Isolation of four phenotypic classes of transfer-proficient, pilus-specific-bacteriophage-resistant mutants of an F-like plasmid of Escherichia coli K12

Summary 173 independent mutants of phenotype Tra⁺ MS2^R were isolated from the derepressed F-like R-plasmid, R192-7, of Escherichia coli K12. Various tests of transfer frequency and phage infectivity of 21 of these mutants grouped them into four clearly distinct phenotypic classes. Class (1): mutants of high transfer frequency proficient in MS2 adsorption but to various extents defective in phage penetration, showing resistance or partial sensitivity to both MS2 and M13 equally. Clss (2): mutants of high transfer frequency which fail to adsorb MS2, resistant to both MS2 and M13. Class (3): mutants of reduced transfer frequency, approximately 10^-2 of that of R192-7, resistant to both MS2 and M13. Class (4): mutants of low transfer frequency, approximately 10^-3 to 10^-4 of that of R192-7 and resembling that of the repressed wild type R192, resistant to both MS2 and M13. Pili of mutants of classes (1) and (2) showed structural abnoramlities in electron microscopy.     

Transduction in Streptomyces hygroscopicus mediated by the temperate bacteriophage SH10

Summary The temperate actinophage SH10 mediates generalized transduction in Streptomyces hygroscopicus at low frequency. The efficiency of transduction depends on the average phage input, age of outgrowing spores of the recipient and on the selective marker. The highest EOT was found for the auxotrophic mutants 21 (phe^-) and 5(try^-)(4.1x10^-6 and 2.7x10^-6, respectively). Transduction of the thermosensitive mutant NG14-216 ts 35 was two orders of magnitude lower (2.5x10^-8). The transductant colonies segregated into stable and unstable clones. Stable transductants were never found to be lysogenic for phage SH10.     

Temperature sensitive mutants of Escherichia coli for tRNA synthesis

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3⁺-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA^Tyr was demonstrated. Electrophoretic fractionation of ³²P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

A search for allelic recombination in Chinese hamster cell hybrids

Summary Mutants resistant to 6-thioguanine were selected from CHO cells which were either temperature sensitive or proline requiring. These mutants were stable and had low levels of hypoxanthine guanine phosphoribosyl transferase (HGPRT). Hybrids were selected which were heteroallelic at the hgprt locus and complementation between the mutants used was not observed. Interallelic recombination at this locus would generate hgprt ⁺ cells which could be selected in Littlefield's HAT medium. Selection experiments with hybrids containing three different pairs of mutants yielded no recombinants among populations of 4x10⁶-2x10⁷ cells. After treatment with the recombinagen mitomycin C, 3 putative recombinants were detected amongst 1.4x10⁷ surviving cells from one hybrid. One of these strains was examined and shown to have a normal level of HGPRT and its heterozygosity at this locus was demonstrated by the segregation of colonies resistant to 6-thioguanine. It cannot be excluded that the rare hgprt ⁺ colonies seen arose by mutation rather than by recombination. Mitotic allelic recombination therefore appears to be a much less frequent event in CHO cells than it is in lower eukaryotes. It is possible that mitotic recombination is effectively suppressed in mammalian cells to prevent the expression of deleterious recessive mutants.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis: Evidence for a Plasmid Determinant that Prevents Phage Adsorption

A phage-insensitive strain of Streptococcus lactis, designated ME2, was used as a prototype strain for the study of mechanisms and genetics of phage resistance in the lactic streptococci. Mutants sensitive to a Streptococcus cremoris phage, ϕ18, were isolated at a level of 17% from cultures of ME2 after sequential transfer at 30°C. Phage-sensitive mutants of ME2 were not fully permissive to ϕ18. The efficiency of plating of ϕ18 on the mutants was 5 × 10⁻⁷ as compared with <10⁻⁹ for ϕ18 on ME2. Further characterization of the mutants showed that they efficiently adsorbed ϕ18 at levels of >99.8%, whereas ME2 adsorbed only 20 to 40% of ϕ18. These results suggest that increased phage susceptibility of the mutants may result from the loss of a mechanism that inhibits phage adsorption. Moreover, the high frequency of spontaneous mutation in ME2 indicates the involvement of an unstable genetic determinant in this phage defense mechanism. ME2 was shown to possess 13 plasmids ranging in size from 1.6 to 34 megadaltons. Of 40 mutants examined that had increased efficiencies of plating, all were missing a 30-megadalton plasmid, pME0030. These data suggest that pME0030 codes for a function that prevents phage adsorption. Further phenotypic characterization of the phage-sensitive mutants showed that some mutants were deficient in the ability to ferment lactose (Lac) and hydrolyze milk proteins (Prt). However, the Lac⁺ and Prt⁺ phenotype segregated independently of the phage-sensitivity phenotype. One phage-sensitive adsorption mutant, designated N1, was tested for susceptibility to 14 different phages. N1 showed increased capacity to adsorb 4 and to replicate 2 of these 14 phages, thereby indicating a phage resistance mechanism in ME2 that generalizes to phage interactions other than the specific ϕ18-ME2 phage-host interaction. These data provide evidence for a unique plasmid-linked phage defense mechanism in phage-insensitive strains of lactic streptococci.     

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

Summary E. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cm^r lysogens. Lysates of P1CmSmSu lysogens transduced Cm^rSm^rSu^r at high frequency and they formed plaques with an efficiency of 10^-4 to 10^-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. Cm^rSm^rSu^r transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, Cm^rSm^rSu^r transductants isolated after infection at low multiplicity appeared to carry the Cm^rSm^rSu^r markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA ^- condition. The mechanism of formation of plaque forming P1Cm is discussed.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

Biochemical and genetic characterization of dehydrobiotin resistant mutants of Escherichia coli

Summary Dehydrobiotin (DHB) resistant mutants were isolated from strains of Escherichia coli K-12 and were classified into two groups; dhbA and dhbB.In dhbB mutants the structural genes for enzymes of the biotin pathway are expressed constitutively at a high rate. The dhbB gene is co-transducible with argE at a frequency of about 50% by P1 transduction and maps on the chromosome between arg EC BH and rif. The dhbB ⁺ gene is trans-dominant over the mutant allele indicating that the dhbB ⁺ gene controls the production of a diffusible substance such as a repressor molecule.The dhbA mutants show biotin biosynthetic activity comparable to the wild type and are as sensitive to repression by biotin as the parent strain. The mutants appear to be deficient in DHB transport as suggested by the findings that the ability of the mutants to take up biotin is reduced significantly and that DHB, a competitive inhibitor of biotin uptake, is much less inhibitory to biotin uptake in the mutants than in the wild type.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996     

Directional alignment of two Salmonella phage P22 maps constructed from temperature sensitive or Amber mutants

Summary Two linkage maps of Salmonella phage P22, one constructed with temperature sensitive mutants and the other from amber mutants, were aligned with respect to directional sequence of the genes. Alignment was determined by use of a three-factor cross.The results are in agreement with two independent complementation tests conducted between temperature sensitive and amber mutants which had been employed in constructing the linkage maps.     

Complementation experiments between conditional lethal mutants of bacteriophage ?X174

Summary Complementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage X174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.