Synthesis and overproduction of the 5A protein of insertion sequence IS5

Chernak, Jeffrey M.; Schlaffer, E J; Smith, Hamilton O.

doi:10.1128/jb.170.11.5368-5370.1988

Cited by 4 publications

(7 citation statements)

References 19 publications

(18 reference statements)

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“…The plasmids used for complementation, preparation of probes, and subcloning segments of thefuc region into M13 sequencing vectors were previously described (11). Plasmid pJE100, a pBR322 derivative containing the entire IS5 element, was provided by Hamilton Smith (12 (11). The open boxes under the map represent DNA fragments used as probes.…”

Section: Methodsmentioning

confidence: 99%

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

1989

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L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of thefuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controliing region shared by the operonsfucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression offucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.Following the discovery that a genetic derepression of ribitol dehydrogenase in Klebsiella pneumoniae conferred a growth ability on xylitol (27, 33), we tried to find other models of experimental evolution for assessing the importance of regulatory mutations in the acquisition of novel functions (for a review, see reference 30). During this search, we discovered that Escherichia coli can give rise to mutants that utilize L-1,2-propanediol aerobically as a sole source of carbon and energy. Serial selection on the compound resulted in the emergence of a mutant (ECL3) which grew on the novel carbon and energy source at a rate close to that on glycerol. This mutant produced constitutively an NAD+-linked oxidoreductase active on L-1,2-propanediol (46). The proximity of the locus specifying this enzyme activity and the fuc locus (at min 60) specifying the growth ability on L-fucose led us to ask whether there was a link between the metabolism of the two compounds. When the propanediol-positive mutant was tested with fucose as a carbon and energy source, growth no longer occurred (13).Previous work, together with our further studies (13), revealed the biochemical connection between the two metabolic pathways (Fig. 1). The dissimilation of fucose by wild-type E. coli requires the sequential action of fucose permease (encoded byfucP), fucose isomerase (encoded by fucf), fucose kinase (encoded by fucK), and fuculose-lphosphate aldolase (encoded by fucA). The last enzyme catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Under anaerobic conditions, the aldehyde is reduced to L-1,2-propanediol by an NAD+-linked oxidoreductase (encoded by fucO) and excreted into the medium. Under aerobic conditions, the aldehyde is oxidized to Llactate and then to pyruvate which enters the general metabolic pool. The inducer of the fucose syst...

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Section: Methodsmentioning

confidence: 99%

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

1989

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“…The observed molecular weight of the 5B protein differed somewhat from the predicted value of 11.1 kDa. In previous work, we have been unable to confirm the synthesis of a 12.3-kDa species from an intact copy of IS5, even when the 5B ORF was located downstream of a strong tac promoter (3,12). This study demonstrates the in vitro and in vivo synthesis and overproduction of the 5B protein using a specially-designed synthetic ribosome binding site (SRBS) in conjunction with a tac promoter and a subcloned copy of the SB ORF.…”

Section: Introductionmentioning

confidence: 80%

“…Plasmids pBR322 (20), ptacll (14), and pMLB1034 (18) have been previously described; pSK10 (21) carries a promoterless galactokinase gene downstream of a tac promoter and contains a laciq gene cloned into the EcoRI site of pDR540 (22). The plasmids pDI15 and pDI40 contain IS5 at the same site but in opposite orientations within the pBR322 derivative pDI14 (11), and pJC100 contains IS5 inserted downstream of the pBR322 tet promoter (3). Plasmids pJC200, pJC201, pJC251, pJC281, pJC287 and pJC57 are described below.…”

Section: Bacteria and Plasmidsmentioning

confidence: 99%

“…1) has a compact genetic structure which includes three ORFs (9)(10)(11) and associated promoter and terminator signals (4,8,10). The longest ORF has been shown to encode a 37-kilodalton (kDa) 5A protein in vitro and in Escherichia coli minicells or maxicells (3,4), and the smaller ORFs have been reported to encode a 12.3-kDa 5B pro- 37.8 kd 5A…”

Section: Introductionmentioning

confidence: 99%

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Use of synthetic ribosome binding site for overproduction of the 5B protein of Insertion sequence IS5

Chernak

Hamilton

1989

Nucl Acids Res

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Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Althoclh there was no detectable expression from the 5B open reading fra.ne when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. 1-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.

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“…By PCR using two oligonucleotides complementary to the IS1106 inverted-repeat sequences (IRs), we isolated a wild-type element from serogroup B strain BL859 (Fig. 1A) (27,8) (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Identification, Characterization, and Variable Expression of a Naturally Occurring Inhibitor Protein of IS 1106 Transposase in Clinical Isolates of Neisseria meningitidis

et al. 2001

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Transposition plays a role in the epidemiology and pathogenesis of Neisseria meningitidis. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS1106 controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS1106 transposase lacking the NH 2 -terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS1106, as shown by gel shift and in vitro transposition assays. IS1106Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.Many studies have pointed out the importance of mobile genetic elements in microbial pathogenesis and adaptation to changing environmental conditions. Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins, capsules, pili, resistance determinants, or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids, or bacteriophages (16, 50). Insertion sequence (IS) elements are known to be involved in microevolution of bacterial genomes by several mechanisms (1, 32). (i) In the chromosomes of both gram-negative and gram-positive bacteria, virulence genes are often clustered in "virulence blocks" or "pathogenicity islands," surrounded by IS elements that promote their transposition and lead to changes in virulence in the course of evolution. Pathogenicity islands are invariably found in pathogenic strains of a given species but are either absent or rarely present in nonpathogenic variants of the same species (12,20,21,23,30,33,34). (ii) In addition, two copies of certain IS elements flanking a DNA segment are able to act in concert, mobilizing the intervening region. (iii) Programmed insertion and excision of IS elements and of invertible DNA sequences may control the expression of several virulence factors by a mechanism of phase variation (24, 58). (iv) "Jumping" of DNA sequences (transposition) and subsequent recombination events may also cause gene activation (5) and antigenic variation of virulence factors, leading to the emergence of novel pathogenic variants (48). (v) IS-related DNA rearrangements do occur in resting bacterial cultures and confer plasticity on the genome under conditions of nutritional deprivation, thereby playing an adaptive role (1,3,18,35,36).With the development of studies of the mechanisms of bacterial pathogenesis and the advent of whole-genome sequencing technologies in recent years, the finding of association between IS elements and pathogenic and virulence functions has become increasingly evident. Such associations have been observed in a variety of animal pathogens (6,9,14,17,31,52).Whole-genome sequence analysis and subtractive hybridization ...

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Synthesis and overproduction of the 5A protein of insertion sequence IS5

Cited by 4 publications

References 19 publications

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

Use of synthetic ribosome binding site for overproduction of the 5B protein of Insertion sequence IS5

Identification, Characterization, and Variable Expression of a Naturally Occurring Inhibitor Protein of IS 1106 Transposase in Clinical Isolates of Neisseria meningitidis

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