High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell

OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.

大肠埃希菌trp operon的克隆与表达

色氨酸操纵子所表达酶的高效表达和酶活性的提高,从而构建高产色氨酸菌株。利用PCR的方法从大肠埃希菌基因组中直接克隆色氨酸操纵子,并将其连接到原核表达载体pBV220中,得到重组质粒pBV220-trp operon,转化大肠埃希菌DH5α,温度诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。通过凝胶电泳观察PCR扩增产物大小约为7 kb,SDS-PAGE鉴定目的蛋白得到了高效表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了3.4倍和2.5倍。成功构建了重组质粒pBV220-trp operon,邻氨基苯甲酸合成酶和色氨酸合成酶的表达量和表达活性在大肠埃希菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。

Cloning Whole Cellulose-Synthesizing Operon (ayacs Operon) from Acetobacter xylinum and Transforming It into Cultivated Cotton Plants

The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose-synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acs operon sequence of A. xylinum ATCC 53582. Alignment of the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302-3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen-tube-pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.

Enhanced Riboflavin Production by Expressing Heterologous Riboflavin Operon from B.cereus ATCC14579 in Bacillus subtilis

Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

MamZ protein plays an essential role in magnetosome maturation process of Magnetospirillum gryphiswaldense MSR-1

Based on analysis of gene structure of mamXY operon in Magnetospirillum gryphiswaldense strain MSR-1,we constructed a mamZ deletion mutant strain(ΔmamZ)and four complemented strains with different mamZ fragment lengths.Various cell phenotypic and physiological parameters were evaluated and compared among the wild-type(WT),mutant,and complemented strains.Cell growth rates were not notably different;however,magnetic response(Cmag)and iron uptake ability were significantly lower inΔmamZ.High-resolution transmission electron microscopy(HR-TEM)showed that magnetosomes inΔmamZ were small and irregular,and rock magnetic measurements suggested that they contained immature particles.In comparison to WT of MSR-1,intracellular iron content ofΔmamZ and the complemented strains cultured with 20mmol/L iron source was similar or slightly higher.The complemented strains were unable to synthesize mature or normal amounts of magnetosomes,apparently because of abnormal expression of the transmembrane domain of MamZ protein.Real-time reverse transcription polymerase chain reaction(RTqPCR)analysis showed that relative transcription levels of mamX and ftsZ-like genes inΔmamZ were higher at 18 h than at 12 h,suggesting that MamXY proteins play cooperative functional roles in the magnetosome maturation process.Transcription level of mms6 was significantly upregulated inΔmamZ(incubated at 12 h)and the complemented strains(incubated at 12 and 18 h),refl ecting possible interaction between MamXY and Mms6 proteins during magnetosome biosynthesis.These findings,taken together,demonstrate the essential role of MamZ in the magnetosome maturation process in MSR-1.

Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system

Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.

Effect of Riboflavin Operon Dosage on Riboflavin Productivity in Bacillus Subtilis

After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.

Structural analyses of the interactions of SoxY and SoxZ from thermo-neutrophilic <i>Hydrogenobacter thermophilus</i>

Microbial redox reactions of inorganic sulfur compounds are one of the important reactions responsible for the recycling of this element to maintain the environmental sulfur balance. These reactions are carried out by phylogenetically diverse set of microorganisms. The sulfur oxidizing gene cluster (sox) of thermo-neutrophilic bacterium Hydrogenobacter thermophilus consists of soxYZAXB. The bacterium shows optimal thiosulfate oxidation activity at 60°C. There are practically no reports regarding the structural biology of the sulfur oxidation proc- ess in this organism. In the present context, we employed homology modeling to construct the three dimensional structures of SoxY and SoxZ from Hydrogenobacter thermophilus. With the help of docking simulations we have identified the amino acid residues of these proteins in- volved in the interactions. The thermodynamics of the protein-protein interactions have also been analyzed. The probable biochemical mechanism of the binding of thiosulfate has been elucidated. Our study provides a rational framework to understand the molecular mechanism of the sulfur oxidation biochemistry.

Legionella dumoffii Tex-KL Mutated in an Operon Homologous to traC-traD is Defective in Epithelial Cell Invasion

Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.

Modification of the rib operon derived from Bacillus subtilis and its expression in Escherichia coli

A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.

Collaborative impact of bacterial exometabolites governing root microbiota formation

The majority of the root microbiota formation derives from soil-dwelling microorganisms.The limited extent of thorough investigation leads to a dearth of knowledge concerning the intricate mechanisms of microbe-microbe interaction implicated in the establishment of root microbiota.Therefore,the taxonomic signatures in bacterial inhibition profiles were determined by in vitro testing of 39,204 binary interbacterial interactions.However,findings from genetic and metabolomic studies elucidated that co-functioning of the antimicrobial 2,4-d iacetylphloroglucinol(DAPG)and the iron chelator pyoverdine as exometabolites has significantly contributed to the potent inhibitory activities of the highly antagonistic Pseudomonas brassicacearum R401.Microbiota restoration with a core of Arabidopsis thaliana root commensals showed that these exometabolites possess a root niche-specific function in establishing root competence and inducing anticipated changes in root surroundings.Both biosynthetic operons are abundant in roots in natural habitats,indicating that these exometabolites co-functioning is an adaptive feature that helps Pseudomonad dominate the root microbiota.

A DnaA-dependent riboswitch for transcription attenuation of the his operon

Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule.In this study,we first report a possible example of the DnaA‐dependent riboswitch for transcription attenuation in Escherichia coli.We show that(i)DnaA regulates the transcription of the structural genes but not that of the leader hisL gene;(ii)DnaA might bind to rDnaA boxes present in the HisL‐SL RNA,and subsequently attenuate the transcription of the operon;(iii)the HisL‐SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important;and(iv)the translating ribosome is required for deattenuation of the his operon,whereas tRNA^(His) strengthens attenuation.This mechanism seems to be phylogenetically conserved in Gram‐negative bacteria and evolutionarily important.

