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

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.

scholarly journals High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980

2020 ◽  
Author(s):  
XingYa Zhao ◽  
JianYong Xu ◽  
Ming Tan ◽  
Jie Zhen ◽  
WenJu Shu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Alkaline Protease ◽  
Copy Number ◽  
Insertion Site ◽  
Pectate Lyase ◽  
Expression Vectors ◽  
Transformation Rate ◽  
E Coli ◽  
Insertion Sites ◽  
Alkaline Pectate Lyase

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: 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. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids. Keywords: Expression vectors; pUC980; Bacillus subtilis; Alkaline pectate lyase; Alkaline protease; Pullulanase.

scholarly journals Genome-Wide Identification and Analysis of High-Copy-Number LTR Retrotransposons in Asian Pears

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 156
Author(s):  
Shuang Jiang ◽  
Xiaoqing Wang ◽  
Chunhui Shi ◽  
Jun Luo
Keyword(s):  
Copy Number ◽  
Long Terminal Repeat Retrotransposons ◽  
High Copy Number ◽  
Pyrus Pyrifolia ◽  
Origin And Evolution ◽  
Asian Pear ◽  
Copy Numbers ◽  
Genome Wide ◽  
History Of ◽  
Insertion Sites

A large proportion of the genome of ‘Suli’ pear (Pyrus pyrifolia) contains long terminal repeat retrotransposons (LTR-RTs), which suggests that LTR-RTs have played important roles in the evolution of Pyrus. Further analysis of retrotransposons, particularly of high-copy-number LTR-RTs in different species, will provide new insights into the evolutionary history of Pyrus. A total of 4912 putative LTR-RTs classified into 198 subfamilies were identified in the ‘Suli’ pear genome. Six Asian pear accessions, including cultivars and wild species, were resequenced. The comparison of copy number for each LTR-RT subfamily was evaluated in Pyrus accessions, and data showed up to four-fold differences for some subfamilies. This contrast suggests different fates for retrotransposon families in the evolution of Pyrus. Fourteen high-copy-number subfamilies were identified in Asian pears, and more than 50% of the LTR-RTs in the genomes of all Pyrus accessions were from these 14 identified LTR-RT subfamilies. Their average insertion time was 3.42 million years ago, which suggests that these subfamilies were recently inserted into the genome. Many homologous and specific retrotransposon insertion sites were identified in oriental and occidental pears, suggesting that the duplication of retrotransposons has occurred throughout almost the entire origin and evolution of Pyrus species. The LTR-RTs show high heterogeneity, and their copy numbers vary in different Pyrus species. Thus, our findings suggest that LTR-RTs are an important source of genetic variation among Pyrus species.

scholarly journals Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

Gene ◽  
1995 ◽  
Vol 166 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Eriko Takano ◽  
Janet White ◽  
Charles J. Thompson ◽  
Mervyn J. Bibb
Keyword(s):  
Copy Number ◽  
High Copy Number ◽  
Expression Vectors ◽  
Streptomyces Spp

scholarly journals Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 657-666 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Valeria R. Parreira ◽  
Russell E. Bishop ◽  
Carlton L. Gyles
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Lipid A ◽  
High Copy Number ◽  
Peptide Antibiotics ◽  
Significant Similarity ◽  
E Coli ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
K 12

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.

scholarly journals Selection against transposable elements in D. simulans and D. melanogaster

1996 ◽  
Vol 68 (1) ◽  
pp. 9-15 ◽  
Author(s):  
C. Vieira ◽  
C. Biémont
Keyword(s):  
In Situ Hybridization ◽  
Transposable Elements ◽  
Copy Number ◽  
Insertion Site ◽  
Natural Populations ◽  
Lower Proportion ◽  
Major Mechanism ◽  
Insertion Sites ◽  
Copy Number Regulation

SummaryThe insertion site numbers of the transposable elements (TEs) copia, mdgl, 412 and gypsy were determined in various natural populations of Drosophila melanogaster and D. simulans by in situ hybridization. We showed that, while all elements except gypsy had many insertion sites scattered over the chromosomes in D. melanogaster, only the 412 element in D. simulans presented a high number of insertions, and this number was lower than in D. melanogaster. This low 412 site number per genome in D. simulans was associated with a lower proportion of insertions on the X chromosome in comparison with D. melanogaster, as determined in diploid genomes (0·090 for D. simulans against 0·137 for D. melanogaster) and in haploid genomes (0·102 against 0·146), each value being, moreover, lower than the value of 0·20 expected on the hypothesis of no selection against insertional mutations. These results suggest that selection is a major mechanism explaining 412 copy number regulation in Drosophila, and is stronger in D. simulans than in D. melanogaster.

scholarly journals Sequence Analysis of Tn10 Insertion Sites in a Collection of Escherichia coli Strains Used for Genetic Mapping and Strain Construction

1998 ◽  
Vol 180 (23) ◽  
pp. 6408-6411 ◽  
Author(s):  
Brian P. Nichols ◽  
Obaid Shafiq ◽  
Victoria Meiners
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Hot Spots ◽  
Insertion Site ◽  
Inverse Pcr ◽  
E Coli ◽  
Chromosome Sequence ◽  
Tn10 Insertion ◽  
Insertion Sites ◽  
Recombination Hot Spots

ABSTRACT The chromosomal insertion sites of Tn10-containingEscherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.

scholarly journals Insertion Site Occupancy by stx2 Bacteriophages Depends on the Locus Availability of the Host Strain Chromosome

2007 ◽  
Vol 189 (18) ◽  
pp. 6645-6654 ◽  
Author(s):  
Ruth Serra-Moreno ◽  
Juan Jofre ◽  
Maite Muniesa
Keyword(s):  
Shiga Toxin ◽  
Insertion Site ◽  
Site Occupancy ◽  
Host Strain ◽  
Host Chromosome ◽  
Shiga Toxins ◽  
E Coli ◽  
Emergent Pathogen ◽  
Insertion Sites ◽  
Host Strains

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is an emergent pathogen characterized by the expression of Shiga toxins, which are encoded in the genomes of lambdoid phages. These phages are infectious for other members of the Enterobacteriaceae and establish lysogeny when they integrate into the host chromosome. Five insertion sites, used mainly by these prophages, have been described to date. In the present study, the insertion of stx 2 prophages in these sites was analyzed in 168 STEC strains isolated from cattle. Additionally, insertion sites were determined for stx 2 phages which (i) converted diverse laboratory host strains, (ii) coexisted with another stx 2 prophage, and (iii) infected a recombinant host strain lacking the most commonly used insertion site. Results show that depending on the host strain, phages preferentially use one insertion site. For the most part, yehV is occupied in STEC strains while wrbA is preferentially selected by the same stx phages in E. coli laboratory strains. If this primary insertion site is unavailable, then a secondary insertion site is selected. It can be concluded that insertion site occupancy by stx phages depends on the host strain and on the availability of the preferred locus in the host strain.

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