Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.     

Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

A general method for isolation of Mu d1-8(Aprlac) operon fusions in Salmonella typhimurium LT2 from Tn10 insertion strains: chlC::Mu d1-8

Summary A general method was developed for the isolation of Salmonella thyphimurium LT2 Mu d1–8 (Ap^r lac) operon fusions in a gene displacing a Tn10 insertion. Random Mu d1–8 fusion pools were prepared to grow phage P22 lysates which transduced chlC::Tn10 to Ap^rTet^s on fusaric acidampicillin plates. Among these Ap^rTet^s potential chlC::Mu d1–8 fusions, a simple spot test identified the fusions that were closely linked to the Tn10 insertion in chlC. Out of 68 Ap^rTet^s isolates 7 chlC::Mu d1–8 fusions with a nitrate-induced Lac⁺ phenotype were obtained. When oxrA::Tn10 was transduced into these chlC::Mu d1–8 fusions, they became Lac^- even in the presence of nitrate, confirming that they were chlC::Mu d1–8 fusions.     

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac⁺ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac⁺ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac⁺ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac⁺ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac⁺ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac⁺ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac⁺ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.     

Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu

A general procedure is described for transposing the lac genes to selected locations on the Escherichia coli chromosome. These transpositions were designed so that the lac² genes could be fused to nearby promoters. In particular, the lac genes were fused to the promoters for the araBAD, araC and leu genes. In these fusions the lac genes are regulated by the controls of the genes to which they are fused. These fusions are therefore useful in discovering new types of regulation of gene expression, as was found in the case of the araC gene. λ transducing phage carrying the fusion as well as nearby genes can easily be isolated. Some of these fusions may result in the formation of hybrid proteins.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Genetic fusions that help define a transcription termination region in Escherichia coli

The trpA gene product was analyzed from a class of strains of Escherichia coli K12 in which the lac operon has been fused by deletion to the trp operon. These are strains that have retained the ability to synthesize tryptophan. Two of these strains are shown to make a wild-type trpA product; these strains retain intact all structural genes of the ttrp operon. It is proposed that the lac operon in these strains is fused to a region of the trp operon between trpA, the last gene in the operon, and the region where trp messenger RNA synthesis terminates. The region where trp messenger RNA synthesis terminates thus is distinct from the trp structural genes.     

Use of the Mud(Ap,lac) bacteriophage to study the regulation of L-proline biosynthetic genes inEscherichia coli K-12

One operon fusion to the promoter of either theproA orproB genes of the proline biosynthetic pathway was obtained by the use of the Mud(Ap,lac) bacteriophage. This operon fusion was further stabilized by transformation with the plasmid pGW600 containing the wild type Mu repressor gene. The level of β-galactosidase in this strain was not affected by the presence of high concentrations of NaCl in the growth medium. Mutations affecting the regulation of thispro-lac genetic fusion were generated by the insertion of Tn5; β-galactosidase levels in these mutants were higher than in the parental strain when proline was present at a high level. In some of these mutants we observed either repression or maintenance of β-galactosidase levels whenpro-lac (F′proAB ⁺) merodiploids were constructed.     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.     

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac⁺) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac⁺ triggers exponential cell growth leading to a stable Lac⁺ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac⁺ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac⁺ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.     

Donor Strains of the Soft-Rot Bacterium Erwinia chrysanthemi and Conjugational Transfer of the Pectolytic Capacity

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F′lac⁺ plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F′lac⁺ heterogenote by repeated platings of single Lac⁺ colonies on lactose minimal agar, do not segregate (as does the parent F′lac⁺ heterogenote) into Lac⁻ or F⁻ clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade⁺, gal⁺, gtu⁺ (utilization of galacturonate), his⁺, lac⁺, leu⁺, lys⁺, mcu⁺ (multiple carbohydrate utilization), pat⁺ (production of polygalacturonic acid trans-eliminase), thr⁺, and trp⁺ in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade⁺, leu⁺, and thr⁺ transfer were higher than those of the other markers tested to date. This donor strain transfers lac⁺ genes during a 6-h mating on membranes; most of the Lac⁺ recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr⁺ and leu⁺ enter the recipient as proximal markers and that lac⁺ enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F′lac⁺ into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat⁺ recombinants and not in Pat⁻ recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.     

