IS186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

Cloning, nucleotide sequence, and expression of the HincII restriction-modification system.

Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.     

Identification, distribution, and sequence analysis of new insertion elements in Caulobacter crescentus.

We describe two insertion elements isolated from Caulobacter crescentus that are designated IS298 and IS511. These insertion elements were cloned from spontaneous flagellar (fla) gene mutants SC298 and SC511 derived from the wild-type strain CB15 (ATCC 19089), in which they were originally identified as insertions in the flbG operon of the hook gene cluster (N. Ohta, E. Swanson, B. Ely, and A. Newton, J. Bacteriol. 158:897-904, 1984). IS298 and IS511 were each present in C. crescentus CB2 and CB15 in at least four different positions, but neither was present in strain CB13 or in several Caulobacter species examined, including C. vibrioides, C. leidyia, and C. henricii. Nucleotide sequence analysis across the chromosome-insertion element junctions showed that IS298 is located 152 base pairs (bp) upstream from the ATG translation start of the hook protein gene flaK, where it is bounded by a 4-bp direct repeat derived from the site of insertion, and that IS511 is inserted at codon 186 of the flaK coding sequence, where it is also bounded by a 4-bp direct repeat duplicated from the site of insertion. The ilvB102 mutation in strain SC125 was also shown to result from insertion sequence IS511, but no duplication of the genomic sequence was present at the insertion element junctions. IS298 contains an imperfect terminal inverted repeat 16 bp long, and IS511 contains a 32-bp inverted repeat at the termini. IS298 and IS511 are the first insertion elements described in C. crescentus.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

Analysis of replication region of the cryptic plasmid pAG20 from Acetobacter aceti 3620

The DNA sequence of small cryptic plasmid pAG20 in Acetobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence 1 and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E. coli, Acetobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp.     

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

Nucleotide sequence analysis of the complement resistance gene from plasmid R100

The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.     

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.     

The IS1 elements in Shigella boydii: horizontal transfer,vertical inactivation and target duplication

IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii. The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E. coli K12 suggest that these IS1 elements had been horizontally transferred among S. boydii and E. coli O157:H7 and K12. The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity. Analysis of the flanking sequences of IS1(SB) and its two variants in S. boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.     

Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747

The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96-98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.     

The Evolution of Insertion Sequences within Enteric Bacteria

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.