Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

Imipenem Resistance Mediated by blaOXA-913 Gene in Pseudomonas aeruginosa

Treatment of infectious diseases caused by carbapenem-resistant Pseudomonas aeruginosa is becoming a greater challenge. This study aimed to identify the imipenem resistance mechanism in P. aeruginosa isolated from a dog. Minimum Inhibitory Concentration (MIC) was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute recommendations. We performed polymerase chain reaction and whole-genome sequencing to detect carbapenem resistance genes. Genomic DNA of P. aeruginosa K19PSE24 was sequenced via the combined analysis of 20-kb PacBio SMRTbell and PacBio RS II. Peptide-Peptide Nucleic Acid conjugates (P-PNAs) targeting the translation initiation region of blaOXA-913 were synthesized. The isolate (K19PSE24) was resistant to imipenem and piperacillin/tazobactam yet was susceptible to most of the tested antimicrobials. Whole-genome sequencing revealed that the K19PSE24 genome comprised a single contig amounting to 6,815,777 base pairs, with 65 tRNA and 12 rRNA genes. K19PSE24 belonged to sequence type 313 and carried the genes aph(3)-IIb, fosA, catB7, crpP, and blaOXA-913 (an allele deposited in GenBank but not described in the literature). K19PSE24 also carried genes encoding for virulence factors (exoenzyme T, exotoxin A, and elastase B) that are associated with adhesion, invasion, and tissue lysis. Nevertheless, we did not detect any of the previously reported carbapenem resistance genes. This is the first report of the blaOXA-913 gene in imipenem-resistant P. aeruginosa in the literature. Notably, no viable colonies were found after co-treatment with imipenem (2 µg/mL) and either of the P-PNAs (12.5 µM or 25 µM). The imipenem resistance in K19PSE24 was primarily due to blaOXA-913 gene carriage.

ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. ΔGunfold leaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5' UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

Switching at the ribosome: riboswitches need rProteins as modulators to regulate translation

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

Targeting of the Essential acpP, ftsZ, and rne Genes in Carbapenem-Resistant Acinetobacter baumannii by Antisense PNA Precision Antibacterials

Infections by carbapenem-resistant A. baumannii (CRAB), a widespread nosocomial pathogen, are becoming increasingly difficult to prevent and treat. Therefore, there is an urgent need for discovery of novel antibiotics against CRAB. Programmable, precision antisense antibiotics, e.g., based on the nucleic acid mimic PNA (peptide nucleic acid) have shown promise in this respect in the form of PNA-BPP (bacteria penetrating peptide) conjugates targeting essential bacterial genes. In the present study, we designed and synthesized a series of PNA-BPPs targeting the translation initiation region of the ftsZ, acpP, or rne gene of CRAB strains. The antimicrobial activity of the compounds and effects on gene expression level was compared to that of analogous mismatch PNA controls. Three antisense conjugates (KFF)3K-eg1-(acpP)PNA (5639), (KFF)3K-eg1-(ftsZ)PNA (5612), and (KFF)3-K-eg1-(rne)PNA (5656) exhibited complete growth inhibition against several CRAB strains at 1–2, 2–8, and 2 µM, respectively, and the compounds were bactericidal at 1–2× MIC. The bactericidal effect was correlated to reduction of target gene mRNA level using RT-qPCR, and the compounds showed no bacterial membrane disruption activity at 1–2× MIC. PNA5612 was tested against a series of 12 CRAB isolates and all were sensitive at 2–8 µM. In addition, the conjugates exhibited no cellular toxicity in the HepG2 cell line (up to 20 μM) and did not shown significant antibacterial activity against other Gram negatives (E. coli, P. aeruginosa). These results provide a starting point for discovery of antisense precision designer antibiotics for specific treatment of CRAB infections.

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

ABSTRACTTwo-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. They mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis.In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent controls. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second control requires only the very 5’ end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.

Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

The Evolution of G-quadruplex Structure in mRNA Untranslated Region

The RNA G-quadruplex (rG4) is a kind of non-canonical high-order secondary structure with important biological functions and is enriched in untranslated regions (UTRs) of protein-coding genes. However, how rG4 structures evolve is largely unknown. Here, we systematically investigated the evolution of RNA sequences around UTR rG4 structures in 5 eukaryotic organisms. We found universal selection on UTR sequences, which facilitated rG4 formation in all the organisms that we analyzed. While G-rich sequences were preferred in the rG4 structural region, C-rich sequences were selectively not preferred. The selective pressure acting on rG4 structures in the UTRs of genes with higher G content was significantly smaller. Furthermore, we found that rG4 structures experienced smaller evolutionary selection near the translation initiation region in the 5′ UTR, near the polyadenylation signals in the 3′ UTR, and in regions flanking the miRNA targets in the 3′ UTR. These results suggest universal selection for rG4 formation in the UTRs of eukaryotic genomes and the selection may be related to the biological functions of rG4s.