The Escherichia coli dnaW mutation is an allele of the adk gene

Summary A dnaW mutant, isolated on the basis of inability to effect conjugal DNA transfer at high temperature, has been shown by complementation and enzyme assay to be defective in the adk (adenylate kinase; EC 2.7.4.3) locus. The adk mutant, known to have reduced ATP concentration at the nonpermissive temperature (Cousin and Belaich 1966), was used to demonstrate a donor energy requirement for stable aggregate formation and for chromosome transfer in conjugation.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.     

The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase.

The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature. We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase. We show that ts8 and a second fda mutation, h8, isolated and characterized by A. B?ck and F. C. Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.     

radC102 of Escherichia coli is an allele of recG

The radC102 mutation causes mild UV and X-ray sensitivity and was mapped previously to near pyrE and recG at 82 min on the Escherichia coli chromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615-625, 1984). We report that radC102 has two striking phenotypes characteristic of recG mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with ruv mutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the radC and recG genes in radC102 strains revealed that the radC102 mutation creates a stop codon in recG that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the radC(+) gene did not affect the UV sensitivity of a wild-type strain, a radC102 strain, or a recG258::Tn10mini-kan strain. We conclude that radC102 is an allele of recG and that the function of the RadC protein remains to be determined.     

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.     

Genetic mapping and DNA sequence analysis of mutations in the polA gene of Escherichia coli

DNA polymerase I of Escherichia coli provides an excellent model for the study of template-directed enzymatic synthesis of DNA because it is a single subunit enzyme, it can be obtained in large quantities and the three-dimensional structure of the polymerizing domain (the Klenow fragment) has recently been determined (Ollis et al., 1985). One approach to assigning functions to particular portions of the structure is to correlate the altered enzymatic behavior of mutant forms of DNA polymerase I with the change in the primary sequence of the protein. Towards this end we have developed a rapid procedure for mapping any polA mutation to a region no larger than 300 base-pairs within the polA gene. Two series of polA deletion mutants with defined end-points were constructed in vitro and cloned into bacteriophage lambda. These phages can then be used to map precisely E. coli polA mutants. Twelve polA- alleles have been mapped in this way and for nine of them the nature of the mutational change has been determined by DNA sequence analysis. Two of the mutations, polA5 and polA6, which affect the enzyme-DNA interaction, provide evidence for the location of the DNA binding region on the polymerase three-dimensional structure.     

ftsW is an essential cell-division gene in Escherichia coli

In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD . We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW , in the presence of the plasmid-borne wild-type ftsW gene under the control of P_BAD. In the absence of arabinose, the ftsW -null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW -null filaments, the FtsZ ring is absent in 50–60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P_BAD is sufficient for complementation of the ftsW -null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli , and that it plays a role in the stabilization of the FtsZ ring during cell division.     

Characterization of the ftsB gene as an allele of the nrdB gene in Escherichia coli.

A temperature-sensitive, salt-rescuable ftsB cell division mutant, MFT84, was found to be hydroxyurea sensitive on low-salt medium. Complementation studies with plasmids and a marker rescue study with bacteriophage M13 nrd indicated that ftsB is an allele of nrdB and that the mutation occurs in the region corresponding to nucleotides 6729 to 7032 of the nrdB gene. Enzymatic characterization demonstrated that the B2 subunit of ribonucleoside-diphosphate reductase encoded by ftsB was responsible for the decreased activity and the thermolability of the enzyme. The ftsB-encoded B2 subunit was activated by the addition of 0.1 M NaCl to an in vitro assay, corroborating the in vivo temperature-dependent salt requirement was a result of a defective B2 subunit.     

Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I

We report the nucleotide sequence of 3.2 kilobase pair region of the Escherichia coli polA gene, comprising the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S. (1982) J. Biol. Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our sequence. Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem. Biophys. Res. Commun. 37, 982-989; Klenow, H., and Henningsen, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polA1 and wild type alleles, we have identified the polA1 mutation as a change from Trp (TGG) to amber (TAG) at residue 342.     

Ultraviolet-Sensitive Mutator mutU4 of Escherichia coli Inviable with polA

Attempts to transduce the ultraviolet-sensitive mutator lesion mutU4 into strains deficient in deoxyribonucleic acid polymerase I (polA) were unsuccessful. Mutator recombinants were found when the polA recipient had first been reverted to Pol(+) by selection for resistance to methyl methanesulfonate. The inviability of the mutU4 polA double mutant was demonstrated by a reduction in the absolute number of transductants when the recipient was polA as compared with Pol(+), and selection was made for markers very close to mutU4. Double mutants containing mutU4 and polA4, which determines a cold-sensitive polymerase, were unable to grow at 24 C, the nonpermissive temperature.     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Escherichia coli parA is an allele of the gyrB gene

Summary A thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.     

Isolation of Haemophilus influenzae genes that suppress Escherichia coli polA mutations

Haemophilus influenzae was found to produce a DNA polymerase that was similar to polymerase I of Escherichia coli. E. coli polA mutants were used as backgrounds for the selection of H. influenzae polA suppressor genes. Six different H. influenzae fragments were isolated that could suppress E. coli polA mutations. None of the suppressors appeared to encode the H. influenzae equivalent of the E. coli polA gene. One type of clone, represented by pGW41, caused a polymerase I activity to appear in a suppressed polA1 mutant. Plasmids from the pGW41 class contained two genes (pol-2 and pol-3) that were both required for polA suppression. Mutated nonsuppressing derivatives of the pGW41 class were used to create H. influenzae mutants that were deficient in polymerase I.     

ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.