Genomic characterization of Aeromonas spp. isolates from striped catfish with motile Aeromonas septicemia and human bloodstream infections in Vietnam.

Aeromonas spp. are commonly found in the aquatic environment and have been responsible for motile Aeromonas septicemia (MAS) in striped catfish, resulting in significant economic loss. These organisms also cause a range of opportunistic infections in humans with compromised immune systems. Here, we conducted a genomic investigation of 87 Aeromonas isolates derived from diseased catfish, healthy catfish and environmental water in catfish farms affected by MAS outbreaks in eight provinces in Mekong Delta (years: 2012-2022), together with 25 isolates from humans with bloodstream infections (years: 2010-2020). Genomics-based typing method precisely delineated Aeromonas species while traditional methods such as aerA PCR and MALDI-TOF were unable identify A. dhakensis. A. dhakensis was found to be more prevalent than A. hydrophila in both diseased catfish and human infections. A. dhakensis sequence type (ST) 656 followed by A. hydrophila ST251 were the predominant virulent species-lineages in diseased catfish (43.7 and 20.7 %, respectively), while diverse STs were found in humans with bloodstream infections. There was evidence of widespread transmission of ST656 and ST251 on striped catfish in the Mekong Delta region. ST656 and ST251 isolates carried a significantly higher number of acquired antimicrobial resistance (AMR) genes and virulence factors in comparison to other STs. They, however, exhibited several distinctions in key virulence factors (i.e. lack of type IV pili and enterotoxin ast in A. dhakensis), AMR genes (i.e. presence of imiH carbapenemase in A. dhakensis), and accessory gene content. To uncover potential conserved proteins of Aeromonas spp. for vaccine development, pangenome analysis has unveiled 2202 core genes between ST656 and ST251, of which 78 proteins were in either outer membrane or extracellular proteins. Our study represents one of the first genomic investigations of the species distribution, genetic landscape, and epidemiology of Aeromonas in diseased catfish and human infections in Vietnam. The emergence of antimicrobial resistant and virulent A. dhakensis strains underscores the needs of enhanced genomic surveillance and strengthening vaccine research and development in preventing Aeromonas diseases in catfish and humans, and the search for potential vaccine candidates could focus on Aeromonas core genes encoded for membrane and secreted proteins.

Anti-Staphylococcus aureus potential of compounds from Ganoderma sp.: A comprehensive molecular docking and simulation approaches.

In this study, a series of secondary metabolites from Ganoderma sp. were screened against Staphylococcus aureus protein targets, including as phosphotransacetylase, clumping factor A, and dihydrofolate reductase, using molecular docking simulations. The chemicals that showed the strongest binding energy with the targeted proteins were ganodermanontriol, lucidumol B, ganoderic acid J, ergosterol, ergosterol peroxide, 7-oxoganoderic acid Z, ganoderic acid AM1, ganosinoside A, ganoderic acid D, and 24R-ergosta-7,2E-diene-3ß,5α,6ß-triol. Interestingly, ganosinoside A showed the greatest affinity for the protein clumping factor A, a result validated by molecular dynamic simulation. Additionally, three natural Ganoderma sp. Strains as Ganoderma lingzhi VNKKK1903, Ganoderma lingzhi VNKK1905A2, and Amauroderma subresinosum VNKKK1904 were collected from Kon Ka Kinh National Park in central land of Vietnam and evaluated for their antibacterial activity against Staphylococcus aureus using an agar well diffusion technique. These results suggest that the fungal extracts and secondary metabolites may serve as valuable sources of antibiotics against Staphylococcus aureus. These findings provided an important scientific groundwork for further exploration of the antibacterial mechanisms of compounds derived from Ganoderma sp. in future research.

Biological Characterization and Genomic Analysis of Novel Phages DLDT_So2 and BHDT_So9 Against Pseudomonas solanacearum, an Infectious Agent in Tomato in Vietnam.