Bacillus sp.CDB3 isolated from cattle dip-sites possesses two ars gene clusters

Contamination of soil and water by arsenic is a global problem. In Australia, the dipping of cattle in arsenic-containing solution to control cattle ticks in last centenary has left many sites heavily contaminated with arsenic and other toxicants. We had previously isolated five soil bacterial strains （CDB 1-5） highly resistant to arsenic. To understand the resistance mechanism, molecular studies have been carried out. Two chromosome-encoded arsenic resistance （ars） gene clusters have been cloned from CDB3 （Bacillus sp.）. They both function in Escherichia coli and cluster i exerts a much higher resistance to the toxic metalloid. Cluster 2 is smaller possessing four open reading frames （ORFs） arsRorf2BC, similar to that identified in Bacillus subtilis Skin element. Among the eight ORFs in cluster 1 five are analogs of common ars genes found in other bacteria, however, organized in a unique order arsRBCDA instead of arsRDABC. Three other putative genes are located directly downstream and designated as arsTIP based on the homologies of their theoretical translation sequences respectively to thioredoxin reductases, iron-sulphur cluster proteins and protein phosphatases. The latter two are novel of any known ars operons. The arsD gene from Bacillus species was cloned for the first time and the predict protein differs from the well studied E. coli ArsD by lacking two pairs of C-terminal cysteine residues. Its functional involvement in arsenic resistance has been confirmed by a deletion experiment. There exists also an inverted repeat in the intergenic region between arsC and arsD implying some unknown transcription regulation.

Comparative study of catheter-mediated gene transfer into heart

OBJECTIVE: To investigate the feasibility and features of 3 catheter-mediated approaches of gene transfer into heart, including direct myocardial injection (DMI), coronary artery perfusion (CAP), and intrapericardial cavity injection (ICI). METHODS: Fifteen dogs were used, and 0.3 ml (1 x 10(9) pfu) of an adenovirus (Adex1SR LacZ) was injected into the heart by 3 methods. The dogs were killed 5 days following injection, and gene expressions in heart and liver were evaluated by histochemical analysis. RESULTS: The results showed that (1) the CAP method was relatively less damaging and induced sparse LacZ expression in the myocardium, and the gene expression was also found in both vessels within the myocardium and liver; (2) gene transfer by DMI resulted in intense LacZ expression around the injection accompanied by a local inflammatory response; (3) LacZ expression elicited by ICI was detected in either the inner surface of the parietal pericardium or epicardial surface of the heart, and also in the myocardium underlying the visceral pericardium. CONCLUSION: Three catheter-mediated methods of gene transfer into the heart may be used and a reasonable approach should be chosen according to purpose.

Arsenic Bioremediation Potential of Arsenite-Oxidizing Micrococcus sp. KUMAs15 Isolated from Contaminated Soil

Environmental arsenic(As) contamination,considered as the largest mass poisoning of the human population,has become a serious health concern for the people of South East Asia,including those living in the Bengal delta.An As-resistant strain,KUMAs15,was isolated from the As-contaminated fields in Nadia Ddistrict in West Bengal,India and was evaluated for its possible use in environmental As bioremediation.Molecular identification based on 16S rDNA gene sequencing revealed that the strain belonged to the genus Micrococcus.The strain tolerated high levels of As and oxidized arsenite under the culture condition.The strain also accumulated large quantities of As when exposed to a wide range of environmentally relevant concentrations of inorganic As.Analysis of the aoxB,arsB,and arsC gene expression explained the underlying cause of arsenite oxidation and As accumulation by KUMAs15.The As-resistant strain KUMAs15 of Micrococcus sp.was suggested to be a potential environmental As decontaminant.

Development of a whole-cell biosensor based on an ArsR-Pars regulatory circuit from Geobacter sulfurreducens

In this study,an Escherichia coli(E.coli)whole-cell biosensor for the specific detection of bioavailable arsenic was developed by placing a green fluorescent protein(GFP)reporter gene under the control of the ArsR1(GSU2952)regulatory circuit from Geobacter sulfurreducens.E.coli cells only emitted green fluorescence in the presence of arsenite and were more sensitive to arsenite when they were grown in M9 supplemented medium compared to LB medium.Under optimal test conditions,the Geobacter arsR1 promoter had a detection limit of 0.01 mM arsenite and the GFP expression was linear within a range of 0.03-0.1 mM(2.25-7.5 mg/l).These values were well below World Health Organization’s drinking water quality standard,which is 10 mg/l.The feasibility of using this whole-cell biosensor to detect arsenic in water samples,such as arsenic polluted tap water and landfill leachate was verified.The biosensor was determined to be just as sensitive as atomic fluorescence spectrometry.This study examines the potential applications of biosensors constructed with Geobacter ArsR-Pars regulatory circuits and provides a rapid and cost-effective tool that can be used for arsenic detection in water samples.

Molecular level biodegradation of phenol and its derivatives through dmp operon of Pseudomonas putida:A bio-molecular modeling and docking analysis

Participation of Pseudomonas putida-derived methyl phenol（dmp） operon and Dmp R protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has Dmp R protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon,phenol hydroxylase encoded by dmp N gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from Dmp R protein and of the DNA sequences from the two Upstream Activation Sequences（UAS）present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation.

Conservation of ribosomal protein gene ordering in 16 complete genomes

The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and ar-chaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacte-ria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variations in some operons across different organisms within each domain, and these variations are informative on the evolutionary relations among the organisms. This method provides a new potential for studying the origin and evolution of old species.