rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene.

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity.     

Gene lon and plasmid inheritance in Escherichia coli K-12.

Lon- mutants of Escherichia coli K-12 are deficient in the inheritance of F-plasmids by conjugation. This deficiency is distinct from the conjugation deficiency caused by overproduction of capsular polysaccharide which decreases donor-recipient pair formation.     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Synthesis of linear plasmid multimers in Escherichia coli K-12.

Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322. A mutation in sbcB increases the proportion of plasmid DNA as linear multimers. A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis. Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication. Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers. The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.     

Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.     

Random replication of the stringent plasmid R1 in Escherichia coli K-12.

The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.     

Reactivation of ultraviolet-irradiated phage lambda and recombination in Escherichia coli K-12 cells containing plasmid ColIb-P9

It was shown that the presence of colicinogenis plasmid ColIb-P9 increased the survival of UV-irradiated bacteriophage lambda cI857 in non-irradiated cells of Escherichia coli K-12. The effect of this plasmid was retained in the polA and recB mutants, being sharply reduced in the uvrA and recB recC sbcB recF mutants. This effect strongly depended on recA+ and lexA+ genotype. The W-reactivation efficiency was slightly higher in the cells containing ColIb-P9 than in those lacking the plasmid. No significant effect of the plasmid on recombination during transduction, after conjugation under usual conditions and in the case when a conjugation mixture or recipient cells were irradiated, was observed. The data demonstrate that the effect of ColIb-P9 plasmid on DNA repair is not mediated by its influence on recombination.     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid.

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Mapping of chromosomal IS5 elements that mediate type II F-prime plasmid excision in Escherichia coli K-12.

Three IS5 elements were mapped in overlapping chromosomal segments on a series of F-prime plasmids by restriction analysis and hybridization. IS5A was located clockwise of proA near 6 min, IS5B was located clockwise of purE near 12 min, and IS5C was tentatively located near 14 min on the Escherichia coli K-12 map. The physical structures of nine type II F-prime plasmids that contain chromosomal DNA from this region indicated that these plasmids were excised from the chromosome by recombination between pairs of IS5 elements.     

Evidence for two distinct pyruvate kinase genes in Escherichia coli K-12

A strain of Escherichia coli K-12 defective in pyruvate kinase F has been produced. The existence of this mutant, in conjunction with earlier results, strongly suggests that the two pyruvate kinases in this bacterium are distinct forms and not interconvertible. Either form of pyruvate kinase appeared to be equally effective in the glycolytic conversion of phosphoenolpyruvate to pyruvate. Genes specifying pyruvate kinase A and pyruvate kinase F were present on the small F-prime F506 and the locus for pyruvate kinase F was found to be at minute 36.5 on the E. coli genetic map.     

The ColIb-CA53 plasmid suppresses umuC- and umuD-mutations in Escherichia coli K-12

The presence of the ColIa-CA53 plasmid in umuC and umuD mutant Escherichia coli K-12 cells restores their mutability under UV irradiation to a level that even exceeds that of the isogenic umuC+umuD+ strains, as well as increases their resistance to the lethal effects of UV irradiation. The ColIb-P9 plasmid which suppresses the umuC mutant phenotype, as we have shown earlier, acts in the same manner with respect to the umuD mutant cells. The results of the study demonstrate that both plasmids encode products that are functionally similar to those of the chromosomal genes umuC and umuD. The plasmids ColIa-CA53, ColIb-P9 and pKM101 are shown to have practically the same effect upon the mutagenesis and survival of the umuC, umuD mutant cells.     

Escherichia coli K-12 mutants with an increased efficiency of plasmid transformation

Escherichia coli K-12 mutants with an enhanced efficiency of plasmid transformation were obtained. In all the mutants, the efficiency of transfection with lambda vir phage DNA was changed, in comparison to the parent strain. However, these changes did not always correlate strictly with plasmid transformation alterations. For instance, two mutants with an increased plasmid transformation efficiency demonstrated 50-fold decrease in the level of transfection with lambda phage DNA. Polyacrylamide gel electrophoresis points to both quantitative and qualitative differences in protein composition of the mutant cell envelopes, as compared with the parent strain.     

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.Abbreviations IRS isoleucyl-tRNA synthetase - VRS valyl-tRNA synthetase - ARS arginyl-tRNA synthetase - cAMP cyclic adenosine-3,5-monophosphate - Cya adenyl cyclase Gene - CRP cAMP receptor protein - O.D. optical density     

Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.     

Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12.

Replicating DNA molecules of the nonconjugative R plasmid RSF1010 (Smr Sur) were cleaved with the EcoRI restriction endonuclease and examined with the electron microscope. Results of this analysis indicated that replication is initiated from an origin located at about 19% of total genome size from one of the EcoRI ends. Replication proceeded either unidirectionally or bidirectionally with equal frequency. Results of the analysis of replicative intermediates of RSF1010 containing the Apr-transposable sequence (Tn) are also presented.     

Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA

A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.     

Endonuclease V (nfi) Mutant of Escherichia coli K-12

Endonuclease V (deoxyinosine 3′ endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A · T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or X174 nor of Uracil-containing bacteriophage λ. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.