Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12.

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.     

Novel incompatibility and partition loci for the REPI replication region of plasmid ColV-K30.

The minimum pColV-K30 REPI region necessary for replication was located within a ca. 1.3-kilobase DNA segment. Adjacent to the essential replication sequences, there are two DNA regions that express incompatibility with plasmids containing the F secondary replicon of the F EcoRI fragment f7. One of these regions corresponds to incE, already described in that F plasmid fragment which expresses incompatibility with f7-containing plasmids. The other is a novel sequence that we designated incF, which confers incompatibility with REPI, P307, and f7 derivatives, cis-acting pColV-K30 sequences conferring stability to REPI-containing plasmids were also identified and localized noncontiguous to REPI, ca. 20 kilobases downstream from the aerobactin iron transport genes, which were thus flanked by REPI and its partition (par) sequences.     

Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.     

Fragments of LINE-1 retrotransposons flanked by inverted telomeric repeats are present in the bovine genome. Homology with human LINE-1 elements

In the bovine genome we found two intrachromosomal DNA fragments flanked by inverted telomeric repeats (GenBank Accession Nos. AF136741 and AF136742). The internal parts of the fragments are homologous exclusively to the human sequences and to the consensus sequence of the L1MC4 subfamily of LINE-1 retrotransposons which are widespread among mammalian genomes. We found that distribution of homologous human sequences within our fragments is not random, reflecting a complicated pattern of insertion mechanisms of and maintenance of retrotransposons in mammalian genomes. One of the possible explanations of the origin of LINE-1 truncated elements flanked by inverted telomeric repeats in the bovine genome is that extrachromosomal DNA fragments may be modified by telomerase and subsequently, transferred into chromosomal DNA.     

Composite IS1 elements encoding hydroxamate-mediated iron uptake in FIme plasmids from epidemic Salmonella spp.

Eleven FIme plasmids representative of those identified in epidemic strains of Salmonella wien and Salmonella typhimurium isolated in North Africa, Europe, and the Middle East have been examined for the presence of determinants of toxigenicity, adherence, and iron-sequestering mechanisms. Chemical and genetic data indicated that all plasmids code for a hydroxamate-mediated iron assimilation system. Detailed analysis of derivative plasmids and cloned fragments of FIme plasmid pZM61 demonstrated that the general genetic and structural organization of the DNA region containing the genes for hydroxamate biosynthesis and cloacin DF13 receptor was virtually identical to that described for the aerobactin-mediated iron uptake system of pColV-K30. This DNA region is part of a composite element that is 16.7 kilobases long and carries its IS1 modules as inverted repeats. A very similar element is present in either orientation in all nine FIme plasmids analyzed.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.     

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.     

Critical sequences in the core of the P1 plasmid replication origin.

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.     

Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications.

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.     

Hoppel-family of mobile elements of Drosophila melanogaster, flanked by short inverted repeats and having preferential localization in the heterochromatin regions of the genome

A mobile element (ME) having 91% homology with Dm1360 (Kholodilov et al., 1987) has been cloned from the Drosophila melanogaster genome and sequenced. The family of ME was designated hoppel. The members of this family are flanked by short inverted repeats likewise P, hobo and HB. The hoppel is hybridized with 10-30 euchromatic sites of polytene chromosomes of different Drosophila stocks. Abundant hybridization with heterochromatic regions of chromosomes-chromocenter, pericentric heterochromatin, the 4 chromosome and telomeres was observed in all stocks of D. melanogaster examined and in D. simulans. At least six genomic variants of ME differing in length of the central part were revealed. Hoppel possesses ARS activity similar to the P element. Two ME hoppel were shown to be arranged as a direct repeat in the recombinant phage.     

Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.

A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC. Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate. Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.     

Two functions of the E protein are key elements in the plasmid F replication control system.

By using a plasmid carrying a translational fusion between the E gene of the IncFI plasmid F and the lacZ gene, we located the operator of the autogenously regulated E gene to an inverted repeat overlapping the E-gene promoter and showing perfect homology to part of the sequence found in all the direct repeats of two regions exerting an inhibitory effect on F replication, incB and incC. Excess E protein provided in trans to an F plasmid increased the replication frequency of the F plasmid. This stimulatory effect was counteracted by increased dosages of incB or incC. A model is proposed for the replication control system of F in which the key elements are autoregulation of E-gene expression and titration of E protein by incB and incC.     

A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells

It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation. Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids. According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II. Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids.     

Nonidentity between plasmid and chromosomal copies of ISS1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes.

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS1 from Lc. lactis subsp. lactis ML3. The IS was named ISS1RS. The chromosome of this strain contains several copies of ISS1-like IS as assessed by hybridization. One of these copies was cloned and named ISS1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.     

Easily unwound DNA sequences and hairpin structures in the Epstein-Barr virus origin of plasmid replication.

The Epstein-Barr virus (EBV) origin of plasmid replication (oriP) includes two known cis-acting components, the dyad symmetry region and the family of repeats. We used P1 nuclease, a single-strand-specific endonuclease, to probe EBV oriP for DNA sequences that are intrinsically easy to unwind on a negatively supercoiled plasmid. Selective nuclease hypersensitivity was detected in the family of repeats on an oriP-containing plasmid and in the dyad symmetry region on a plasmid that lacks the family of repeats, indicating that the DNA in both cis-acting components is intrinsically easy to unwind. The hierarchy of nuclease hypersensitivity indicates that the family of repeats is more easily unwound than the dyad symmetry region, consistent with the hierarchy of helical stability predicted by computer analysis of the DNA sequence. A specific subset of the family of repeats is nuclease hypersensitive, and the DNA structure deduced from nucleotide-level analysis of the P1 nuclease nicks is a cruciform near a single-stranded bubble. The dyad symmetry region unwinds to form a broad single-stranded bubble containing hairpins in the 65-bp dyad sequence. We propose that the intrinsic ease of unwinding the dyad symmetry region, the actual origin of DNA replication, is an important component in the mechanism of initiation.     

tDNA(ser) sequences are involved in the excision of Streptomyces griseus plasmid pSG1.

Plasmid pSG1 is maintained in some derivatives of Streptomyces griseus NRRL3851 mainly in the chromosomally integrated form (pSG1int). In others, the integrated plasmid is co-maintained with free pSG1 [Cohen et al., Plasmid 13 (1985) 41-50]. The pSG1 plasmid integration site (attP) and the pSG1int-chromosome boundaries (attL and attR) were cloned and sequenced. The results indicate that pSG1int is flanked at attL by a functional tRNA(ser) gene and at attR by a 60-nt sequence of the 3' end of the same tRNA(ser) gene. A single mismatch distinguishes the 60-nt sequence at attR from its direct repeat at attL. The attP site contains a single copy of the 60-nt repeat, identical to the one at attL. This observation indicates that pSG1, like integrating plasmids of other Actinomycetes, is integrated in a tRNA gene and suggests that the exact excision of pSG1int occurs by a recombinational crossing-over event at the first 43 nt of the 60-nt repeat.