Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

Transfer-deficient cointegrates of Flac and lambda prophage

Summary Twelve transfer-deficient mutants of the plasmid Flac were obtained by insertion of prophage lambda into secondary attachment sites within the transfer region. Insertions into eight different tra genes were identified. These mutations were strongly polar on expression of tra genes previously mapped downstream, and thus confirmed that the genes traA through traD form a single operon. However, some continued expression of traI suggested that this was transcribed in part from a promoter located between traD and tral, and in part from the transfer operon promoter. One insertion early in the transfer operon produced a plasmid-specific tra mutation not complemented by R100-1 or R1-19: this insertion was into a new gene (traY), located before traA as the first member of the transfer operon. Partial tra deletion mutants were obtained as 42° C — survivors from several of the Flac tra:: ED4 plasmids, and their properties are described.     

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli

Summary Strains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize -galactosidase constitutively in anaerobiosis; (ii) mutants in which -galactosidase synthesis is no longer repressed by oxygen.Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of -galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Early and late transfer of F genes by Hfr donors of E. coli K-12

Summary The results of short interrupted matings between an Hfr donor and a recipient strain carrying a temperature-sensitive replication mutant (frp ^–) of Flac demonstrate that the Hfr strain transfers this frp gene of F early in conjugation. This frp gene was also shown to function in the maintenance of mutant F plasmids which appear to be generated from the DNA transferred early in conjugation by Hfr donors. In the course of these experiments, it was further demonstrated that certain Hfr strains which had been described as transferring the tra genes early in fact transfer that region of F late in conjugation.     

Synthetic multifunctional proteins

Summary Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and -galactosidase. The mutants were isolated as TonB^- Lac⁺ derivatives of a phenotypically Lac^- TrpR^- strain carrying a lac I ⁺-Z⁺ fusion on a 80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA^- and 3 Trp^- strains produce triple chimeras. The chimeras from the two TrpA^- strains were further examined. They consist of tryptophan synthetase -subunit, lac repressor and -galactosidase. In crude extracts of these strains the tryptophan synthetase -subunit part can be identified by its ability to aggregate with the -subunit since some of the -subunit activity can be precipitated with antiserum against -galactosidase. Furthermore -galactosidase precipitates with antiserum against tryptophan synthetase -subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The -galactosidase part is as unaffected as in the original lac repressor--galactosidase chimera. The molecular weigths of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.     

Early transfer of genes determining transfer functions by some Hfr strains in Escherichia coli K12

Summary Sixty-eight Hfr strains were examined for their ability to transfer early in conjugation the transfer genes carried by the integrated sex factor. This was measured by mating these strains with F^- phenocopied recipient cultures of strains carrying transfer-deficient Flac ⁺ factors, and then measuring the ability of the recipient strains to transfer lac ⁺ to a further recipient strain. Most Hfr strains did not complement the missing transfer functions, though in some strains complementation was observed. It is concluded that on the sex factors of different Hfr strains either the site at which integration occurs or the origin of transfer must vary.     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.     

Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coli

Summary The expression of a newly transferred lacZ ⁺ gene in lacZ recipients carrying various mutations in the recA and recB genes was studied by measuring the rates of induced synthesis of -galactosidase in zygotes formed after mating with either F or Hfr donors. The ability to synthesize -galactosidase decreases with time in both recA and recB zygotes when the lacZ ⁺ gene is transferred from an Hfr donor, but not when the lacZ gene is transferred from an F donor. There is no such inactivation of the newly transferred lacZ ⁺ gene in Rec⁺ zygotes. We conclude that the functioning of the transferred DNA is progressively inactivated in rec recipients unless the DNA is contained in an episome such as F.     

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli

Summary The frequency of lac ^- mutations induced in an F lacI ^S plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, pro lac recipients, is 2-3 fold higher than that typical of non-mating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication.     

Detection of developmentally regulated genes of the myxobacterium Stigmatella aurantiaca with the transposon Tn5lacZ

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. Insertional mutations were introduced at random sites into the Stigmatella aurantiaca genome with the promotor probe Tn5lacZ derived from Tn5lac by deleting non-essential sequences. 638 transconjugants were obtained with a frequency of 1×10^-7. In 260 of the transconjugants isolated the -glactosidase gene of Tn5lacZ is fused to vegetative promotors of Stigmatella aurantiaca. In 65 of the strains -galactosidase is induced by starvation; in 14 of the transconjugants -galactosidase activity is observed after chemical induction of sporulation by 3-methyl-indole. Thirtytwo of the mutants are affected in fruiting body formation and morphology.     

