SD Sequence Research Articles

STnc1280, a trans-coding sRNA is involved in virulence modulation via targeting gldA mRNA in Salmonella Typhimurium.

Introduction. Salmonella Typhimurium (STM) is a food-borne Gram-negative bacterium, which can infect humans and a wide range of livestock and poultry, causing a variety of diseases such as septicaemia, enteritis and abortion.Hypothesis/Gap Statement. We will decipher the impacts of sRNA STnc1280 on STM virulence and provide a theoretical basis to reveal the regulatory role and molecular mechanism of STnc1280.Aim. The main objective of this study was to clarify whether sRNA STnc1280 exerts regulatory roles on STM pathogenicity.Methodology. The STnc1280 gene was amplified and its molecular characteristics were analysed in this study. Then, STnc1280 gene deletion strain (STM-ΔSTnc1280) and the complementary strain (ΔSTnc1280/STnc1280) were constructed by λ-Red homologous recombination method, respectively, to analyse of adhesion and invasive ability and pathogenicity of different strains. Subsequently, the potential target gene regulated by STnc1280 was predicted using target RNA2 software, followed by the verification of the interaction between STnc1280 and target mRNA using the dual plasmid reporter system (DPRS). Furthermore, the mRNA and protein level of target gene was determined using qRT-PCR and Western blot, respectively.Results. The results revealed that the cell adhesion and invasive ability and pathogenicity of STM-ΔSTnc1280 were significantly reduced compared to STM-SL1344 strain, indicating that the deficiency of STnc1280 gene significantly influenced STM pathogenicity. The DPRS results showed that STnc1280 can interact with the mRNA of target gene gldA, thus suppressing the expression of lacZ gene. Furthermore, the level of gldA mRNA was not influenced in STM-ΔSTnc1280, but the expression of GldA protein decreased significantly.Conclusion. Combining the bioinformatic analysis, these findings suggested that STnc1280 may bind to the SD sequence of gldA mRNA, hindering the binding of ribosomes to gldA mRNA, thereby inhibiting the expression of GldA protein to modulate the virulence of STM.

Effect of expansion of Shine-Dalgarno sequence for overexpression of nicotinamide adenine dinucleotide oxidase gene from Deinococcus geothermalis in Escherichia coli

Thermostable enzymes have applications in biosensors and bioreactors. Overexpression of thermostable enzyme-encoding genes from thermophiles in Escherichia coli is necessary to produce sufficient quantities of such enzymes. Deinococcus gerthermalis is a moderately thermophilic bacterium and has moderately thermostable enzymes. Nicotinamide adenine dinucleotide (NADH) is an important coenzyme for various dehydrogenases. NADH oxidase (Nox) catalyze the oxidation of NADH. Therefore, Nox is a versatile enzyme that can be used in combination with many types of biosensors. This study reveals the effect of expansion of the SD sequence in the leader sequence of NADH oxidase gene from D. geothermalis for overexpression in E. coli, and shows that while 1- or 2-bases expansion of the Shine-Dalgarno sequence is effective. This study should be contributed to the expression of many genes in E. coli.

The gatekeeper of Yersinia type III secretion is under RNA thermometer control.

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.

Regulation of the ThiM riboswitch is facilitated by the trapped structure formed during transcription of the wild-type sequence.

The ThiM riboswitch from Escherichia coli is a typical mRNA device that modulates downstream gene expression by sensing TPP. The helix-based RNA folding theory is used to investigate its detailed regulatory behaviors in cells. This RNA molecule is transcriptionally trapped in a state with the unstructured SD sequence in the absence of TPP, which induces downstream gene expression. As a key step to turn on gene expression, formation of this trapped state (the genetic ON state) highly depends on the co-transcriptional folding of its wild-type sequence. Instead of stabilities of the genetic ON and OFF states, the transcription rate, pause, and ligand levels are combined to affect the ThiM riboswitch-mediated gene regulation, which is consistent with a kinetic control model.

