Min Lin

Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco.

ABSTRACT A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.

Improved Osmotic Tolerance and Ethanol Production of Ethanologenic Escherichia coli by IrrE, a Global Regulator of Radiation-Resistance of Deinococcus radiodurans

Successful fermentations to produce ethanol using ethanologenic Escherichia coli require tolerance to high concentrations of sugars. Here we demonstrate that irrE, encoding a regulatory protein for radiation-resistance in Deinococcus radiodurans, conferred improved osmotic stress tolerance to E. coli. Expression of the gene protected E. coli cells against 25% glucose or xylose, acid shock. It also markedly improved cellular viability, the transcriptional levels of trehalose biosynthetic genes (otsBA) and trehalose content in the IrrE-expressing strain compared with the control strain. IrrE expression also enhanced the expression levels and enzymatic activities of PDC and ADHB as well as ethanol production. Our results suggest that IrrE could potentially be used to improve osmotic stress tolerance and ethanol production in ethanologenic strains.

Benzoate Catabolite Repression of the Phenol Degradation in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 exhibited a delayed utilization of phenol in the presence of benzoate. Benzoate supplementation completely inhibited phenol degradation in a benzoate 1,2-dioxygenase knockout mutant. The mphR encoding the transcriptional activator and mphN encoding the largest subunit of multi-component phenol hydroxylase in the benA mutant were significantly downregulated (about 7- and 70-fold) on the basis of mRNA levels when benzoate was added to the medium. The co-transformant assay of E. coli JM109 with mphK::lacZ fusion and the plasmid pETR carrying mphR gene showed that MphR did not activate the mph promoter in the presence of benzoate. These results suggest that catabolite repression of phenol degradation by benzoate in A. calcoaceticus PHEA-2 is mediated by the inhibition of the activator protein MphR.

Identification of a New Gene Encoding EPSPS with High Glyphosate Resistance from the Metagenomic Library

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.

Purification of green fluorescent protein using a two-intein system

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 2.41xa0mg from 1xa0l 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.

Halomonas huangheensis sp. nov., a moderately halophilic bacterium isolated from a saline–alkali soil

A novel, Gram-stain-negative, aerobic, rod-shaped, non-motile and moderately halophilic bacterium, designated strain BJGMM-B45(T), was isolated from a saline-alkali soil collected from Shandong Province, China. Growth of strain BJGMM-B45(T) occurred at 10-45 °C (optimum, 30 °C) and pH 5.0-12.0 (optimum, pH 7.0) on Luria-Bertani agar medium with 1-20u200a% (w/v) NaCl (optimum, 7-10u200a%). The predominant respiratory quinone was Q-9. The major cellular fatty acids (>5u200a%) were C18u200a:u200a1ω7c, C16u200a:u200a0, C19u200a:u200a0 cyclo ω8c, summed feature 3, C12u200a:u200a0 3-OH and C12u200a:u200a0. The genomic DNA G+C content was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BJGMM-B45(T) belonged to the genus Halomonas in the class Gammaproteobacteria. The closest relatives were Halomonas cupida DSM 4740(T) (98.2u200a% 16S rRNA gene sequence similarity) and Halomonas denitrificans M29(T) (97.8u200a%). Levels of DNA-DNA relatedness between strain BJGMM-B45(T) and Halomonas cupida CGMCC 1.2312(T) and Halomonas denitrificans DSM 18045(T) were 57.0 and 58.9u200a%, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BJGMM-B45(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas huangheensis sp. nov. is proposed. The type strain is BJGMM-B45(T) (u200a=u200aACCC 05850(T)u200a=u200aKCTC 32409(T)).

Resistance of Deinococcus radiodurans to Mutagenesis Is Facilitated by Pentose Phosphate Pathway in the mutS1 Mutant Background

MutS1 is a key protein involved in mismatch repair system for ensuring fidelity of replication and recombination in Deinococcus radiodurans. The zwf gene encodes glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate (PP) pathway, which provides adequate metabolites as precursors of DNA repair. In this study, mutS1 and zwf were disrupted by homologous recombination. The zwf mutant (Δzwf) and the zwf/mutS1 double mutant (Δzwf/mutS1) were sensitive to ultraviolet (UV) light, H2O2, and DNA cross-linking agent mitomycin C (MMC), whereas the mutS1 mutant (ΔmutS1) showed resistance to UV light, H2O2 and MMC as the wild-type strain. Inactivation of mutS1 resulted in a 3.3-fold increase in frequency of spontaneous rifampicin-resistant mutagenesis and a 4.9-fold increment in integration efficiency of a donor point–mutation marker during bacterial transformation. Although inactivation of zwf had no obvious effect compared with the wild-type strain, dual disruption of zwf and mutS1 resulted in a 4.7-fold increase in mutation frequency and a 7.4-fold increase in integration efficiency. These results suggest that inactivation of the PP pathway decreases the resistance of D. radiodurans cells to DNA damaging agents and increases mutation frequency and integration efficiency in the mutS1 mutant background.

Falsirhodobacter deserti sp. nov., isolated from sandy soil.

A Gram-stain-negative, non-motile, heterotrophic, rod-shaped bacterium, designated strain W402(T), was isolated from an enrichment culture of a Tamarix ramosissima rhizosphere soil sample from the Xinjiang desert in the PR China. Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to a species of the genus Falsirhodobacter, having a close relationship to Falsirhodobacter halotolerans JA744(T) (97 % similarity). However, DNA-DNA relatedness between W402(T) and F. halotolerans JA744(T) was 43.2±1.2 %. Strain W402(T) grew in 0-10 % (w/v) NaCl. The temperature and pH ranges for growth were 10-40 °C and pH 5.5-10.0, respectively. Optimal growth occurred at 1-3 % (w/v) NaCl, 30 °C and pH 7.0. The predominant cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c, 61.4 %), C18 : 0 (9.8 %) and 11-methyl C18 : 1ω7c (8.2 %). The major quinone of strain W402(T) was Q-10. Phosphatidylethanolamine was predominant in the polar lipid profile. The DNA G+C content of strain W402(T) was 67.3 mol%. Taken together, these results confirm that W402(T) represents a novel species of the genus Falsirhodobacter, for which the name Falsirhodobacter deserti sp. nov. is proposed. The type strain is W402(T) (u200a= ACCC 05851(T)u200a= KCTC 32408(T)).

Transmembrane structure and function ofdctPQM encoding C4-dicarboxylate transport proteins from nitrogen-fixingP. stutzeri A1501

C4-dicarboxylate transport proteins of diazotrophPseudomonas stutzen were encoded bydctPQM genes. Nucleotide sequence analysis indicated thatdctP, dctQ, anddctM grouped together. Its nucleotide and amino acid sequence shared high homology with that ofdctP gene encoding periplasmic C4-dicarboxylate-binding protein anddctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixingAotobacter vinelandii. Structural analysis showed that DctP ofP. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively. The fragment containing the completedctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ21. The resultant plasmid was named pSZY6. By triparental mating, Tn5 transposon carrying thedctPQM genes inserted into the genome of the wild type strain A1501, randomly. The recombinant strain A-142 which harboured an extra copy ofdctPQM genes was constructed and identified by PCR amplification ofnpt II gene. When A-142 was grown in minimal medium with different concentrations (20, 10 and 5 mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A1501. This result was established that an extra copy ofdctPQM genes could increase the activity of nitrogen fixation ofP. stutzeri strain A1501.