TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization

Wilmes-Riesenberg, Mary R.; Wanner, Barry L.

doi:10.1128/jb.174.14.4558-4575.1992

Cited by 82 publications

(53 citation statements)

References 42 publications

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“…Positive LacZ fusions represent fusions to cytoplasmic domains, while positive PhoA fusions represent fusions to periplasmic domains. Genetic tricks have been developed which make it possible to exchange the LacZ reporter molecules in positive fusions for the PhoA molecule and vice versa (117,200), resulting in fusions with complementary enzymatic properties (76,170). The use of PhoA as a reporter molecule in these studies is advantageous in principle because a positive result (i.e., a highactivity fusion) requires the active export of the mature reporter enzyme moiety into the periplasm.…”

Section: Enzyme Tagsmentioning

confidence: 99%

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Geest

Lolkema

2000

Microbiol Mol Biol Rev

178

169

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SUMMARY Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.

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Section: Enzyme Tagsmentioning

confidence: 99%

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Geest

Lolkema

2000

Microbiol Mol Biol Rev

178

169

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“…pWM4, pWM5, and pWM6 contain cassettes with a promoterless uidA and different antibiotic resistance markers (35). The sources of pEG5294 (32) and pSKS114 (55) have been reported. RZ5phoA-lacZ DK110 has a transcriptional fusion of lacZ to the E. coli phoA promoter (2).…”

Section: Methodsmentioning

confidence: 99%

“…Transposon mutagenesis was carried out with suicide vectors carrying TnphoA (capable of forming phoA gene fusions), TnphoAЈ-1 (capable of forming lacZ transcriptional fusions), or TnphoAЈ-4 (capable of forming lacZ translational fusions) essentially as described elsewhere (48,55). To do this, BW17272 carrying pKL248, pKL253, pKL257, or pWM67 was infected with one of these phages, Kan r transductants were selected, and these were replica-mated with BW16945, BW19615, or BW19715.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

et al. 1995

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Two pathways exist for cleavage of the carbon-phosphorus (C-P) bond of phosphonates, the C-P lyase and the phosphonatase pathways. It was previously demonstrated that Escherichia coli carries genes (named phn) only for the C-P lyase pathway and that Enterobacter aerogenes carries genes for both pathways (K.-S. Lee, W. W. Metcalf, and B. L. Wanner, J. Bacteriol. 174:2501-2510, 1992). In contrast, here it is shown that Salmonella typhimurium LT2 carries genes only for the phosphonatase pathway. Genes for the S. typhimurium phosphonatase pathway were cloned by complementation of E. coli ⌬phn mutants. Genes for these pathways were proven not to be homologous and to lie in different chromosomal regions. The S. typhimurium phn locus lies near 10 min; the E. coli phn locus lies near 93 min. The S. typhimurium phn gene cluster is about 7.2 kb in length and, on the basis of gene fusion analysis, appears to consist of two (or more) genes or operons that are divergently transcribed. Like that of the E. coli phn locus, the expression of the S. typhimurium phn locus is activated under conditions of P i limitation and is subject to Pho regulon control. This was shown both by complementation of the appropriate E. coli mutants and by the construction of S. typhimurium mutants with lesions in the phoB and pst loci, which are required for activation and inhibition of Pho regulon gene expression, respectively. Complementation studies indicate that the S. typhimurium phn locus probably includes genes both for phosphonate transport and for catalysis of C-P bond cleavage.Phosphonates (Pn) are a large class of molecules containing a direct carbon-phosphorus (C-P) bond in place of the more familiar carbon-oxygen-phosphorus bond of phosphoesters. Although Pn do not exist in all organisms, they do exist in many different ones. Pn have been found in organisms as diverse as the intracellular procaryote Bdellovibrio species, the filamentous bacteria streptomycetes, the protozoans Tetrahymena and Trypanosoma spp., mollusks, insects, and others. In these organisms, Pn have been isolated as constituents of glycolipids, glycoproteins, polysaccharides, or phosphonolipids. In Bacteroides fragilis, Pn were identified as components of capsular polysaccharide (6); in streptomycetes, Pn are synthesized as antibiotics such as fosfomycin (25); in Tetrahymena spp., Pn exist as phosphonolipids (19); in Trypanosoma cruzi, Pn are components of lipopeptidophosphoglycan (the major cell surface glycoconjugate [10]); in a sea anemone, Pn are the major P compounds (38); and in a locus, a Pn is the principal P compound of hemolymph (20). Yet, in spite of their widespread natural occurrence, the biological role of natural Pn is poorly understood (17). In phosphonolipids, 2-aminoethylphosphonate (AEPn) exists in place of its analog ethanolamine phosphate.No doubt because of the abundance of PN in nature, bacteria have acquired pathways for Pn breakdown. Bacteria capable of breaking the C-P bond may use Pn as a sole P source, for which two pathways exist, the C-P l...

