A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184

This study shows that lipid A of Escherichia coli O157 : H7 differs from that of E. coli K-12 in that it has a phosphoform at the C-1 position, which is distinctively modified by a phosphoethanolamine (PEtN) moiety, in addition to the diphosphoryl form. The pmrC gene responsible for the addition of PEtN to the lipid A of E. coli O157 : H7 was inactivated and the changes in lipid A profiles were assessed. The pmrC null mutant still produced PEtN-modified lipid A species, albeit in a reduced amount, indicating that PmrC was not the only enzyme that could be used to add PEtN to lipid A. Natural PEtN substitution was shown to be present in the lipid A of other serotypes of enterohaemorrhagic E. coli and absent from the lipid A of E. coli K-12. However, the cloned pmrC O157 gene in a high-copy-number plasmid generated a large amount of PEtN-substituted lipid A species in E. coli K-12. The occurrence of PEtN-substituted lipid A species was associated with a slight increase in the MICs of cationic peptide antibiotics, suggesting that the lipid A modification with PEtN would be beneficial for survival of E. coli O157 : H7 in certain environmental niches. However, PEtN substitution in the lipid A profiles was not detected when putative inner-membrane proteins (YhbX/YbiP/YijP/Ecf3) that show significant similarity with PmrC in amino acid sequence were expressed from high-copy-number plasmids in E. coli K-12. This suggests that these potential homologues are not responsible for the addition of PEtN to lipid A in the pmrC mutant of E. coli O157 : H7. When cells were treated with EDTA, the amount of palmitoylated lipid A from the cells carrying a high-copy-number plasmid clone of pmrC O157 that resulted in significant increase of PEtN substitution was unchanged compared with cells without PEtN substitution, suggesting that the PEtN moiety substituted in lipid A does not compensate for the loss of divalent cations required for bridging neighbouring lipid A molecules.

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Construction of high copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980

10.21203/rs.2.18382/v1 ◽

2019 ◽

Author(s):

XingYa Zhao ◽

JianYong Xu ◽

Ming Tan ◽

Jie Zhen ◽

WenJu Shu ◽

...

Keyword(s):

Copy Number ◽

Insertion Site ◽

Pectate Lyase ◽

High Copy Number ◽

Expression Vectors ◽

Upstream Region ◽

Transformation Rate ◽

E Coli ◽

Shuttle Plasmid ◽

Insertion Sites

Abstract Background: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli - B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence ( ori ) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1~pUC980-8) containing all possible insertion sites and directions were constructed.Results: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN , an alkaline protease spro1 and a pullulanase gene pulA11 , respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were upto 5200 U/mL, 21537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports.Conclusion: In this work we can conclude that the optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis . And the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

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