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ABSTRACT: An Escherichia coli bioreporter harboring the phenol-inducible mphK promoter (P
mphK
) from Acinetobacter calcoaceticus PHEA-2 fused to a β-galactosidase gene (lacZ) and the regulator gene (mopR) of A. calcoaceticus NCIB8250 was constructed to detect phenol. P
mphK
containing three inverted repeats (IR1, IR2 and IR3) upstream of mphK was activated by the regulator MopR in the presence of phenol. Deletion analysis of P
mphK
revealed that IR2 and IR3 were essential for promoter activity, while IR1 was involved in transcriptional repression. The
sensitivity of the bioreporter for the detection of phenol (0.1–5μM) was improved by about 100% through deletion of IR1 in
P
mphK
.
KeywordsBioreporter-
Escherichia coli
-Inverted repeat sequence-MopR-
mphK promoter
Biotechnology Letters 04/2012; 32(9):1265-1270. · 1.68 Impact Factor
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Haiying Yu,
Zixin Peng,
Yuhua Zhan,
Jin Wang,
Yongliang Yan,
Ming Chen,
Wei Lu,
Shuzhen Ping,
Wei Zhang, Zhonglin Zhao,
Shuying Li,
Masahiro Takeo,
Min Lin
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ABSTRACT: Acinetobacter calcoaceticus PHEA-2 utilizes phenol as its sole carbon and energy source and has a multi-component phenol hydroxylase-encoding gene operon (mphKLMNOP) for phenol degradation. Two additional genes, mphR and mphX, were found upstream and downstream of mphKLMNOP, respectively. The mphR gene encodes a XylR/DmpR-type regulator-like protein and is transcribed in the opposite direction to mphKLMNOP. The mphX gene is transcribed in the same direction as mphKLMNOP and encodes a protein with 293 amino acid residues showing weak identity with some unknown proteins encoded in the meta-cleavage pathway gene clusters for aromatic compound degradation. Disruption of mphR by homologous recombination resulted in the loss of phenol degradation while disruption of mphX caused significantly faster phenol degradation than in the wild type strain. Transcriptional assays for mphK, mphR, and mphX revealed that mphR activated mphKLMNOP transcription in the presence of phenol, but mphX partially repressed this activation. Gel mobility-shift assay demonstrated a direct interaction of MphR with the mphK promoter region. These results indicate the involvement of a novel repressor protein MphX in transcriptional regulation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR.
PLoS ONE 01/2011; 6(3):e17350. · 4.09 Impact Factor
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ABSTRACT: An Escherichia coli bioreporter harboring the phenol-inducible mphK promoter (P(mphK)) from Acinetobacter calcoaceticus PHEA-2 fused to a beta-galactosidase gene (lacZ) and the regulator gene (mopR) of A. calcoaceticus NCIB8250 was constructed to detect phenol. P(mphK) containing three inverted repeats (IR1, IR2 and IR3) upstream of mphK was activated by the regulator MopR in the presence of phenol. Deletion analysis of P(mphK) revealed that IR2 and IR3 were essential for promoter activity, while IR1 was involved in transcriptional repression. The sensitivity of the bioreporter for the detection of phenol (0.1-5 microM) was improved by about 100% through deletion of IR1 in P(mphK).
Biotechnology Letters 09/2010; 32(9):1265-70. · 1.68 Impact Factor
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ABSTRACT: During ethanol fermentation, bacterial strains may encounter various stresses, such as ethanol and acid shock, which adversely affect cell viability and the production of ethanol. Therefore, ethanologenic strains that tolerate abiotic stresses are highly desirable. Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation, ultraviolet light, and desiccation, and therefore constitute an important pool of extreme resistance genes. The irrE gene encodes a general switch responsible for the extreme radioresistance of D. radiodurans. Here, we present evidence that IrrE acting as a global regulator confers high stress tolerance to a Zymomonas mobilis strain. Expression of the gene protected Z. mobilis cells against ethanol, acid, osmotic, and thermal shock. It also markedly improved cell viability, the expression levels and enzyme activities of pyruvate decarboxylase and alcohol dehydrogenase, and the production of ethanol under both ethanol and acid stress. These data suggest that irrE is a potentially promising gene for improving the abiotic stress tolerance of ethanologenic bacterial strains.
Journal of Microbiology and Biotechnology 07/2010; 20(7):1156-62. · 1.38 Impact Factor
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ABSTRACT: A mutant of green fluorescent protein (GFPmut3*) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of GFPmut3* was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear GFPmut3*. The circular GFPmut3* was 5 degrees C more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular GFPmut3* also displayed increased relative fluorescence intensity. In addition, chemical stability of GFPmut3* against GdnHCl revealed more stability of the circular form compared with the linear form.
Journal of Microbiology and Biotechnology 03/2010; 20(3):460-6. · 1.38 Impact Factor
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ABSTRACT: The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is an attractive target for drugs and herbicides. Here we identified a novel RPMXR motif that is strictly conserved among class II EPSP synthases. Site-directed mutational analysis of this motif showed that substitutions of the four strictly conserved amino acid residues, Arg127, Pro128, Met129, and Arg131, resulted in complete loss of enzymatic activity, whereas changes in the non-conserved Asn130 residue strongly influenced glyphosate resistance (all numbering according to Pseudomonas stutzeri A1501 EPSP synthase). These experimental results, combined with 3D structure modeling of the location and interaction of the RPMXR motif with phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P), demonstrate that the novel motif is required for enzymatic activity and glyphosate resistance of class II EPSP synthases.
Journal of biotechnology 09/2009; 144(4):330-6. · 2.88 Impact Factor
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ABSTRACT: A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.
Applied Microbiology and Biotechnology 02/2008; 77(5):1175-80. · 3.42 Impact Factor
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Dan Jin,
Wei Lu,
Shuzhen Ping,
Wei Zhang,
Jian Chen,
Baoqing Dun,
Ruiqiang Ma, Zhonglin Zhao,
Jiying Sha,
Liang Li,
Zhirong Yang,
Ming Chen,
Min Lin
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ABSTRACT: Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 mM: glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 mM: , was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein.
Current Microbiology 11/2007; 55(4):350-5. · 1.82 Impact Factor
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Baoqing Dun,
Wei Lu,
Wei Zhang,
Shuzhen Ping,
Xujing Wang,
Ming Chen,
Yuquan Xu,
Dan Jin,
Jin Wang, Zhonglin Zhao,
Aimin Liang,
Songna Hou,
Ming-Qun Xu,
Min Lin
[show abstract]
[hide abstract]
ABSTRACT: A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted
mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12
sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal
and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296lc. Co-transformation of plasmids,
pMEPSN295IN and pKEPSc296lc, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were
bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.
Chinese Science Bulletin 06/2006; 51(13):1652-1654. · 1.32 Impact Factor
