Weak Promoter Research Articles

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systemsmetabolic strategies.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine β-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

CLCN5 5’UTR isoforms in human kidneys: differential expression analysis between controls and patients with glomerulonephritis

ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed...

Sudden death in Dakar

Sudden adult death is an instantaneous or rapid death of natural, unexpected or unexpected origin. As a public health issue, the global incidence of sudden death would be 20100 per 100,000 population.The main objective of our study was to assess the incidence of sudden death in Dakar. The specific objectives were to establish the epidemiological profile of sudden death, identify the circumstances of its occurrence and the medical and surgical causes, and identify the factors associated with sudden deaths from cardiovascular causes.It is an observational, descriptive, retrospective study covering a period of 5 years, from January 1, 2014 to December 31, 2018. Included in the study were all records of adults (18 years of age and over) who died suddenly, in a health facility or outside it and were autopsied in the morgues of Aristide le Dantec Hospital (HALD) and Idrissa Pouye General Hospital (HOGIP).We found 1133 cases of sudden adult death, or 28 % of autopsies. The annual average was 226.6 cases. Our series consisted of 918 men and 215 women, for a sex ratio of 3.27. The sudden death of the adult was predominantly cardiovascular in origin with 561 cases or 49.5 %. The MI occurred on average at 51.95 years of age with 164 males (84.5 % of MIs) and 30 females (15.5 % of MIs). With regard to pulmonary causes, pulmonary tuberculosis was the leading cause of pulmonary origin with 66 cases or 26.2 %. For digestive causes, generalized acute peritonitis (GAP) was the most frequent cause with 29 cases (38.7 %).The prevention of sudden death is essentially based on cardiovascular disease treatment and prevention. The African context has to deal with weak health promotion, a failure of pre-hospital care and the daily difficulties of hospital care.

Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

BackgroundTransposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony.ResultsWe analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

Exons as enhancers.

A study in Cell identifies a mechanism by which exon splicing can increase gene expression through the activation of weak upstream promoters.

Exon-Mediated Activation of Transcription Starts

The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, ourfindings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution ofalternative promoters and gene expression in mammals.

Discrete and continuous models of probability flux of switching dynamics: Uncovering stochastic oscillations in a toggle-switch system.

The probability flux and velocity in stochastic reaction networks can help in characterizing dynamic changes in probability landscapes of these networks. Here, we study the behavior of three different models of probability flux, namely, the discrete flux model, the Fokker-Planck model, and a new continuum model of the Liouville flux. We compare these fluxes that are formulated based on, respectively, the chemical master equation, the stochastic differential equation, and the ordinary differential equation. We examine similarities and differences among these models at the nonequilibrium steady state for the toggle switch network under different binding and unbinding conditions. Our results show that at a strong stochastic condition of weak promoter binding, continuum models of Fokker-Planck and Liouville fluxes deviate significantly from the discrete flux model. Furthermore, we report the discovery of stochastic oscillation in the toggle-switch system occurring at weak binding conditions, a phenomenon captured only by the discrete flux model.

Weak MGMT gene promoter methylation confers a clinically significant survival benefit in patients with newly diagnosed glioblastoma: a retrospective cohort study.

Quantitative methylation specific PCR (qMSP) is a frequently used technique to assess MGMT gene promoter methylation in glioblastoma patients. The optimal technical cut-off value to distinguish methylated from unmethylated samples is nevertheless still undetermined. In literature, a "grey zone" of diagnostic uncertainty has been described. We performed a retrospective analysis of newly diagnosed glioblastoma patients treated according to the Stupp protocol. Epidemiological data were gathered from the individual patient files. MGMT gene promoter methylation status was determined on stored tumour samples using qMSP. A strong, weak or absent promoter methylation was determined based on Cq values (quantification value) of the MGMT and ACTB primers as well as a positive control sample. In total, 181 patient files were reviewed and included for statistical analysis. MGMT promoter hypermethylation was detected in 38.7% of glioblastoma patients. The median overall survival of unmethylated and strongly methylated patients was 10.1months and 19.7months respectively. Furthermore, 11% of the total patient cohort had a weak MGMT gene promoter methylation. The median OS in this subgroup was 15.4months, significantly better compared to the unmethylated cohort (P < 0.001). Multivariate Cox regression analysis showed weak MGMT promoter methylation as an independent prognostic parameter for overall survival. Glioblastoma patients with weak promoter methylation show a statistically significant longer overall survival when compared to clearly unmethylated patients. Patients with grey zone qMSP test results should receive additional molecular analysis in future to further direct individual therapy strategies.