Isolation and complementation of mutants of Streptomyces coelicolor “Müller” DSM3030 deficient in lysozyme production

Summary Streptomyces coelicolor Müller is known to excrete the lysozyme N-acetylmuramidase. Culture filtrates of this strain form a characteristic halo on agar plates containing freeze-dried Micrococcus luteus cells (lysoplate technique). The halo consists of a clear inner zone and a turbid outer ring. Simulation experiments showed that the turbid outer ring is most probably produced by lysozyme whereas the clear inner zone can be considered to be due to an additional protease action. Using the lysoplate technique UV- and NTG-mutagenized strains of S. coelicolor Müller were screened for mutants defective in lysozyme production. Two mutants, SC11 and SC12, were identified. The mutant SC11 was selected for complementation studies. First, a transformation system was established. The use of a soft-agar overlay method was necessary to yield high regeneration rates of SC11 protoplasts. The plasmid vector pEB15 could be transferred into this mutant strain with an efficiency of 10⁵ transformants/g DNA. The high efficiency allowed shot-gun cloning experiments. Genomic DNA of S. coelicolor Müller digested with Sau3A and inserted into pEB15 was introduced into the mutant SC11. A complemented mutant was identified. A 2.9 kilobase pair (kb) DNA fragment was found which restored the lysozyme production of both mutants, SC11 and SC12. According to the diameter of the produced halos the complemented mutant SC11 was suggested to produce more lysozyme than the wildtype strain.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

Catabolite repression of the lac operon: effect of operator-constitutive mutations

Summary We have constructed and tested three lac diploid strains in an attempt to show whether operator-constitutive mutations relieve catabolite repression of the lac operon. Each of these carries a different operator mutation on the chromosome, and all three have the genotype I⁺P⁺O^cZ⁺Y^-polar/Flac I⁺P⁺O⁺Z^delY⁺A⁺. When these strains were grown in medium containing glucose plus gluconate, synthesis of -galactosidase (directed by a gene cis to a mutant operator) and of thiogalactoside transacetylase (directed by a gene cis to an intact operator) suffered equal catabolite repression. We conclude that the operator-constitutive mutations have no effect on catabolite repression. Since it has been shown in analogous experiments that all promoter mutations tested do alleviate catabolite repression, these results are consistent with the view that the operator and promoter are functionally distinct.     

Conjugal transfer of E. coli F''lac from Erwinia chrysanthemi to Pseudomonas syringae pv. glycinea and the apparent stable incorporation of the plasmid into the pv. glycinea chromosome

Summary The E. coli Flac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv. glycinea recipient. Transfer occurred at a frequency of approximately 10^-5/donor. Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the Flac plasmid in detectable frequency to other pv. glycinea or E. coli recipients. The plasmid DNA was shown to be integrated into the pv. glycinea chromosome (Fig. 1).     

Chromosome mobilization and integration of F-factors in the chromosome of RecA strains of E. coli under the influence of bacteriophage Mu-1

Summary Transfer of chromosome promoted by an F⁺ or an F needs a recombinational event between episome and chromosome and is therefore very low in RecA strains (Wilkins, 1969).When F⁺ or F RecA cells are mated with F^- cells and simultaneously infected with bacteriophage Mu-1 the transfer of chromosome is greatly stimulated and seems to take place on any site of the chromosome even when homology is present between the F and the chromosome.A spot-test based on Mu-1 promoted chromosome-mobilization was developed to search for mutants of Mu-1 that had lost the ability to promote chromosome-mobilization. Two conditional lethal amber mutants falling in different complementation groups were found to have lost this property.The integration of an Ftslac ⁺ in the chromosome of a RecA strain is also strongly stimulated by Mu-1 and not site-specific. The resulting Hfr's are very stable and of the clockwise and counterclockwise types. Some of the integrated F-primes are sterile and not able to transfer chromosome or do not form F-pilli.Also the Mu-1 stimulated integration of an Fts(nadA-chlA)⁺ in the chromosome of a RecA strain which has a deletion on the chromosome from nadA-chlA was studied. It was found that when the F is integrated under the influence of Mu-1 also episomal genes carried by that F can be inactivated.     

