Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein

Sharpe, Pamela L.; Craig, Nancy L.

doi:10.1093/emboj/17.19.5822

Cited by 42 publications

(34 citation statements)

References 38 publications

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“…Some of these diverse proteins were MukB, a protein involved in the partition of chromosomes between daughter cells; SecA, a key protein translocation component; and SpoT, a protein required for the adaptation of cell physiology to nutrient limitation. Moreover, ACP had previously been reported to be an essential cofactor for the synthesis of a periplasmic oligosaccharide in vitro (49,50), to stimulate the nicking reaction of transposon Tn3 (26), and to give improved binding of Tn7 transposase to its target sequence (44).…”

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confidence: 99%

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Lay¹,

Cronan

2006

J Bacteriol

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Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.Two systems for fatty acid biosynthesis, called types I and II, are found in nature. Type I systems occur in the cytosols of mammalian and plant cells, in some plant plastids, and in a few bacteria. In these systems, the steps of fatty acid biosynthesis are catalyzed by one or two very large polypeptides that contain multiple active sites (47). In contrast, the type II systems which occur in bacteria, mitochondria, most plant plastids, and protozoan apicoplasts produce fatty acids using a series of soluble enzymes in which each protein has a discrete enzymatic activity (38). A key component of both systems is acyl carrier protein (ACP) which constitutes a domain of the type I multifunctional proteins but is a very small, soluble, and highly acidic protein in type II systems. ACP is functional only in fatty acid biosynthesis after it has been posttranslationally modified by covalent attachment of a 4Ј-phosphopantetheinyl (4Ј-PP) moiety. The 4Ј-PP prosthetic group is attached to the hydroxyl group of a centrally located serine residue by the AcpS 4Ј-PP transferase. Acyl intermediates are bound to the 4Ј-PP thiol in a thioester linkage that allows ACP to shuttle intermediates among the fatty acid synthetic enzymes. The thioester linkage also serves to facilitate chemistry at acyl chain ␤-carbons.Escherichia coli contains a single ACP encoded by the acpP gene (35). This was the first ACP discovered (40, 52) and remains the best studied of these proteins. Despite the breadth and depth of studies of E. coli ACP and its acylated derivatives, no mutant strain encoding a mutant ACP has been reported. However, ACP seemed certain to be an essential protein since mutants in several f...

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confidence: 99%

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Lay¹,

Cronan

2006

J Bacteriol

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“…A satisfying aspect of this hypothesis is that, since the abundance of nucleoid-associated proteins varies during cell growth, their relative availability would accordingly serve as a read-out of the cell's environment (4). The involvement of other host proteins in the transposition process (for example, proteases, chaperones, DnaA, ribosome-associated proteins) (13,29,31,49,57) suggests that this form of "cellular input" plays a much greater role than has currently been defined. Our unpublished results and the findings described here support this idea, by defining additional host factors that modulate transposition.…”

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Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

et al. 2005

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Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.Transposons are defined as distinct regions of DNA that have the ability to move from one genomic location to another, a process mediated by element-encoded transposases (8, 9). While the main focus of transposition research has been in determining the detailed mechanisms of transposition, relatively little attention has been paid to how transposition is regulated in vivo or the in vivo requirements for the process (35). Transposon movement can result in gene inactivation (by insertional inactivation or deletion) or activation (by creation or introduction of promoters upstream of genes). In addition, intramolecular transposition can result in extensive DNA rearrangements, including deletions, inversions, and duplications of chromosome segments; these events are thought to play an important role in facilitating genome evolution (7-9). As the consequences of transposition can be either dire or favorable for the host organism, it is intuitive that the host has the capability to regulate this process. There are multiple examples of how transposons regulate their own movement, but very little work has focused on the role played by the host in regulating transposition. To address this, we have generated an insertion mutant library of Escherichia coli, which we have used to screen for host genes involved in regulation of IS903 transposition (E. Twiss, A. Coros, N. Tavakoli, and K. M. Derbyshire, unpublished data).The first comprehensive genetic screen for host mutants affecting transposition was carried out in 1985; it revealed a role for dam methylation in decreasing the rate of IS10, IS903, and IS50 transposi...

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“…Others, exemplified by Tn7, specify two different proteins that form a more complex transposase. With Tn7, each transposase protein has a distinct role in transposition, and additional Tn7-encoded proteins help select insertion sites and affect the efficiency of transposition, in part through interactions with host proteins (9,31). A third type of element, exemplified by IS605 of the gastric pathogen Helicobacter pylori, contains just two transposition-related genes, each of which has protein level homology to the single putative transposase genes of other simpler onegene ISs and thus may be of different phylogenetic origin (18,19).…”

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Transposable Element IS Hp608 of Helicobacter pylori : Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity

et al. 2002

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A new member of the IS605 transposable element family, designated ISHp608, was found by subtractive hybridization in Helicobacter pylori. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (orfA and orfB), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes; orfB is also related to the Salmonella virulence gene gipA. PCR and hybridization tests showed that ISHp608 is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. ISHp608 also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively; P ‫؍‬ 0.04). Two ISHp608 types differing by ϳ11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by <2% in DNA sequence, but several recombinant elements were also found. ISHp608 marked with a resistance gene was found to (i) transpose in Escherichia coli; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5-TTAC without duplicating target sequences; and (iv) require orfA but not orfB for its transposition. ISHp608 represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.

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Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein

Cited by 42 publications

References 38 publications

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

Transposable Element IS Hp608 of Helicobacter pylori : Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity

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