Agglomeration and removal characteristics of fine particles from coal combustion under different turbulent flow fields

Turbulent agglomeration is a promising pretreatment technology for improving the removal of fine particles in industrial flue gas, which can improve the particle removal effect of dust removal equipment safely and economically. However, due to the complexity of turbulence mechanisms, the relationship between turbulent flow fields and the agglomeration of fine particles is not known with precision, resulting a weak promotion effect for particle removal with this pretreatment technology. In this work, three kinds of turbulent agglomerators were constructed to investigate the agglomeration and removal characteristics of fine particles under different turbulent flow fields. The results demonstrated that the turbulent agglomerator with small-scale and three-dimensional vortexes in the flow field had the best effect in improving the agglomeration and removal of fine particles. Two kinds of agglomeration modes in turbulent agglomeration were proposed, one being agglomeration between fine particles in the vortex area, and the other the capture of fine particles by coarse particles. Furthermore, the motion trajectory, relative velocity and residence time of fine particles of different sizes in different flow fields were calculated by numerical simulation to investigate the interaction mechanism of particle agglomeration and turbulent flow fields. The results showed that a flow field with small-scale and three-dimensional vortexes can reduce the Stokes number (StK) and the relative velocity of particles of different sizes, and extend their residence time in a turbulent flow field, so as to obtain a better agglomeration effect for fine particles.

Transcriptional Regulation and Mechanism of SigN (ZpdN), a pBS32-Encoded Sigma Factor in Bacillus subtilis.

Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis also encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to being encoded on the large, low-copy-number plasmid pBS32, which was lost during domestication. DNA damage triggers pBS32 hyperreplication and cell death in a manner that depends on ZpdN, but how ZpdN mediates these effects is unknown. Here, we show that ZpdN is a bona fide sigma factor that can direct RNA polymerase to transcribe ZpdN-dependent genes, and we rename ZpdN SigN accordingly. Rend-seq (end-enriched transcriptome sequencing) analysis was used to determine the SigN regulon on pBS32, and the 5' ends of transcripts were used to predict the SigN consensus sequence. Finally, we characterize the regulation of SigN itself and show that it is transcribed by at least three promoters: PsigN1 , a strong SigA-dependent LexA-repressed promoter; PsigN2 , a weak SigA-dependent constitutive promoter; and PsigN3 , a SigN-dependent promoter. Thus, in response to DNA damage SigN is derepressed and then experiences positive feedback. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon.IMPORTANCE Sigma factors are utilized by bacteria to control and regulate gene expression. Some sigma factors are activated during times of stress to ensure the survival of the bacterium. Here, we report the presence of a sigma factor that is encoded on a plasmid that leads to cellular death after DNA damage.

Identification of prokaryotic promoters and their strength by integrating heterogeneous features

The promoter is a regulatory DNA region and important for gene transcriptional regulation. It is located near the transcription start site (TSS) upstream of the corresponding gene. In the post-genomics era, the availability of data makes it possible to build computational models for robustly detecting the promoters as these models are expected to be helpful for academia and drug discovery. Until recently, developed models focused only on discriminating the sequences into promoter and non-promoter. However, promoter predictors can be further improved by considering weak and strong promoter classification. In this work, we introduce a hybrid model, named iPSW(PseDNC-DL), for identification of prokaryotic promoters and their strength. It combines a convolutional neural network with a pseudo-di-nucleotide composition (PseDNC). The proposed model iPSW(PseDNC-DL) has been evaluated on the benchmark datasets and outperformed the current state-of-the-art models in both tasks namely promoter identification and promoter strength identification. The developed tool iPSW(PseDNC-DL) has been constructed in a web server and made freely available at https://home.jbnu.ac.kr/NSCL/PseDNC-DL.htm

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus.