Tomato (Solanum lycopersicum L.) is an important grown vegetable in Vietnam. Bacterial wilt caused by Pseudomonas solanacearum has been considered to be an important disease resulting in a harvest loss up to 90% and significant economic loss to farmers. In this study, two bacteriophages DLDT_So2 and BHDT_So9 specific to P. solanacearum were isolated. Morphological analysis indicated that DLDT_So2 and BHDT_So9 had podovirus morphology and were classified into Autographiviridae family. The latent period and burst size of DLDT_So2 was found to be approximately 120 min and 20.0 ± 2.4 virions per infected cell. Meanwhile, the latent period of BHDT_So9 was 140 min with a burst size of 11.5 ± 2.8 virions per infected cell. Of the 23 bacterial strains tested, the phages infected 7/11 strains of P. solanacearum and none of the other bacteria tested were susceptible to the phages. Stability of the phages at different temperatures, pHs, solvents was also investigated. The genomes of DLDT_So2 and BHDT_So9 are 41,341 bp and 41,296 bp and long with a total GC content of 63%, contains 48 and 46 predicted protein-encoding CDSs. No virulence or antibiotic resistance genes were found in the genomes, suggesting they would be useful biocontrol agents against P. solanacearum. Classification of the phage using average nucleotide identity, phylogenetic analysis was also carried out. The two phages represented new species when they had overall average nucleotide identity of < 95%. This is first report of the isolation and characterization of P. solanacearum-specific phages from tomato farms in Vietnam. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01090-9.

Phylogenetic analysis and morphology of Ganoderma multipileum, a Ganoderma species associated with dieback of the metropolitan woody plant Delonix regia (Boj. ex Hook.) Raf. in Vietnam.

Ganoderma multipileum, a wood decay mushroom, was initially discovered and classified in Taiwan through the analysis of its morphology and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. In this study, we identified a mushroom associated with the dieback of Delonix regia (Boj. ex Hook.) Raf., a woody ornamental street tree in Vietnam, as Ganoderma multipileum. This classification was based on phylogenetic analysis of ITS, RPB2, and TEF1 sequences, as well as morphology assessment and scanning electron microscope observation of basidiospores. The phylogenetic analysis revealed that the specimens collected in Vietnam formed a monophyletic group of Ganoderma multipileum with a high bootstrap value and posterior probability (100%/1.00). Furthermore, the morphological features consistent with laccate Ganoderma, including a thin pileipellis composed of enlarged and bulbous hyphae, and the basidiomes exhibited two different phenotypes. Notably, scanning electron microscopy of the basidiospores revealed ovoid spores with numerous echinules, providing the first documented evidence of this characteristic for Ganoderma multipileum. This research represents the first recorded instance of Ganoderma multipileum in Vietnam associated with the dieback of Delonix regia.

N-terminal LysSN-His-tag improves the production of intracellular recombinant protein in Bacillus subtilis.

Choosing fusion tags to enhance the recombinant protein levels in the cytoplasm of Bacillus subtilis has been limited. Our previous study demonstrated that His-tag at the N-terminus could increase the expression levels of the low-expression gene egfp, while significantly reducing the high-expression genes gfp+ and bgaB in the cytoplasm of B. subtilis. In this study, we aimed to prove the potential of a fusion tag, the combination of the N-terminal domain of B. subtilis lysyl tRNA synthetase (LysSN) and His-tag with varying numbers of histidine (6xHis, 8xHis, 10xHis) by investigating their effects on the expression levels of egfp, gfp+ and bgaB in B. subtilis. For the low-expression gene, LysSN-xHis-tag could enhance the fluorescent intensity of EGFP 23.5 times higher than EGFP without a fusion tag, and 1.5 times higher than that fused with only His-tag. For high-expression genes, the expression level of BgaB and GFP+ was 2.9 and 12.5 times higher than that of His-tag, respectively. The number of histidines in LysSN-xHis-tag did not influence the expression levels of the high-expression genes but affected the expression levels of the low-expression gene.

Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis.

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The ß-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.

Extract antibody and antigen names from biomedical literature.