Increased production of alkaline polygalacturonate lyase in the recombinant WB43CB by optimizing the medium

Alkaline polygalacturonate lyase (PGL), which was the central enzyme of green environmental enzyme treatment technology, has been widely used in textile, papermaking and beverage production. In the previous work, Bacillus subtilis was used as the expression host, and the PGL gene derived from B. subtilis WSHB04-02 was successfully expressed in the engineered strain WB43CB by regulating of molecular elements such as signal peptide, promoter and SD sequence. In this study, the fermentation medium was optimized from four aspects of carbon source, nitrogen source, liquid volume and inoculum volume, and the extracellular PGL enzyme activity in WB43CB increased from 264.5 U·mL-1 to 461.96 U·mL-1, which laid a solid foundation for the industrial production of PGL.

High PGL productivity in Bacillus subtilis by optimizing the SD sequence

Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) can catalyze the cleavage of a-1, 4-glycosidic bonds of polygalacturonate by a trans-elimination reaction and generate an unsaturated oligogalacturonates. As the critical enzyme of many environmental friendly processes, alkaline PGL has been widely used in many fields including paper, textile and beverage industries. At present, Bacillus subtilis is an ideal strain for producing PGL, but the yield is too low for industrial production. In this study, the effect of different SD sequences on the production of PGL was comparatively investigated, and the strong SD sequence (AGAGAACAAGGAGGG G) directed efficient PGL secretory expression and increased PGL yield to 264.5 U·mL-1 with a high productivity. As a result, the PGL yield in B. subtilis was effecively increased and laid the solid foundation for PGL industrial production.

Global fitness landscapes of the Shine-Dalgarno sequence.

Shine-Dalgarno sequences (SD) in prokaryotic mRNA facilitate protein translation by pairing with rRNA in ribosomes. Although conventionally defined as AG-rich motifs, recent genomic surveys reveal great sequence diversity, questioning how SD functions. Here, we determined the molecular fitness (i.e., translation efficiency) of 49 synthetic 9-nt SD genotypes in three distinct mRNA contexts in Escherichia coli. We uncovered generic principles governing the SD fitness landscapes: (1) Guanine contents, rather than canonical SD motifs, best predict the fitness of both synthetic and endogenous SD; (2) the genotype-fitness correlation of SD promotes its evolvability by steadily supplying beneficial mutations across fitness landscapes; and (3) the frequency and magnitude of deleterious mutations increase with background fitness, and adjacent nucleotides in SD show stronger epistasis. Epistasis results from disruption of the continuous base pairing between SD and rRNA. This “chain-breaking” epistasis creates sinkholes in SD fitness landscapes and may profoundly impact the evolution and function of prokaryotic translation initiation and other RNA-mediated processes. Collectively, our work yields functional insights into the SD sequence variation in prokaryotic genomes, identifies a simple design principle to guide bioengineering and bioinformatic analysis of SD, and illuminates the fundamentals of fitness landscapes and molecular evolution.

A novel heterologous expression strategy for the quorum-quenching enzyme MomL in Lysobacter enzymogenes to the inhibit pathogenicity of Pectobacterium.

Quorum-quenching (QQ) enzymes can block the quorum-sensing (QS) system and prevent the expression of QS-controlled pathogenic factors in bacteria. However, the low expression levels of QQ proteins in the original host bacteria have affected their widespread application. In this study, we heterologously expressed momL, encoding a QQ enzyme with high activity, in Lysobacter enzymogenes. A "yellow-to-white" selection marker and the high-constitutive-expression promoter PgroEL were used in this novel heterologous expression system. In addition, we optimized the spacer between the SD sequence and the initiator to improve the efficiency of the expression system by 1.54-fold. The engineered strain LeMomL degraded the AHL molecule and the virulence factors of Pectobacterium carotovorum subsp. carotovora (Pcc). Additionally, LeMomL significantly decreased the disease caused by Pcc in Chinese cabbages and carrot root tissues. In conclusion, this novel and facile L. enzymogenes expression strategy has good prospects and is an ideal approach for foreign protein expression.