Capsular polysaccharide (CP) biosynthesis in Staphylococcus aureus is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in nongrowing cells. Expression of the corresponding biosynthesis gene cluster is driven by one promoter element (Pcap ). Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat (IR) that is crucial for cap expression. The essentiality of the IR is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for cap repressors such as SaeR, CodY and Rot. Heterogeneous CP synthesis is determined by SigB activity and repressor binding to the upstream region. SigB dependency and regulation by the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when transcription is rendered constitutive, suggesting additional posttranscriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.

Improved Monitoring of Low-Level Transcription in Escherichia coli by a β-Galactosidase α-Complementation System.

Genetically encoded reporter proteins are important and widely used tools for the identification and capture of a promoter, tracking the dynamic behavior of transcription, and the quantification of promoter activity. The sensitivity of the reporter gene is a critical factor for an ideal reporter system because weak transcriptional signal has usually failed to be detected using classical reporters. In this study, we present a novel reporter system for improved monitoring of transcription in E. coli based on β-galactosidase α-complementation. In this reporter system, the β-galactosidase activity resulting from the assembly of a reporter lacZα and an existing α-acceptor in advance serves as a measure of transcriptional activity in vivo. To validate the potential of the lacZα-derived reporter system, a series of artificial operons were constructed, and the moderately strong lac promoter, ara promoter, and weak pbr promoter were chosen as the detection promoters. The response profiles of lacZα was similar to that of wild type lacZ in artificial lac operons. Due to its small size and efficient expression profile, the detection sensitivity of a lacZα-derived reporter system was significantly higher than that of the traditional full-length β-galactosidase and the fluorescent protein mCherry reporter system in artificial ara operons. As expected, the response sensitivity of the lacZα-derived reporter system was also demonstrated to be significantly higher than that of the β-galactosidase and mCherry reporter systems in lead-sensitive artificial pbr operons. The lacZα-derived reporter system may prove to be a valuable tool for detecting promoter activity, especially low-level transcription in vivo.

Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter

Introduction: In basic research, it is essential to use an inducible promoter which can be controlled to express a small amount of protein for studying their roles in the cell. Pspac, a well-known weak promoter for Bacillus subtilis, uses isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. However, plasmids carrying this promoter such as pHCMC05 still have a disadvantage which harbors a repetitive DNA fragment of about 200 bp, resulting in structural instability in Escherichia coli, causing difficulty during cloning.&#x0D; Methods: In this study, we constructed a plasmid that does not carry the repetitive sequences and investigated plasmid structural stability in E. coli, then measured the β-galactosidase reporter gene (bgaB) expression in B. subtilis.&#x0D; Results: The constructed plasmid pHT2002 was stable over 56 generations while pHCMC05-bgaB was structurally instable and ultimately lost after 42 generations. BgaB activities and Western-blot indicated that BgaB-coding gene under control of IPTG-inducible promoter Pspac could be expressed at low levels.&#x0D; Conclusion: The study demonstrated that the new expression plasmid without the repeated sequences retained its structural stability in E. coli facilitating the cloning step. The expression plasmid with Pspac promoter for B. subtilis could be used to express a modest amount of the heterologous protein in the presence of IPTG.&#x0D;

Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis

BackgroundPromoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter.ResultsThe native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between − 35 box and − 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the − 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the − 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as β-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters.ConclusionComparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.

Improved GFP Variants to Study Gene Expression in Haloarchaea.

