A critical combination of operating parameters can significantly increase the electrotransformation efficiency of a gram-positive Dietzia strain

Lu, Shelian; Nie, Yong; Tang, Yue‐Qin; Guan, Xiong; Wu, Xiao‐Lei

doi:10.1016/j.mimet.2014.05.015

Cited by 16 publications

(27 citation statements)

References 54 publications

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“…Positive clones were numbered and picked after 3 days incubation. The method of competent cell preparation and electro-transformation was as previously described (33).…”

Section: Methodsmentioning

confidence: 99%

“…DQ12-45-1b chromosomal DNA with the primers Ku-BF and Ku-HR (Table S1), and then the 1046 bp DNA fragment was ligated into pNV18-PEK using BamHI and HindIII restriction sites to construct a new plasmid, pNV18-PKK (Table 1). Vector pNV18-PKK was introduced into ku mutant cells via electroporation as described previously to obtain the complementary mutant cell (33). The effect of ku gene on the circular function of linear DNA in Dietzia sp.…”

Section: Methodsmentioning

confidence: 99%

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Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

Nie

Wang

et al. 2018

Preprint

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17Non-homologous end-joining (NHEJ) is critical for genome stability because of its roles in Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic 31 organisms, especially for non-model microorganisms. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/300723 doi: bioRxiv preprint first posted online Apr. 13, 2018; IMPORTANCE 35The non-model gram-positive bacteria lack efficient genetic manipulation systems, but they 36 express genes encoding Ku and LigD. The NHEJ pathway in Dietzia sp. DQ12-45-1b was 37 evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic 38 studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in 39 phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination 40 by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse 41 non-model prokaryotic organisms.

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“…Positive clones were numbered and picked after 3 days incubation. The method of competent cell preparation and electro-transformation was as previously described (33).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

Nie

Wang

et al. 2018

Preprint

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“…The resulting vector was designated pXL1821 and was introduced into Dietzia sp. DQ12-45-1b by electroporation, as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

“…The generated linear DNA fragment was introduced into Dietzia sp. DQ12-45-1b cells via electroporation (2,000 V mm Ϫ1 ; 12 ms) as described previously (36), and the Smresistant clones were identified by amplifying and sequencing the cypR fragments with primers idtF and idtR ( Table 2). The correct chromosomal integration of the fused DNA fragments resulted in an Sm-resistant mutant designated Dietzia sp.…”

Section: Methodsmentioning

confidence: 99%

“…The 1,222-bp DNA fragment obtained was then ligated into pK using BamHI and PstI restriction sites to construct a new plasmid, pXL1825 (Table 1). Vector pXL1825 was introduced into cypR mutant cells via electroporation, as described previously (36). Control transformations with the Dietzia-E. coli shuttle vector pNV18-dsRed were also included (Table 1).…”

Section: Methodsmentioning

confidence: 99%

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Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

Liang

Jiangyang

Nie

et al. 2016

Appl Environ Microbiol

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CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the ؊10 and ؊35 regions in Actinobacteria. Further analysis of the ␤-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria.

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Role of the Group 2 Mrp sodium/proton antiporter in rapid response to high alkaline shock in the alkaline- and salt-tolerant Dietzia sp. DQ12-45-1b

Fang

Qin

Zhang

et al. 2018

Appl Microbiol Biotechnol

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The six- and seven-subunit Na/H antiporters (Mrp) are widely distributed in bacteria. They are reported to be integral for pH homeostasis in alkaliphilic bacteria when adapting to high pH environments. In this study, operons encoding for the six-subunit Na/H antiporters were found in the genomes of all studied Dietzia strains, which have different alkaline-resistant abilities. Disruption of the operon in the strain Dietzia sp. DQ12-45-1b which leads to declined growth in presence of hypersaline and alkaline conditions suggested that the six-subunit Na/H antiporter played an important role in hypersaline and alkaline resistance. Although the complexes DqMrp from DQ12-45-1b (strain with high alkaline resistance) and DaMrp from D. alimentaria 72 (strain with low alkaline resistance) displayed Na(Li)/H antiport activities, they functioned optimally at different pH levels (9.0 for DQ12-45-1b and 8.0 for 72). While both antiporters functioned properly to protect Escherichia coli cells from salt shock, only the DqMrp-containing strain survived the high alkaline shock. Furthermore, real-time PCR results showed that the expression of mrpA and mrpD induced only immediately after DQ12-45-1b cells were subjected to the alkaline shock. These results suggested that the expression of DqMrp might be induced by a pH gradient across the cell membrane, and DqMrp mainly functioned at an early stage to respond to the alkaline shock.

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A critical combination of operating parameters can significantly increase the electrotransformation efficiency of a gram-positive Dietzia strain

Cited by 16 publications

References 54 publications

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

Role of the Group 2 Mrp sodium/proton antiporter in rapid response to high alkaline shock in the alkaline- and salt-tolerant Dietzia sp. DQ12-45-1b

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