been obtained through genetic mutations, mutations that suppress a phenotype of streptomycin-dependence.52,53 Loss of carboxy-terminal regions of S4 in such mutants prevents ribosome assembly and cellular growth at high temperatures.54 Such strains also oversynthesize proteins of the S4 regulatory unit, S11, S13, and S4, suggesting the involvement of the carboxy-terminal region of the S4 polypeptide in binding to its mRNA.55,71 In merodiploid strains carrying both mutant and wild type S4 alleles, only the wild type S4 molecule is incorporated into ribosomes while the truncated S4 protein is degraded.54 Thus, the carboxy-terminus of S4 probably contributes to the binding to 16S rRNA as well as to mRNA. Therefore, it appears that the S4 molecule is also subdivided into functionally different domains.
Dual RNA-seq reveals a type 6 secretion system-dependent blockage of TNF-α signaling and BicA as a Burkholderia pseudomallei virulence factor important during gastrointestinal infection
Published in Gut Microbes, 2022
Javier I. Sanchez-Villamil, Daniel Tapia, Nittaya Khakhum, Steven G. Widen, Alfredo G. Torres
All cloning methods were performed using the Gibson assembly system and primers used in this study are listed in Table 1. For the mutant construction, upstream (530 bp) and downstream (530 bp) of Bpm K96243 of bicA gene (BPSS1533) were amplified from genomic DNA and purified before assembly into a linearized PCR product of the suicide vector, pMo130 following the manufacturer’s recommendations. Assembled products were transformed into E. coli NEB-5α (NEB, Massachusetts) for clonal screening and the plasmids were confirmed by Sanger sequencing at GENEWIZ. The plasmid containing the fusion of upstream and downstream regions of BPSS153 were ultimately transformed into E. coli S17-1 λpir donor strain. Mobilizable vector was introduced into Bpm K96243 by biparental mating. Overnight culture (500 μl) of donor E. coli S17-1 λpir containing upstream-downstream/pMo130 plasmid and 12 h culture of a recipient Bpm K96243 (500 μl) were centrifuged separately or combined with equal volume then resuspended the pellet with 100 μl of PBS. The conjugation mixture was spotted on LB agar and incubated at 37°C for 8 h. Following conjugation, the spots were scraped and resuspended with 1 ml of PBS before plating on LB agar supplemented with 500 μg/ml kanamycin and 30 μg/ml of polymyxin B for Bpm transformant selection. Plates were incubated for 48 h at 37°C and the isolated colonies were exposed to 0.45 M pyrocatechol for merodiploid screening. Selected yellow colonies were sub-cultured in LB containing 100 μg/ml of kanamycin then incubated at 37°C with shaking for 12 h. Transformant colonies were grown in YT broth supplemented with 15% sucrose for 4 h at 37°C with shaking followed by serial dilution and plating onto YT agar +15% sucrose for resolved Bpm co-integrant selection. Following 48 h incubation at 37°C, resulting white colonies after exposure to pyrocatechol as mentioned above were analyzed by PCR, and sequenced to confirm gene deletion.