The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters Pfdx of the ferredoxin gene and P2 of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (gvp) genes in Halobacterium salinarum PHH1. The basal promoter strength dropped in the order PpA, PpO > PpF, PpD. All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of PpA and PpD were high, but lower compared to the Pfdx or P2 promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5′-untranslated regions (5′-UTRs) of three different gvp mRNAs. A deletion of the 5′-UTR always resulted in an increased expression, implying a negative effect of the 5′-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.

Polymorphisms of Gene Cassette Promoters of the Class 1 Integron in Clinical Proteus Isolates.

ObjectiveTo describe the polymorphisms of gene cassette promoters of the class 1 integron in clinical Proteus isolates and their relationship with antibiotic resistance.MethodsPolymorphisms of the gene cassette promoter in 153 strains of Proteus were analyzed by PCR and nucleotide sequencing. Variable regions of atypical class 1 integrons were detected by inverse PCR and nucleotide sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyze the phylogenetic relationships of class 1 integron-positive clinical Proteus isolates. Representative beta-lactamase genes (bla), including blaTEM,blaSHV,blaCTX-M-1,blaCTX-M-2,blaCTX-M-8,blaCTX-M-9,blaCTX-M-25 and blaOXA-1, and plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrC, qnrD, qnrS, oqxA, oqxB, qepA, and aac(6′)-Ib were also screened using PCR and sequence analysis.ResultsFifteen different gene cassette arrays and 20 different gene cassettes were detected in integron-positive strains. Of them, aadB-aadA2 (37/96) was the most common gene cassette array. Two of these gene cassette arrays (estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3) have not previously been reported. Three different Pc-P2 variants (PcS, PcWTGN-10, PcH1) were detected among the 96 Proteus strains, with PcH1 being the most common (49/96). Strains carrying the promoters PcS or PcWTGN-10 were more resistant to sulfamethoxazole, gentamicin and tobramycin than those carrying PcH1. Strains with weak promoter (PcH1) harbored significantly more intra- and extra-integron antibiotic resistance genes than isolates with strong promoter (PcWTGN-10). Further, among 153 isolates, representative beta-lactamase genes were detected in 70 isolates (blaTEM-1, 54; blaOXA-1, 40; blaCTX-M-3, 12; blaCTX-M-14, 12; blaCTX-M-65, 5; blaCTX-M-15, 2) and representative PMQR genes were detected in 87 isolates (qnrA, 6; qnrB, 3; qnrC, 5; qnrD, 46; qnrS, 5; oqxA, 7; aac(6′)-Ib, 13; aac(6′)-Ib-cr, 32).ConclusionTo the best of our knowledge, this study provides the first evidence for polymorphisms of the class 1 integron variable promoter in clinical Proteus isolates, which generally contain relatively strong promoters. Resistance genotypes showed a higher coincidence rate with the drug-resistant phenotype in strong-promoter-containing strains, resulting in an ability to confer strong resistance to antibiotics among host bacteria and a relatively limited ability to capture gene cassettes. Moreover, strains with relatively weak integron promoters can “afford” a heavier “extra-integron antibiotic resistance gene load”. Furthermore, the gene cassettes estX, psp and the gene cassette arrays estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 have been confirmed for the first time in clinical Proteus isolates. Beta-lactamase genes and PMQR were investigated, and blaTEM-1 and blaOXA-1 were the most common, with qnrD and aac (6′)-Ib-cr also being dominant.

Piperacillin-Tazobactam (TZP) Resistance in Escherichia coli Due to Hyperproduction of TEM-1 β-Lactamase Mediated by the Promoter Pa/Pb.