BACKGROUND: The roles of antibody and antigen are indispensable in targeted diagnosis, therapy, and biomedical discovery. On top of that, massive numbers of new scientific articles about antibodies and/or antigens are published each year, which is a precious knowledge resource but has yet been exploited to its full potential. We, therefore, aim to develop a biomedical natural language processing tool that can automatically identify antibody and antigen entities from articles. RESULTS: We first annotated an antibody-antigen corpus including 3210 relevant PubMed abstracts using a semi-automatic approach. The Inter-Annotator Agreement score of 3 annotators ranges from 91.46 to 94.31%, indicating that the annotations are consistent and the corpus is reliable. We then used the corpus to develop and optimize BiLSTM-CRF-based and BioBERT-based models. The models achieved overall F1 scores of 62.49% and 81.44%, respectively, which showed potential for newly studied entities. The two models served as foundation for development of a named entity recognition (NER) tool that automatically recognizes antibody and antigen names from biomedical literature. CONCLUSIONS: Our antibody-antigen NER models enable users to automatically extract antibody and antigen names from scientific articles without manually scanning through vast amounts of data and information in the literature. The output of NER can be used to automatically populate antibody-antigen databases, support antibody validation, and facilitate researchers with the most appropriate antibodies of interest. The packaged NER model is available at https://github.com/TrangDinh44/ABAG_BioBERT.git .

ELN missense variant in patient with mid-aortic syndrome case report.

BACKGROUND: Mid-aortic syndrome (MAS) is characterized by the congenital coarctation of the abdominal aorta, abdominal and limb claudication, and hypertension. The etiology of this disorder is very diverse and often manifests in conjunction with Takayasu's arteritis, Williams-Beurens syndrome, and neurofibromatosis. The isolated mid-aortic syndrome is very rare with only a few cases reported in the literature. CASE PRESENTATION: A 45 years old man was admitted to the Emergency Department with sudden muscle weakness and facial paralysis on the left side. Imaging studies reveal right middle cerebral artery infarction at the M1 section. Incidental findings include multiple moderate to severe stenoses in the right internal carotid artery, and total abdominal aorta occlusion. A variant at the ELN gene (Elastin, OMIM*130,160): c.1768G > A/wt (p.Ala590Thr) was identified. CONCLUSION: This is the first reported case of ELN related mid-aortic syndrome in Vietnam which was diagnosed through careful clinical and genetic workup. The finding of mid-aortic syndrome, in this case, was incidental and the decision to reverse the occlusion was postponed as there was no immediate risk of renal failure or reduced blood flow to the lower limb.

Studying the characteristics of nanobody CDR regions based on sequence analysis in combination with 3D structures.

BACKGROUND: Single-domain antibodies or nanobodies have recently attracted much attention in research and applications because of their great potential and advantage over conventional antibodies. However, isolation of candidate nanobodies in the lab has been costly and time-consuming. Screening of leading nanobody candidates through synthetic libraries is a promising alternative, but it requires prior knowledge to control the diversity of the complementarity-determining regions (CDRs) while still maintaining functionality. In this work, we identified sequence characteristics that could contribute to nanobody functionality by analyzing three datasets, CDR1, CDR2, and CDR3. RESULTS: By classification of amino acids based on physicochemical properties, we found that two different amino acid groups were sufficient for CDRs. The nonpolar group accounted for half of the total amino acid composition in these sequences. Observation of the highest occurrence of each amino acid revealed that the usage of some important amino acids such as tyrosine and serine was highly correlated with the length of the CDR3. Amino acid repeat motifs were also under-represented and highly restricted as 3-mers. Inspecting the crystallographic data also demonstrated conservation in structural coordinates of dominant amino acids such as methionine, isoleucine, valine, threonine, and tyrosine and certain positions in the CDR1, CDR2, and CDR3 sequences. CONCLUSIONS: We identified sequence characteristics that contributed to functional nanobodies including amino acid groups, the occurrence of each kind of amino acids, and repeat patterns. These results provide a simple set of rules to make it easier to generate desired candidates by computational means; also, they can be used as a reference to evaluate synthetic nanobodies.

Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis.

The influence of fusion tags to produce recombinant proteins in the cytoplasm of Bacillus subtilis is not well-studied as in E. coli. This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene (gfp+) and low expression gene (egfp) in the cytoplasm of B. subtilis cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the Pgrac212 in B. subtilis. The fusions of His-tags with GFP+ caused a reduction compared to the construct without His-tag. When three His-tags fused with egfp, the EGFP production levels were significantly increased up to 3.5-, 12-, and 15-fold. This study suggested that His-tag at the N-terminus could enhance the protein production for the low expression gene and reduce that of the high expression gene in B. subtilis.

