Isolation and Characterisation of the ylmE Homologue of Thermus thermophilus

Spada, Stefania; Gibert, Yann; Pembroke, J. Tony; Wall, J. Gerard

doi:10.3109/10425170109041334

“…Chromosomal DNA from E. coli strains, for templates in PCR amplifications, was isolated as described [17]. DNA amplification and primer design was performed using standard techniques [18].…”

Section: Methodsmentioning

confidence: 99%

“…For iPCR amplifications, E. coli chromosomal DNA was prepared as described [17]. DNA was digested with Hin dII and Sau 3A (Roche Molecular Biochemicals, East Sussex, UK), for left‐end and right‐end region determination, respectively.…”

Section: Methodsmentioning

confidence: 99%

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 inE. coliK12

McGrath

¹

,

Pembroke

²

2004

FEMS Microbiology Letters

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The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype IncJ element, R391, revealed it to be a mosaic structure, integrated into the prfC gene in E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5' end of prfC at 99.31 min on the E. coli chromosome, and confirmed this for R391. Despite disrupting prfC, the IncJ's encode novel promoter and 5' sequences, restoring function of the disrupted prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their attL and attR sites, with subsequent recombination between the attP sites on the circular forms and the attB sites in the host genomes. The attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.

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“…Chromosomal DNA from E. coli strains, for templates in PCR amplifications, was isolated as described [17]. DNA amplification and primer design was performed using standard techniques [18].…”

Section: Dna Amplification By Pcrmentioning

confidence: 99%

“…For iPCR amplifications, E. coli chromosomal DNA was prepared as described [17]. DNA was digested with HindII and Sau3A (Roche Molecular Biochemicals, East Sussex, UK), for left-end and right-end region determination, respectively.…”

Section: Dna Amplification By Pcrmentioning

confidence: 99%

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 in E. coli K12

McGrath

¹

,

Pembroke

²

2004

FEMS Microbiology Letters

View full text Add to dashboard Cite

The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype IncJ element, R391, revealed it to be a mosaic structure, integrated into the prfC gene in E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5' end of prfC at 99.31 min on the E. coli chromosome, and confirmed this for R391. Despite disrupting prfC, the IncJ's encode novel promoter and 5' sequences, restoring function of the disrupted prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their attL and attR sites, with subsequent recombination between the attP sites on the circular forms and the attB sites in the host genomes. The attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.

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Isolation of a novel Thermus thermophilus metal efflux protein that improves Escherichia coli growth under stress conditions

Spada

¹

,

Pembroke

²

,

Wall

³

2002

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The mechanisms of metal ion transport in thermophilic organisms are poorly understood. Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the identification of a novel metal cation efflux protein. The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species. Expression of the gene in E. coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell. This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt. Furthermore, E. coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein. The molecular basis and potential application of the findings are discussed.

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Isolation and Characterisation of the ylmE Homologue of Thermus thermophilus

Cited by 4 publications

References 23 publications

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 inE. coliK12

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 inE. coliK12

Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 in E. coli K12

Isolation of a novel Thermus thermophilus metal efflux protein that improves Escherichia coli growth under stress conditions

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