scholarly journals Production of Recombinant α-Galactosidases inThermus thermophilus

2001 ◽  
Vol 67 (9) ◽  
pp. 4192-4198 ◽  
Author(s):  
Olafur Fridjonsson ◽  
Ralf Mattes
Keyword(s):  
Insertional Mutagenesis ◽  
Bacillus Stearothermophilus ◽  
Plasmid Stability ◽  
Shuttle Vector ◽  
Thermus Thermophilus ◽  
Phenotypic Selection ◽  
Kanamycin Resistance ◽  
E Coli ◽  
Insertional Inactivation ◽  
Selection Of

ABSTRACT A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene (agaT) ofT. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis ofagaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal+ phenotype was assumed to be essential for growth on melibiose. In a Gal−background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT− strain had to be Kms for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT− Gal+ Kms) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in T. thermophilus, the isogenes agaA and agaBof Bacillus stearothermophilus KVE36 were cloned into anEscherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.

scholarly journals Functional Characterization of Replication and Stability Factors of an Incompatibility Group P-1 Plasmid from Xylella fastidiosa

2010 ◽  
Vol 76 (23) ◽  
pp. 7734-7740 ◽  
Author(s):  
Min Woo Lee ◽  
Elizabeth E. Rogers ◽  
Drake C. Stenger
Keyword(s):  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Plasmid Stability ◽  
Xylella Fastidiosa ◽  
Shuttle Vector ◽  
Functional Characterization ◽  
Replication Initiation ◽  
Incompatibility Group ◽  
Antibiotic Selection ◽  
E Coli

ABSTRACT Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

scholarly journals Macrolide Resistance Gene mreA ofStreptococcus agalactiae Encodes a Flavokinase

2001 ◽  
Vol 45 (8) ◽  
pp. 2280-2286 ◽  
Author(s):  
Gervais Clarebout ◽  
Corinne Villers ◽  
Roland Leclercq
Keyword(s):  
High Performance ◽  
Shuttle Vector ◽  
Sodium Dodecyl ◽  
Macrolide Resistance ◽  
Upstream Region ◽  
Flavin Mononucleotide ◽  
Active Efflux ◽  
Fourfold Increase ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned inEscherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreArevealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

scholarly journals Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

2004 ◽  
Vol 47 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Paramasamy Gunasekaran
Keyword(s):  
Copy Number ◽  
Zymomonas Mobilis ◽  
Northern Blot Analysis ◽  
Shuttle Vector ◽  
Transcript Levels ◽  
E Coli ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Sacb Gene ◽  
In Cells

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB.

Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 203-208 ◽  
Author(s):  
Hongmei Dong ◽  
Xiaohu Xu ◽  
Mohong Deng ◽  
Xiaojun Yu ◽  
Hu Zhao ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Target Genes ◽  
Review Process ◽  
Peer Review Process ◽  
Nitrilotriacetic Acid ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Expression Plasmids ◽  
Selection Of

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28–349 aa), MAC2 (166–369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni–NTA, were ~80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28–349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

2020 ◽  
Author(s):  
Carmen Lopez ◽  
Mingfeng Cao ◽  
Zhanyi Yao ◽  
Zengyi Shao
Keyword(s):  
Yarrowia Lipolytica ◽  
Plasmid Stability ◽  
Critical Role ◽  
Fold Increase ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform ◽  
Regulatory Dna ◽  
Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

scholarly journals Effect of Dimethyl Sulphoxide on the Expression of Nitrogen Fixation in Bacteria

1977 ◽  
Vol 30 (2) ◽  
pp. 141 ◽  
Author(s):  
Mary L Skotnicki ◽  
Barry G Rolfe
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Energy Metabolism ◽  
Membrane Proteins ◽  
Klebsiella Pneumoniae ◽  
Dimethyl Sulphoxide ◽  
Rhizobium Trifolii ◽  
Nif Genes ◽  
E Coli ◽  
Selection Of

Storage in dimethyl sulphoxide (DMSO) of Escherichia coli K12 hybrids carrying nif+ genes from Klebsiella pneumoniae can result in selection of a defective nitrogen-fixing phenotype. Similar results are obtained with E. coli K12 hybrids containing the nitrogep-fixing capacity from Rhizobium trifolii. DMSO appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K12 and four chromosomal regions (chID, chlG, his and unc) are associated with resistance to DMSO.

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