Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.     

Location of DNA methylation genes on the Escherichia coli K-12 genetic map

Summary The genes responsible for DNA adenine methylation (dam) and DNA cytosine methylation (dcm) have been mapped on the E. coli K-12 genetic map. The dam gene is situated at min 65 and the gene order cysG-(trpS, dam)-aro B inferred. The dcm gene is located at min 37.5 and the gene order is supD-dcm-flaA1. In F merodiploids, the dam and dcm alleles are recessive.     

Presence of DNA, encoding parts of bacteriophage tail fiber genes, in the chromosome of Escherichia coli K-12

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.     

Linkage map of Escherichia coli K-12, edition 8.

The linkage map of Escherichia coli K-12 depicts the arrangement of genes on the circular chromosome of this organism. The basic units of the map are minutes, determined by the time-of-entry of markers from Hfr into F- strains in interrupted-conjugation experiments. The time-of-entry distances have been refined over the years by determination of the frequency of cotransduction of loci in transduction experiments utilizing bacteriophage P1, which transduces segments of DNA approximately 2 min in length. In recent years, the relative positions of many genes have been determined even more precisely by physical techniques, including the mapping of restriction fragments and the sequencing of many small regions of the chromosome. On the whole, the agreement between results obtained by genetic and physical methods has been remarkably good considering the different levels of accuracy to be expected of the methods used. There are now few regions of the map whose length is still in some doubt. In some regions, genetic experiments utilizing different mutant strains give different map distances. In other regions, the genetic markers available have not been close enough to give accurate cotransduction data. The chromosome is now known to contain several inserted elements apparently derived from lambdoid phages and other sources. The nature of the region in which the termination of replication of the chromosome occurs is now known to be much more complex than the picture given in the previous map. The present map is based upon the published literature through June of 1988. There are now 1,403 loci placed on the linkage group, which may represent between one-third and one-half of the genes in this organism.     

Inversion induced by temperature bacteriophage mu-1 in the chromosome of Escherichia coli K-12.

Induction of the Mu prophage of a lysogenic HfrP4X strongly stimulates the early transfer of the purE gene, which is located far from the origin of transfer. By using a rec- Mu cts62 X lysogenic donor, it was established that this process reflects the inversion of the origin of transfer in part of the Hfr population. Hfr's with inverted polarity of gene transfer were isolated; their analysis suggests that two Mu genomes in opposite orientation surround the inverted DNA fragment. Due to the presence of the Mu genome of the invertible G segment, homologous regions in the same orientation can appear in Mu genomes in opposite orientation. In a Rec+ background, Hfr's with inverted polarity (i) return to their original polarity of transfer by recomination between the two inverted Mu and (ii) produce new F' strains by recombination between the two similarly oriented G segments.     

The E. coli K-12 chromosome flanked by two IS10 sequences transposes

Summary Transposon are commonly found among prokaryotes and usually range up to 20 kilobases. In this study, we were interested to determine whether a larger DNA segment could transpose. We observed that the E. coli K-12 chromosome, 4,000 kilobases in size, when flanked by two IS10 sequences, could transpose to pACYC177 at a frequency of 10^-8 per cell per generation. We suggest that this transposition event occurs independently of the size and without duplication of the entire DNA sequence flanked by the IS10 elements.     

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.     

Correlation of a subset of the pLC plasmids to the physical map of Escherichia coli K-12.

We determined map positions of the Escherichia coli K-12 portions of a subset of the hybrid E. coli-ColE1 plasmids constructed by Clarke and Carbon. The probe DNA of pLC plasmids was labeled with digoxigenine-dUTP, hybridized to the 476 phage clones of the E. coli ordered clone bank miniset, which was adsorbed on a strip of nylon membrane filters, and detected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed color reaction. The total number of Clarke-Carbon plasmids we analyzed was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed with those reported previously, and the remaining 41 plasmids gave map positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it is presented, as well as a table which correlates the pLC plasmids with the clones of the E. coli ordered clone bank miniset on the basis of the hybridization data. We estimate that approximately one-half of the entire genome of E. coli was covered by the pLC plasmids used in this study.     

A physical map of belomycin-specific fragmentation sites on PM2 bacteriophage DNA

Bleomycin treatment of PM2 DNA results in fragmentation of the genome at several specific sites. Application of restriction endonuclease digestion followed by bleomycin treatment has provided the basis for constructing a physical map of bleomycin fragmentation sites. Eleven sites have been located on the physical map relative toHpa II,Pst I, andHindIII cleavage sites. The fragmentation sites are not clustered in a particular region of the PM2 genome but 3 of the 11 sites do occur between theHpa II andPst I cleavage sites, a segment of DNA which comprises 14% of the PM2 DNA length.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu.

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.