A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis

ABSTRACTCsrA is a widely conserved, abundant small RNA binding protein that has been found in E. coli and other Gram-negative bacteria where it is involved in the regulation of carbon metabolism, biofilm formation and virulence. CsrA binds to single-stranded GGA motifs around the SD sequence of target mRNAs where it inhibits or activates translation or influences RNA processing. Small RNAs like CsrB or CsrC containing 13–22 GGA motifs can sequester CsrA, thereby abrogating the effect of CsrA on its target mRNAs. In B. subtilis, CsrA has so far only been found to regulate one target, hag mRNA and to be sequestered by a protein (FliW) and not by an sRNA. Here, we employ a combination of in vitro and in vivo methods to investigate the effect of CsrA on the small regulatory RNA SR1 from B. subtilis, its primary target ahrC mRNA and its downstream targets, the rocABC and rocDEF operons. We demonstrate that CsrA can promote the base-pairing interactions between SR1 and ahrC mRNA, a function that has so far only been found for Hfq or ProQ.Abbreviations: aa, amino acid; bp, basepair; nt, nucleotide; PAA, polyacrylamide; SD, Shine Dalgarno.

Ethanol→Nicotine & Nicotine→Ethanol drug-sequence discriminations: Conditional stimulus control with two interoceptivedrug elements in rats.

Self-administration of ethanol (E) and nicotine (N) occurs frequently in tandem orders (i.e., N→E vs. E→N) and thereby produces differing interoceptive profiles of subjective effects in humans. If the interoceptive stimulus characteristics of N→E differ from E→N, it is possible that such differences contribute to their co-dependence. The rationale for the present investigation was to determine whether ethanol, when preceded or followed by nicotine, produces different discriminative stimulus effects in rats. In two experiments, using a one-manipulandum operant drug discrimination procedure, rats were trained to discriminate temporal sequential administrations of ethanol (1.0g/kg) that was followed or preceded by nicotine (0.3mg/kg). Sessions alternated between food-reinforcement sessions on a variable interval 30-sec schedule (i.e., SD) and non-reinforcement sessions (i.e., SΔ). In Experiment 1, administrations of ethanol were followed or preceded by a 10-min interval of nicotine. Training sessions took place 10min following the second drug injection. Four groups of rats were trained to discriminate only one sequence from sequential administrations of saline, and each drug sequence was counterbalanced across groups for their roles as SD or SΔ. There was robust stimulus control. N→E and E→N functioned equally well as SD or SΔ. Experiment 2 used two groups of rats. For one group, the E→N sequence functioned as the SD and the N→E sequence functioned as the SΔ. The drug sequences were counterbalanced for the other group. Brief non-reinforcement tests revealed significantly greater responding during the SD sequence compared to the SΔ sequence for both groups. These results suggest that different drug sequences of ethanol followed or preceded by nicotine established reliable discriminative stimulus control over operant responding, potentially because of characteristic differences in the overlapping pharmacokinetic profiles of the NE compound. The results are discussed in terms of: 1) conditional stimulus control among two interoceptive drug states; and 2) the clinical modulation of human alcohol consumption and tobacco smoking.

Molecular and Biochemical Aspects of the Ontogenesis of Eukaryotes Under Global Warming

Global warming is an irreversible process resulting in the deterioration of living conditions for various organisms, including the most important agricultural species. So-called σ32 factor of Escherichia coli is embedded into the RNA thermosensor in the λ cIII gene and plays an important role in the regulation of bacterial response to heightened temperatures. Expression of heat/cold shock genes and some virulence genes in response to temperature changes is coordinated by the genome. There are some known RNA thermosensors with different structures that provide a functional control of the diversity of cellular processes. The most common RNA thermosensor is the ROSE element suppressing expression of heat shock genes. A common feature of all ROSE elements is the presence of the G residue opposite to the SD sequence since this nucleotide is functionally important and its elimination makes the RNA thermosensor insensitive to high temperatures. In this paper, we describe molecular sequences (RNA thermosensors) whose chemical compounds influence on the homeostatic temperature regulation, namely, on the corresponding enzymes. Though the data on RNA thermosensors were obtained for microorganisms, it may be possible in the long run to change the animal genome at the molecular level by the insertion of these sequences or cultivation of symbiotic microorganisms, which may be used for production of biologically active compounds. In addition, such insertions would probably be able to reduce the negative effect of high environmental temperatures on living organisms.

Promoter engineering of cascade biocatalysis for α-ketoglutaric acid production by coexpressing L-glutamate oxidase and catalase.