A New Pathway for the Preparation of Pyrano[2,3-c]pyrazoles and molecular Docking as Inhibitors of p38 MAP Kinase.

We report a new pathway to synthesize pyrano[2,3-c]pyrazoles and their binding mode to p38 MAP kinase. Pyrano[2,3-c]pyrazole derivatives have been prepared through a four-component reaction of benzyl alcohols, ethyl acetoacetate, phenylhydrazine, and malononitrile in the presence of sulfonated amorphous carbon and eosin Y as catalysts. All products were characterized by melting point, 1H and 13C NMR, and HRMS (ESI). The products were screened in silico for their binding activities to both the ATP-binding pocket and the lipid-binding pocket of p38 MAP kinase, using a structure-based flexible docking provided by the engine ADFR. The results showed that eight synthesized compounds had a higher affinity to the lipid pocket than to the other target site, which implied potential applications as allosteric inhibitors. Finally, the most biologically active compound, 5, had a binding affinity comparable to those of other proven lipid pocket inhibitors, with affinity to the target pocket reaching -10.9932 kcal/mol, and also had the best binding affinity to the ATP-binding pockets in all of our products. Thus, our research provides a novel pathway for synthesizing pyrano[2,3-c]pyrazoles and bioinformatic evidence for their biological capability to block p38 MAP kinase pockets, which could be useful for developing cancer or immune drugs.

Construction of a G-quadruplex-specific DNA endonuclease.

We generated a novel G-quadruplex (G4)-specific endonuclease by fusing a G4 recognition domain of the RHAU helicase with a cleavage domain of the Fok1 nuclease. The fusion protein can specifically bind a parallel G4 and cleave a double-stranded DNA (dsDNA) next to it. The new endonuclease could be used to detect a G4 in a long dsDNA, providing a useful tool for mapping the formation of G4s in the genome.

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis.

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the ß-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.

Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells.

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.

Development of a Mobile App for Family Planning Providers.

PURPOSE: To provide an overview of lessons learned during the development process of an app for iOS and Android based on national recommendations for providing quality family planning services. STUDY AND DESIGN: After a review of existing apps was conducted to determine whether an app of clinical recommendations for family planning existed, a team of clinicians, training specialists, and app developers created a resource app by first drafting a comprehensive content map. A prototype of the app was then pilot tested using smart tablets by a volunteer convenience sample of women's healthcare professionals. Outcomes measured included usability, acceptability, download analytics, and satisfaction by clinicians as reported through an investigator-developed tool. RESULTS: Sixty-nine professionals tested a prototype of the app, and completed a user satisfaction tool. Overall, user feedback was positive, and a zoom function was added to the final version as a result of the pilot test. Within 3 months of being publicly available, the app was downloaded 677 times, with 97% of downloads occurring on smart phones, 76% downloads occurring on iOS devices, and 24% on Android devices. This trend persisted throughout the following 3 months. CLINICAL IMPLICATIONS: Clinicians with an interest in developing an app should consider a team approach to development, pilot test the app prior to wider distribution, and develop a web-based version of the app to be used by clinicians who are unable to access smart devices in their practice setting.

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

BACKGROUND: Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. RESULTS: In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The ß-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 104 to 32 × 104 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. CONCLUSIONS: We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

A Novel Method to Predict Highly Expressed Genes Based on Radius Clustering and Relative Synonymous Codon Usage.

