Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.     

Escherichia coli molecular genetic map (1500 kbp): update II

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.     

Assessment of inhomogeneities in an E. coli physical map.

A statistical method based on r-fragments, sums of distances between (r + 1) consecutive restriction enzyme sites, is introduced for detecting nonrandomness in the distribution or too markers in sequence data. The technique is applicable whenever large numbers of markers are available and will detect clumping, excessive dispersion or too much evenness of spacing of the markers. It is particularly adapted to varying the scale on which inhomogeneities can be detected, from nearest neighbor interactions to more distant interactions. The r-fragment procedure is applied primarily to the Kohara et al. (1) physical map of E. coli. Other applications to DAM methylation sites in E. coli and NotI sites in human chromosome 21 are presented. Restriction sites for the eight enzymes used in (1) appear to be randomly distributed, although at widely differing densities. These conclusions are substantially in agreement with the analysis of Churchill et al. (3). Extreme variability in the density of the eight restriction enzyme sites cannot be explained by variability in mono-, di- or trinucleotide frequencies.     

Linkage Map of Escherichia coli K-12, Edition 10: The Physical Map

A physical map, EcoMap10, of the now completely sequenced Escherichia coli chromosome is presented. Calculated genomic positions for the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII are depicted. Both sequenced and unsequenced Kohara/Isono miniset clones are aligned to this calculated restriction map. DNA sequence searches identify the precise locations of insertion sequence elements and repetitive extragenic palindrome clusters. EcoGene10, a revised set of genes and functionally uncharacterized open reading frames (ORFs), is also depicted on EcoMap10. The complete set of unnamed ORFs in EcoGene10 are assigned provisional names beginning with the letter “y” by using a systematic nomenclature.     

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.     

Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12.

The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1. The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E. coli linkage map. Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E. coli DNA. The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E. coli chromosome by probing Southern blots of restriction digests of E. coli DNA with a fragment of the bfr gene. The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein. A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein). BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain. An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene.     

Localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome

The isolation and analysis of two recombinant plasmids containing the kdsA gene from Escherichia coli chromosomal gene libraries is reported. The subfragments obtained from the inserts correspond to the fragment pattern around coordinate 1,282 kilobases of the physical map of the E. coli chromosome (Kohara et al. Cell 50:495-508, 1987). The kdsA gene has been located at coordinates 1,282 through 1,283 kilobases, corresponding to min 26.7 in the classical map coordinates. The kdsA gene is transcribed from this position toward the nearby nar gene.     

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.     

Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon.

Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli. We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes. We have now studied the upstream promoter and the downstream terminator regions of this operon more completely. We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene. Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t. An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t. By comparing our DNA fragments with those prepared from this region of the E. coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E. coli chromosome. We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E. coli chromosomal map.     

The distribution of restriction enzyme sites in Escherichia coli.

A statistical analysis of physical map data for eight restriction enzymes covering nearly the entire genome of E. coli is presented. The methods of analysis are based on a top-down modeling approach which requires no knowledge of the statistical properties of the base sequence. For most enzymes, the distribution of mapped sites is found to be fairly homogeneous. Some heterogeneity in the distribution of sites is observed for the enzymes Pstl and HindIII. In addition, BamHI sites are found to be more evenly dispersed than we would expect for random placement and we speculate on a possible mechanism. A consistent departure from a uniform distribution, observed for each of the eight enzymes, is found to be due to a lack of closely spaced sites. We conclude from our analysis that this departure can be accounted for by deficiencies in the physical map data rather than non-random placement of actual restriction sites. Estimates of the numbers of sites missing from the map are given, based both on the map data itself and on the site frequencies in a sample of sequenced E. coli DNA. We conclude that 5 to 15% of the mapped sites represent multiple sites in the DNA sequence.     

Dynamic programming algorithms for restriction map comparison

For most sequence comparison problems there is a correspondingmap comparison algorithm. While map data may appear to be incompatiblewith dynamic programming, we show in this paper that the rigorand efficiency of dynamic programming algorithms carry overto the map comparison algorithms. We present algorithms forrestriction map comparison that deal with two types of map errors:(i) closely spaced sites for different enzymes can be orderedincorrectly, and (ii) closely spaced sites for the same enzymecan be mapped as a single site. The new algorithms are a naturalextension of a previous map comparison model. Dynamic programmingalgorithms for computing optimal global and local alignmentsunder the new model are described. The new algorithms take aboutthe same order of time as previous map comparison algorithms.Programs implementing some of the new algorithms are used tofind similar regions within the Escherichia coli restrictionmap of Kohara et al.     

Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro.

Operons uxaCA and exuT of the hexuronate system are very closely linked on the Escherichia coli genetic map. Using plasmid vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980), we formed exuT-lacZ and uxaA-lacZ fusions in vitro. The phenotypic properties of the new plasmids allowed us to confirm that the exuT and uxaCA operons are divergently transcribed. An analysis of these fusion plasmids and derivatives in the presence of multiple copies of the exuR regulatory gene demonstrated that the two operons possess separate control regions. The precise location of the operator site relative to endonuclease restriction sites was determined. In addition, deletions of different lengths were generated on exu plasmids by restriction enzymes and were recombined into the chromosome. The expression of the exu regulon genes in the resulting deletion mutants is in agreement with the postulated location of the exuT and uxaCA operators in the fusion plasmids.     

Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci.

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD alpha and GAD beta, and these differed by only five amino acids. Both GAD alpha and GAD beta contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gadA and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.     

The aconitase of Escherichia coli: purification of the enzyme and molecular cloning and map location of the gene (acn)

The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95,000 or 97,500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120,000) enzymes.     

In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus.

The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).     

Molecular analysis of the Escherichia coli phoP-phoQ operon.

The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.     

Cosmid-derived map of E. coli strain BHB2600 in comparison to the map of strain W3110.

A physical map for the genome of E. coli K12 strain BHB2600 was constructed by use of 570 cloned DNA elements (CDEs) withdrawn from a cosmid library. Dot blot hybridisation was applied to establish contig interrelations with subsequent fine mapping achieved by analysis of EcoR1 restriction patterns on Southern blots. The derived map covers nearly 95% of the E. coli genome resulting in 12 minor gaps. It may be compared to the almost complete map for strain W3110 of Kohara et al. (1). Except for one tiny gap (lpp,36.5') remaining gaps in BHB2600 do not coincide with those in W3110 so that both maps complement each other establishing an essentially complete clone represented map. Besides numerous minute differences (site and fragment gains and losses) both strains harbour at differing positions extended rearrangements flanked by mutually inverted repetitive elements, in our case insertion elements (IS1 and IS5).     

A newly identified DNA replication terminus site, TerE, on the Escherichia coli chromosome.

To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences. We found a Ter site, which we named TerE, located at 23.2 min on the linkage map. TerE inhibits only counterclockwise DNA replication. Macroscopically, five Ter sites are located in a periodic arrangement on the genome.