Enzymatic transformation is now an attractive alternative for α-ketoglutaric acid (α-KG) production, but the oxidative deamination from L-glutamic acid to α-KG is along with H2O2 accumulation. To remove the effect of H2O2 on α-KG production, in vitro cascade biocatalysis was designed using the purified L-glutamate oxidase (LGOX) from Streptomyces ghanaensis and catalase (KatG) from Escherichia coli W3110, and the optimal ratio of LGOX:KatG (2.0:1250) was detected in this system. To achieve this ratio, in vivo cascade biocatalysis was constructed by varying promoters and ribosome binding sites (RBSs), and optimized by promoter engineering, such as adjusting the junctions between the SD sequence and start codon ATG and tuning the strengths of RBSs. When the final ratio of LGOX:KatG (2.1:1185) in strain E. coli-(T7)LGOX-(rbs2)KatG was used, α-KG concentration and its conversion rate were increased to 106gL-1 and 96%, respectively. This strategy described here paves the way to the development of cascade biocatalysis for enzymatic production of other chemicals.

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.

BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

Construction of genetic parts from the Corynebacterium glutamicum genome with high expression activities.

To construct effective genetic expression parts controlling transcription and translation initiation for synthetic biology and heterologous expression in Corynebacterium glutamicum. Twelve highly expressed genes were identified from the proteomic data of C. glutamicum. Their related sequences were used to construct bicistronic genetic expression parts. Each part contain promoter, 5'-UTR, N-terminal sequence of the source gene and a conserved SD sequence, associated with target gene, forming the bicistronic expression cassette. The enhanced green fluorescent protein (EGFP) expression levels controlled by these novel parts have 1.4 to 790-fold increase in C. glutamicum compared with corresponding promoter-5'-UTR part. One of the bicistronic parts is 1.35 times the EGFP expression of the constitutive-expression pXMJ19. These bicistronic parts had expression advantage compared with conventional promoter-5'-UTR parts. Various genetic parts for efficient gene expression can be quickly obtained via this new method.

Cloning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene in Anabaena sp. PCC7120

Human granulocyte-macrophage colony stimulating factor(hGM-CSF) is one of a family glycoprotein cytokines that have potent effects in stimulating proliferation,maturation and function of hematopoietic cells.The gene encoding hGM-CSF was PCR amplified from the plasmid pAGMT-8 and modified by addition SD sequence to promote prokaryotic expression.The resultant hGM-CSF was inserted into downstream of the strong promoter,PpsbA of expression vector pRL-439,then ligated with pDC-08 to produce the shuttle expression vector,pDC-GM.The resulting pDC-GM was transferred into the filamentous,heterocystour cyanobacterium,Anabaena PCC7120,by the tri-parental conjugation transfer method.When cells were cultivated without antibiotics for many generations,the transgenic cyanobacteria remained resistant to neomycin.PCR amplification of wild type and transgenic Anabaena cells with primers P_1 and P_2 revealed no band in wild type and plasmid free cells,whereas transgenic cells yielded a about(390 bp) band.This is the first report of expression hGM-CSF in cyanobacteria cells.

Strength and Regulation of Seven rRNA Promoters in Escherichia coli.

The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.

RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis

Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine–Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis.

Initiation Factor 3 (IF3) does not have any Role in the Release of Messenger RNA and Transfer RNA from the Post-Termination Complex during the Ribosome Recycling Reaction Catalyzed by EF-G/GTP and RRF (Ribosome Recycling Factor)

At the end of translation of an ORF, the ribosome meets the termination codon and peptides are released by release factors 1, 2 and 3. The resulting post-termination complex (PoTC) is characterized to have one tRNA and mRNA per ribosome. We showed that it is disassembled by RRF and EF-G/GTP into mRNA, tRNA, and subunits (JBC, 1973, 248, 7580). On the other hand, others claimed that RRF and EF-G split the ribosome but do not release mRNA or tRNA. IF3 was claimed to be necessary to release mRNA and/or tRNA from the complex of 30S/tRNA/mRNA. This wrong conclusion is due to the use of short ORF with strong SD sequence (Mol Cell., 2005, 18, 675; Mol Cell., 2005, 18, 403). We use more natural PoTC which is free of the influence of the SD sequence, and show here that IF3 has no role in the release of mRNA and tRNA from the PoTC. We (RNA, 2005, 8, 1317) and others showed that splitting of subunits occurs due to RRF and EF-G. It has been known that IF3 prevents association of subunits. However, its effect on the rate of ribosomal splitting by EF-G/RRF remains to be elucidated. In this paper, we will present the results which shed lights on this question.