Recombinant proteins play an important role in many aspects of life and have generated a huge income, notably in the industrial enzyme business. A gene is introduced into a vector and expressed in a host organism-for example, E. coli-to obtain a high productivity of target protein. However, transferred genes from particular organisms are not usually compatible with the host's expression system because of various reasons, for example, codon usage bias, GC content, repetitive sequences, and secondary structure. The solution is developing programs to optimize for designing a nucleotide sequence whose origin is from peptide sequences using properties of highly expressed genes (HEGs) of the host organism. Existing data of HEGs determined by practical and computer-based methods do not satisfy for qualifying and quantifying. Therefore, the demand for developing a new HEG prediction method is critical. We proposed a new method for predicting HEGs and criteria to evaluate gene optimization. Codon usage bias was weighted by amplifying the difference between HEGs and non-highly expressed genes (non-HEGs). The number of predicted HEGs is 5% of the genome. In comparison with Puigbò's method, the result is twice as good as Puigbò's one, in kernel ratio and kernel sensitivity. Concerning transcription/translation factor proteins (TF), the proposed method gives low TF sensitivity, while Puigbò's method gives moderate one. In summary, the results indicated that the proposed method can be a good optional applying method to predict optimized genes for particular organisms, and we generated an HEG database for further researches in gene design.

Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli.

BACKGROUND: In general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis. This raises the problem that basal level of expression of the recombinant gene could be harmful for E. coli cells. Based on the Pgrac promoter, we optimized the UP element, the -35, 15, -10 and the +1 region to enhance the promoter activity in B. subtilis after induction. However, detailed investigations for a promoter to develop expression vectors that allows high protein production levels in B. subtilis and a relatively low basal expression levels in E. coli has not been studied yet. RESULTS: We screened the previously constructed library of E. coli - B. subtilis shuttle vectors for high level expression in B. subtilis and low basal level in E. coli. Promoter Pgrac100 turned out to meet these criteria, in which ß-galactosidase expression level of Pgrac100-bgaB is about 9.2 times higher than Pgrac01-bgaB in B. subtilis and the ratio of those in induced B. subtilis over un-induced E. coli from Pgrac100-bgaB is 1.3 times higher than Pgrac01-bgaB. Similarly, GFP expression level of Pgrac100-gfp is about 27 times higher than that of Pgrac01-gfp and the ratio from Pgrac100-gfp is 35.5 times higher than Pgrac01-gfp. This promoter was used as a basis for the construction of three novel vectors, pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) and pHT255 (MCS-Strep-tag). Expression of the reporter proteins BgaB and GFP using these expression vectors in B. subtilis at a low IPTG concentration were measured and the fusion proteins could be purified easily in a single step by using Strep-Tactin or IMAC-Ni columns. CONCLUSIONS: This paper describes the construction and analysis of an IPTG-inducible expression vector termed Pgrac100 for the high level production of intracellular recombinant proteins in B. subtilis and a relatively low basal expression level in E. coli. Based on this vector, the derivative vectors, Pgrac100-His-tag-MCS, Pgrac100-MCS-His-tag and Pgrac100-MCS-Strep-tag have been constructed.

Construction of a 5'-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis.

Different mRNA stabilizing elements including the 3'-stem-loop, the ribosome binding sites (RBS), the 5'-stem-loop and the spacer region between the RBS and the 5'-stem-loop were analysed in detail to increase mRNA stability resulting in enhanced expression of heterologous proteins. In addition, in combination with mRNA stabilizing elements, we propose a new class of 5'-mRNA controllable stabilizing element (CoSE) which is composed of a transcriptional operator such as lacO of the Escherichia coli lac operon and a suitable RBS followed by an optimal spacer length. Such a CoSE allowed Bacillus subtilis cells to synthesize extraordinary stable transcripts with a half-life of the model bgaB reporter transcript (codes for an ß-galactosidase gene derived from Bacillus stearothermophilus) of more than 60 min. This CoSE will be an important tool to control mRNA stability in cells for both research and biotechnological applications. For example, this CoSE can be used in inducible expression vectors offering two major advantages: (i) controlling transcription of target genes by the inducer and (ii) enhancing the stability of the transcript allowing the production high levels of recombinant proteins.

Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements.

Transcription efficiency of inducible promoters remains a bottleneck in recombinant protein production in Bacillus subtilis cells. Here, we present experimental data how to generate strong IPTG-inducible promoters by optimization of nucleotides at the conserved regions of the groESL promoter including the UP element, the -35, -15, -10 and the +1 region. Combination of these changes into one promoter enhanced the amount of recombinant proteins accumulating intracellularly up to about 30% of the total cellular protein.