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“…An overnight culture of CC118(pCU1) grown in L broth was infected with the Pam phage (25) carrying the TnphoAЈ-1 transposon element essentially as described by Wilmes-Reisenberg and Wanner (40). After overnight incubation at 37ЊC, the cells were spread on LB plates supplemented with kanamycin, streptomycin, spectinomycin, and ampicillin and incubated overnight.…”

Section: Methodsmentioning

confidence: 99%

“…We wished to monitor the fate of the plasmid in the population of Klebsiella recipients by screening rather than selecting transconjugants. To do this, we constructed a derivative of pCU1 called pCU1-lacZ carrying the Tn5-based transposable element TnphoAЈ-1 described by Wilmes-Reisenberg and Wanner (40). In this element, phoA of TnphoA is replaced by lacZ.…”

Section: ϫ7mentioning

confidence: 99%

Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids

1995

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The transmission of plasmid pCU1 (or other IncN group plasmid) into a population of Klebsiella oxytoca cells reduces the viability of the population. A 2,400-bp region adjacent to traA is responsible for this phenotype and includes two regions, called kikA and kikC. Klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. To obtain insight into the mode of this apparent lethality, an appropriate pCU1lacZ derivative was constructed. It could be introduced with high efficiency into Klebsiella cells. Analyses of the resultant colonies indicate that the loss of viability is not a consequence of the death of plasmid-free segregants. On the contrary and unlike postsegregational killing by plasmids, cells survived by losing the plasmid or by acquiring, secondarily, a chromosomal mutation which confers immunity to killing.All conjugative antibiotic resistance plasmids of the incompatibility group N of gram-negative bacteria confer on their hosts a phenotype called Kik ϩ . The phenotype consists of a marked reduction of the viability of Klebsiella oxytoca (previously Klebsiella pneumoniae [30,31,42]) recipients but not of Escherichia coli K-12 recipients following matings with the donor hosts on solid surfaces, a condition that is necessary for the efficient conjugative transfer of the plasmid. The phenotype was shown to have an intracellular basis and was initially called Kil (30,31), but following the use of the designation kil for plasmid loci conditionally lethal in E. coli (6, 41) and to indicate its host specificity, it was renamed Kik (38). Further studies were undertaken with a plasmid of this group called pCU1 that was the subject of detailed studies in our laboratory. It was shown by transposon Tn5 mutagenesis that a locus called kikA had an important role in this phenotype. The locus mapped close to one end of the conjugative transfer (tra) region of the plasmid, but the Tn5 mutants did not alter the efficiency of conjugative transfer between E. coli organisms (38). Hengen et al. (12) cloned and sequenced kikA and constructed a plasmid derivative in which kikA is controlled by the tac promoter. This cloned plasmid could be transformed efficiently into and maintained in Klebsiella cells. Upon induction with isopropyl-␤-D-thiogalactopyranoside (IPTG), the Klebsiella cells lost their viability. This showed that mating was not essential to bring about Klebsiella cell lethality and that loss of viability of Klebsiella cells could be mediated by events that occurred in them. While these observations showed that kikA has an important intracellular role in determining Klebsiella lethality, previous observations (11, 12, 18) have shown that there are loci on pCU1 and the closely related plasmid pKM101 that are lethal to E. coli (kil loci) in the absence or dysfunction of other, cognate loci (kor loci). Conceivably, such loci could also contribute to lethality of pCU1 in Klebsiella cells.The...

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TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization

Cited by 82 publications

References 42 publications

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids

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