TEM-1, mediated by plasmid and transposon, is the most commonly encountered β-lactamase in Gram-negative bacteria. Four different promoters upstream of blaTEM-related genes have been identified: the weak P3 promoter, and the strong promoters Pa/Pb, P4, and P5. In this study, we investigated the genetic basis of a clinical strain of Escherichia coli (RJ904), which was found to be resistant to BLBLIs (β-lactam/β-lactamase inhibitors), including amoxicillin-clavulanate, ticarcillin-clavulanate (TCC), and piperacillin-tazobactam (TZP) but sensitive to third-generation cephalosporins. The conjugation test and S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) demonstrated that transfer of this resistance was mediated by a ca. 100 kb plasmid. The transformant with TZP resistance was screened out with the shortgun cloning. Sequence analysis revealed that the recombinant plasmid contained a blaTEM-1b gene with the strong promoter Pa/Pb. Different plasmids were cloned based on the clone vector pACYC184 with the insertion of the blaTEM-1b gene with promoters Pa/Pb or P3. Susceptibility to TZP was determined by the E-test, agar dilution, and broth microdilution. The level of blaTEM-1b-specific transcription was determined by quantitative real-time PCR. Substitution of Pa/Pb for P3 resulted in a 128-fold decline of the MIC value of TZP, from >1024 mg/L to 8 mg/L, and a significantly lower blaTEM-1b expression level. Hyperproduction of TEM-1 β-lactamase mediated by the promoter Pa/Pb was responsible for high resistance to TZP in E. coli. Our data show possible risks of resistance development in association with the clinical use of TZP. The blaTEM promoter modifications should be considered for whole genome whole-genome sequencing-inferred bacterial antimicrobial susceptibility testing.

Evolvability of orthologous genes

The project aims to generate a library of laboratory orthologous genes for studying evolvability. In this study, we have taken one of histidine biosynthetic genes hisA from Salmonella enterica through alternating rounds of weak selection (simulated by random mutagenesis through error-prone PCR and screens for partial loss of hisA function) followed by strong purifying selection (simulated by additional rounds of random mutagenesis and screens for restored hisA function). We have started from the wild-type hisA gene expressed from relatively weak arabinose inducible promoter ParaBAD. Twenty different single nucleotide polymorphisms (SNPs) that cause slow growth in the absence of histidine were isolated. In twelve of these deleterious mutants the activity was restored to wild-type levels by additional SNP after another round of random mutagenesis. In this study, in some of the compensated his A variants it was difficult to isolate further deleterious mutations, suggesting that these compensating mutations can mask the effects of additional deleterious mutations. By combining the compensating mutations with known deleterious mutations, complete or partial compensation was seen. Orthologs from one of the diverging lineages were also compared for their evolvability toward TrpF activity (ability to synthesize tryptophan) using fluctuation test. Two orthologs in this lineage showed higher evolvability compared to the wild type hisA.

Review on Factors Affecting Production and Marketing of Spices in Ethiopia

Spices are produced in different agro-ecologies of Ethiopia that generate income for producers and traders involved in marketing. They are very important cash crops used for food flavor and medication purpose. Even though, their importance the sector is constrained by different factors. The objectives of this review paper are to review factors affecting production of spices, to review factors affecting marketing of spices and to review opportunities of spices production in Ethiopia. Traditional way of farming, absence of seeds and planting materials, high frequency of weeding, high input requirement, high input cost, lack of drying facility, taking long time to dry, shortage of access to credit and extension, different disease and pests, wild animal competition, poor quality of output, absence of proper post harvest handling practices and others are factors affecting production of spices in Ethiopia. Low output price, poor market access and imperfect market information, capital constraints, limited processing of spices, adulteration, mismatch between demand and spices, transportation problems, unlicensed traders, theft, low government support, lack of value addition, price volatility, weak market research and promotion, poor market infrastructure, lack of effective linkage of stakeholders, lack of capacity building, low bargaining power of producers, limited ability of producers to enter in market due to limited time, skill, and resources are some of the factors that affect spices marketing in Ethiopia. Ethiopia is suitable area for spices production due to suitable climate condition and presence of various agro-ecologies, abundant land, cheap labor and favorable policy environment. By reducing different production and marketing constraints the country can boost production and become competitor in the world spices market within a short period of time. Keywords: Spices; constraints; opportunities DOI : 10.7176/JBAH/9-6-02 Publication date :March 31 st 2019