Concomitant Conjugal Transfer of Reduced-Bacteriophage-Sensitivity Mechanisms with Lactose- and Sucrose-Fermenting Ability in Lactic Streptococci

Ten previously reported lactose-positive (Lac⁺) transconjugants from Streptococcus lactis, S. cremoris, and S. lactis subsp. diacetylactis and one sucrose-positive (Suc⁺) transconjugant from S. lactis were examined for their sensitivity to prolate- and small isometric-headed bacteriophages. Four of the Lac⁺ transconjugants showed a 10- to 100-fold reduction in the efficiency of plating (EOP) as well as a reduced plaque size for the prolate phage c2 and were insensitive to the small isometric phage 712. A fifth Lac⁺ transconjugant demonstrated a similar reduced sensitivity to phage c2; however, this transconjugant was able to plaque phage 712, but with a reduced plaque size and EOP. The other five Lac⁺ transconjugants were sensitive to both c2 and 712 phages. The Suc⁺ transconjugant plaqued phage 712 with a reduced plaque size and EOP, but no reduction in plaque size or EOP was observed for phage c2. The Lac⁺ and reduced bacteriophage sensitivity (Rbs⁺) phenotypes were correlated with specific plasmids in the Lac⁺ transconjugants. As four of the Lac⁺ transconjugants exhibited a phenotypically indistinguishable Rbs⁺, one (AB001) was selected for further study. The Rbs⁺ in AB001 for both small isometric- and prolate-headed phages was not related to adsorption, and the reduced EOP for phage c2 was not related to the presence of a restriction and modification system. The latent period for phage c2 was unchanged, but the burst size was reduced 80%. The presence of the plasmid coding for Rbs⁺ retarded the lysis of a mitomycin C-induced prophage-containing strain. The Rbs⁺ mechanism appears to be abortive phage infection. This study supports previous observations that Rbs⁺ and conjugal transfer ability are physically linked among some group N streptococci. The results presented have implications in the identification of plasmids coding for Rbs⁺ and may also aid in explaining the dissemination of Rbs⁺ genes among lactic streptococci.     

Genetic and molecular study of the inability of the yeast Kluyveromyces lactis var. drosophilarum to ferment lactose

The fermentation of lactose (Lac⁺) in the dairy yeast Kluyveromyces lactis var. lactis is controlled by the LAC4 (β-galactosidase) and LAC12 (lactose permease) genes. The complementation analysis of twelve Kl. lactis var. drosophilarum natural homothallic Lac^? strains of different origin was carried out using the genetic heterothallic lines of Kl. lactis var. lactis of the lac4LAC12 and LAC4lac12 genotypes. It was shown that the natural Lac^? strains did not possess the LAC4LAC12 gene cluster. Southern hybridization of chromosomal DNA with LAC4 and LAC12 probes, as well as recombination analysis, showed that Kl. lactis var. drosophilarum yeasts do not have even silent copies of these genes. As distinct from this yeast, natural Lac^? strains of the yeast Kl. marxianus are mutants impaired in the lactose permease gene (lac12 analogue), but possess an active β-galactosidase gene (LAC4 analogue). The origin of the LAC4LAC12 gene cluster of the dairy yeasts Kl. lactis is discussed.     

Lac messenger RNA in lac Z gene mutants of Escherichia coli caused by insertion of bacteriophage Mu

The messenger RNA made under conditions of induction of the lac operon has been studied in various lac mutants in which the mutation was caused by integration of bacteriophage Mu into the Z gene. The percentage of RNA hybridizing specifically to lac DNA is proportional to the distance from the beginning of the gene at which a given mutation is located. It thus appears that only lac RNA proximal to the site of insertion is transcribed in these mutants. This may account for the complete polarity of Mu-induced lacZ mutants.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

We have worked out a system to obtain mutations that map in the promoter region of the Escherichia coli galactose operon. In order to easily detect small changes in gal promoter activity, we constructed a plasmid containing an operon fusion in which the lactose operon structural genes were controlled by the galactose operon promoter region. In cells harbouring this plasmid, even modest variations in the expression of the lac genes could be detected on MacConkey lactose indicator plates.Enrichment for mutations that map in the promoter segment of the galactose operon was achieved by mutagenesis in vitro of a small fragment of DNA covering the promoter region. After insertion of the mutagenized gal promoter fragment into the gal-lac fusion plasmid, lac^?1 cells were transformed and screened for an altered Lac⁺ phenotype on indicator plates. Several mutants were isolated due to lesions mapping in the small fragment covering the galactose promoter. In these mutants, the level of β-galactosidase was between 15 and 50% of the wild-type level.The mutant promoters were subsequently reinserted into a plasmid containing the intact galactose operon. Cells harbouring such plasmids, reconstituted with mutant galactose promoters, contained decreased levels of galactokinase that paralleled the decreases in β-galactosidase. The biochemical properties of these mutants are reported in the accompanying paper (Busby et al., 1982).