R Factor Proteins Synthesized in
            <i>Escherichia coli</i>
            Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Levy, Stuart B.

doi:10.1128/jb.120.3.1451-1463.1974

Cited by 53 publications

(26 citation statements)

References 35 publications

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“…The E. coti minicell strain DS410 {minA. minB) was transformed with plasmid DNA, and the segregated, chromosomedeficient minicells were purified by segregation through sucrose gradients (Levy, 1974). The plasmid-encoded proteins were labelled in the presence of lO^Ci of ^H-methlonine in minimal salts medium and, after lysis of the minicells in sample buffer (Laemmli, 1970), were subjected to electrophoresis in a 12.5% SDS-PAGE and fluorographed (Laskey and Mills, 1975).…”

Section: Preparation Of Miniceiismentioning

confidence: 99%

Cloning and genetic characterization of a Hellcobacter pylori flagellin gene

Leying

Suerbaum

Geis

et al. 1992

Molecular Microbiology

173

123

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Helicobacter pylori produces polar sheathed flagella, which are believed to be essential for the bacterial colonization of the human gastric mucosa. Here we report on the cloning and genetic characterization of a H. pylori gene encoding the subunit of the flagellar filament, the flagellin. Screening of a genomic library of H. pylori with an oligonucleotide probe derived from the N-terminal amino acid sequence of purified flagellin resulted in a recombinant plasmid clone carrying the flagellin-encoding gene flaA on a 9.3 kb Bg/II fragment. The nucleotide sequence of flaA revealed an open reading frame of 1530 nucleotides, encoding a protein with a predicted molecular mass of 53.2 kDa, which is similar in size with the purified flagellin protein in SDS-polyacrylamide gel electrophoresis. Sequence alignment of H. pylori flagellin (FlaA) with other bacterial flagellins demonstrates a high degree of similarity in the amino-terminal and carboxy-terminal regions, including those of the closely related genus Campylobacter (56% overall identity with Campylobacter coli flaA), but little homology in the central domain. Southern hybridizations of chromosomal DNA with flaA-specific probes did not reveal the presence of additional homologous flagellin genes in H. pylori. Sequence analysis of the flaA flanking regions and mapping of the flaA mRNA start site by a primer extension experiment indicated that transcription of the gene is under the control of a sigma 28-specific promoter sequence in H. pylori. The region upstream of the flaA promoter is subject to local DNA modification, resulting in the masking of two out of three closely linked HindIII restriction sites in the chromosome of strain 898-1. Escherichia coli strains harbouring the recombinant plasmid did not produce full-length flagellin and data obtained with FlaA fusion proteins using an E. coli plasmid expression system suggest that a distinct nucleotide sequence in the gene interferes with productive translation of this protein in E. coli.

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Section: Preparation Of Miniceiismentioning

confidence: 99%

Cloning and genetic characterization of a Hellcobacter pylori flagellin gene

Leying

Suerbaum

Geis

et al. 1992

Molecular Microbiology

173

123

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“…E. coli DH5a, E131 {recA\ phage fd gene-2) and E145 (polAI; phage fd gene-2) were used for cloning and transposon mutagenesis (Haas et al, 1993). Strain DS410 was used for the minicell experiments (Levy, 1974). The E. co//plasmid library of MS11 chromosomal DNA (L3) consists of 3-4 kb fragments produced by partial digests using faqi and Mbo\ and inserted between the the Cla\ and BglW sites of vector pBA (Stern etal., 1986).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation

Facius

Meyer

1993

Molecular Microbiology

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A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA-uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site-specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74 kDa. The comA gene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative sigma 54-type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross-reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane alpha-helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non-piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.

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“…Isolation of plasmid-containing minicells. The procedure foilowed for the isolation of minicells was essentially that described by Frazer and Curtiss (9), with the addition of a penicillin G selection as described by Levy (20). The penicillin treatment of the partially purified minicells was in complex medium, YET or SLBH.…”

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confidence: 99%

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12

Gayda¹,

Markovitz²

1978

J Bacteriol

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Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.The prominent phenotypes of the capR (Ion) mutants are overproduction of capsular polysaccharide, characterized by mucoid colonies on minimal medium, and increased sensitivity to UV radiation (for review, see 27). In an attempt to clone the capR (Ion) gene, an EcoRI restriction fragment of Escherichia coli K-12 DNA was cloned using plasmid pSC101. Three independent recombinant plasmids, designated pMC44, pMC52, and pMC303, were isolated that, when transformed into capR (Ion) mutant bacteria, inhibited the overproduction of capsular polysaccharide but did not affect the UV sensitivity of the capR (Ion) mutant (4). Analysis of the EcoRI-digested pMC44, pMC52, and pMC303 DNA by agarose gel electrophoresis revealed that the three plasmids each contained a 2-megadalton (Mdal) DNA fragment in addition to a 6.1-Mdal DNA fragment (pSC101; 4). The 2-Mdal fragment does not contain the capR+ gene (36; B. Zehnbauer and A. Markovitz, manuscript in preparation).The proteins coded by a plasmid can be selectively labeled using plasmid-containing minicells; the latter exclude the bacterial chromosome (10). To identify the proteins specified by plasmid pMC44, minicells that contained pMC44 or pSC101 were purified and labeled with [35S]methionine. The 'S-labeled polypeptides were then analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). We expected that one or more plasmid pMC44-specified proteins could be membrane associated, since pMC44 decreased the polysaccharide synthesis in capR (ion) mutant bacteria. Polysaccharide synthesis occurs in the bacterial envelope (17) similar to the synthesis of lipopolysaccharide (18,29). Also, polysaccharide polymerase activity in capR mutants has been detected in membrane preparations (17; M. M. Lieberman, Ph.D. thesis, University of Chicago, Chicago, Ill., 1969). Therefore, ['S]methionine-labeled, pMC44-containing minicell membrane preparations were isolated and fractionated based on solubility in 1% sodium lauryl sarcosinate (Sarkosyl, a detergent that solubilizes the inner membrane proteins but not the "major" outer membrane p...

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R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Cited by 53 publications

References 35 publications

Cloning and genetic characterization of a Hellcobacter pylori flagellin gene

Cloning and genetic characterization of a Hellcobacter pylori flagellin gene

Novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12

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