Genetic analysis of mutants producing defective beta-galactosidase which can be activated by specific antibodie

Summary Eleven lac ^- mutants have been isolated producing -galactosidase mutant proteins, which can be activated to enzyme activity upon addition of anti -galactosidase antibodies (lac _aba ^- -mutants). The mutants have been mapped using P1 transduction and deletion mapping. Seven of them fall into one group (1), two others into another group (2). Two mutants map at sites different from the two groups (Fig. 2). Lac _aba ^- -mutant sites farthest apart correspond to a distance of about 3/4 of the z gene.     

Host controlled restriction and modification of bacteriophage Mu and Mu-promoted chromosome mobilization in Citrobacter freundii

Summary Bacteriophage Mu grown on Escherichia coli K12 (Mu. K) is restricted by wild type Citrobacter freundii. In two C. freundii mutants, where the restriction of foreign F factors is absent (de Graaff and Stouthamer, 1971), the restriction for Mu. K, although at a lower level, still exists. Consequently two host specificity systems exist in C. freundii, one affecting mainly the acceptance of foreign plasmidal and chromosomal DNA and one affecting foreign DNA of bacteriophage Mu. Mu is able to lysogenize C. freundii and to induce mutations at random in its chromosome. Furthermore Mu is able to promote the mobilization of the C. freundii chromosome in strains carrying F factors. Mu promoted integration of F ts 114 lac ⁺ into the C. freundii chromosome was observed, resulting in the formation of stable Hfr strains. In this way it is possible to devise a method for chromosome transfer in other genera than E. coli to which plasmids of E. coli can be transferred, but in which no chromosome mobilization is possible because of poor DNA homology between the foreign plasmid and the host chromosome.     

Two classes of Flac mutants insensitive to transfer inhibition by an F-like R factor

Summary Mutants of Flac episomes whose transfer is no longer inhibited by the fi ⁺ R factor R100 are shown to be of at least two types. One is recessive in transient heterozygotes containing Fhis and R100 in addition to the Flac mutant. The other is dominant. The occurrence of recessive mutants suggests that inhibition of F transfer by R100 requires an F-specified gene product in addition to that produced by the R factor. Synthesis of the F product or its interaction with the R100 product to give the true inhibitor seems to be a slow process. Since the inhibitor and mutations of the transfer gene traJ both affect a plasmid-specific transfer product, F-pilus formation, and surface exclusion, we propose that the inhibitor prevents the synthesis or function of the traJ product.Supported by a George Murray Scholarship from the University of Adelaide, Australia.     

R factor-mediated tetracycline resistance in Escherichia coli K12 dominance of some tetracycline sensitive mutants and relief of dominance by deletion

Summary Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed. They carried the wild-type Tet^r genes in the chromosome and single site Tet^s mutations on plasmids. Some heterozygotes could not express tetracycline resistance fully after induction. The mutant tet allele was thus partially dominant.When heterozygotes carrying the dominant tet mutant were plated on agar containing 50 g/ml tetracycline, mutants which grew normally occurred at a frequency of 1–4×10^-4. Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra⁺ or Tra^- deletion mutants of the plasmid. The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture.     

Feeding in the rotifer Brachionus calyciflorus

Summary The laboratory feeding behavior of Brachionus calyciflorus varies depending upon the type of food cell available in suspension. When feeding on the yeast Rhodotorula glutinis, rotifers show a continuous increase in ingestion with increased cell density between 0.01 and 1000 g dry weight ml^-1. Effective clearance rates drop from ca. 50 l animal^-1 h^-1 to less than 0.5 l animal^-1 h^-1 over this food density range. When feeding on Englena gracilis, B. calyciflorus ingestion rates are constant between 1.0 and 100 g ml^-1 of available food, averaging close to 25 ng animal^-1 h^-1. The decrease in clearance rate is more striking than with R. glutinis, dropping from 45 l animal^-1 h^-1 at 0.1 g ml^-1 to 0.13 l animal^-1 h^-1 at 100 g ml^-1. Differences between the patterns obtained with the two food types indicate fundamental dissimilarities in the feeding behavior of this rotifer species when presented with these different foods.