High efficiency transformation of E.coli by high voltage electroporation

Dower, William J.; Miller, Jeff F.; Ragsdale, Charles W.

doi:10.1093/nar/16.13.6127

Cited by 2,653 publications

(1,611 citation statements)

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“…Plasmids were introduced into E. coli strains by chemical transformation (Sambrook et al, 1989) or electroporation (Dower et al, 1988). H. influenzae strain DB117 was transformed using the MIV method of Herriott et al (1970).…”

Section: Bacterial Strains Bacteriophage and Plasmidsmentioning

confidence: 99%

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Hendrixson

Morena

Stathopoulos

et al. 1997

Molecular Microbiology

102

139

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SummaryHaemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap s ). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the Cterminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap ␤ ) predicted formation of a ␤-barrel with an N-terminal transmembrane ␣-helix followed by 14 transmembrane ␤-strands. Additional analysis revealed that the final ␤-strand contains an amino acid motif common to other ␤-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap s . These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of ␤-barrel proteins to the bacterial outer membrane.

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Section: Bacterial Strains Bacteriophage and Plasmidsmentioning

confidence: 99%

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Hendrixson

Morena

Stathopoulos

et al. 1997

Molecular Microbiology

102

139

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“…After 24 h, DNA was extracted from the protoplasts according to Werr and L~rz (1986). Four microliter aliquots were used to electroporate (Dower et a/., 1988) Transformed bacteria were plated on LB agar containing X-Gal (40 I~g ml-1), IPTG (0.3 mM), ampiciUin (100 tlg ml-1). One blue colony was recovered among 104 transformants, representing the transformation with the whole DNA sample extracted from the protoplasts.…”

Section: Ds Excision Takes P/ace In the Target Plasmidmentioning

confidence: 99%

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast‐derived cells

1994

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SummaryNicotiana plurnbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposese. It is shown that Ds can efficiently transpose from extrachromosomal DNA to IV. plurnbaginifolia chromosomes when the Ac transposese gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 trensgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was pert of the transposese construct integrated. By segregation analysis of tranegenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplaste had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

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“…Bacterial transformations were performed by electroporation (Dower et al, 1988). For mating experiments, donor and recipient strains were grown overnight in LB broth containing appropriate antibiotics for plasmid selection.…”

Section: Methodsmentioning

confidence: 99%

Genes involved in conjugative DNA processing of plasmid R6K

Núñez

Avila

Cruz

1997

Molecular Microbiology

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SummaryThe conjugative transfer region of the IncX plasmid R6K (TRA X ) was analysed by transposon mutagenesis and DNA sequencing. Tn5 tac1 insertional mutations localized TRA X to a 14.8 kb segment containing the ␣ origin of transfer (oriT␣), genes involved in conjugative DNA-processing (Dtr X ) and genes involved in pilus synthesis and assembly (Mpf X ). A second functional oriT, oriT ␤, was located at a distance of 5.3 kb from oriT␣ and was outside TRA X . Mpf X occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilusspecific phage X-2. At both sides of Mpf X there were insertions that were Tra ¹ but X-2 sensitive, suggesting that the mutations were in Dtr X . This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT␣-taxA-taxC-Mpf X -taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein , which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein for conjugation.

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High efficiency transformation of E.coli by high voltage electroporation

Cited by 2,653 publications

References 8 publications

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast‐derived cells

Genes involved in conjugative DNA processing of plasmid R6K

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