Mary L. Droffner

Prolonged environmental stress via a two step process selects mutants of Escherichia, Salmonella and Pseudomonas that grow at 54°C

A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48°C with nalidixic acid selected mutants (T48) able to grow at 48°C. A prolonged incubation at 54°C of the T48 mutants selected mutants (T54) able to grow at 54°C. These mutants were susceptible to the same bacteriophages as the original mesophilic strains. Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48°C or 54°C or on a minimal agar with glucose at 37°C. The T48 alleles mapped in the gyrA region of E. coli or S. typhimurium chromosome. In S. typhimurium the T54 alleles, which permit growth at 54°C, were shown by cotransductional analysis to be linked to gyrA.

Procedure for isolation of Escherichia, Salmonella, and Pseudomonas mutants capable of growth at the refractory temperature of 54°C

Abstract When fully grown cultures of the genetically well characterized genera Escherichia, Salmonella and Psuedomonas were placed at 48°C for at least 4 days a few (frequency ≈ 10 −8 ) mutants able to grow continuously at 48°C were isolated. None were ever obtained immediately, implying mutation by heat stress but not selection of preexisting mutants. These mutants (thermotolerant or T48 mutants) capable of growth at 48°C had a generation time of ≈35 min at 48°C. Incubation of the T48 mutants at 54°C for at least 4 days yielded a few (frequency ≈ 10 −8 ) mutants (mesothermophilic or T54 mutants) able to grow continuosly with division every 30–40 min at 54°C. Thus, by the stepwise procedure of prolonged thermal stress, mutants which grow at 54°C were obtained from these three mesophilic genera. Isolation of mesothermophilic Salmonella (T54) mutants was also achieved directly from mesophilic parents at a frequency of −10 when the mesophilic strain was incubated for at least 10 days at 54°C suggesting two stepwise mutational events can take place during a very prolonged exposure to thermal stress. T48 and T54 mutants were found to require cellobiose as an energy source for growth on minimal agar and exhibition of parental auxotrophic genotypes in the thermal environment.

Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.

Role of nalidixic acid in isolation of Salmonella typhimurium strains capable of growth at 48 degrees C.

Salmonella typhimurium thermotolerant mutants dependent on the presence of nalidixic acid for growth at 48°C were isolated and designated nalidixic acid-dependent, thermotolerant mutants,naldttl. Genetic mapping revealed thatnaldttl alleles map within thegyrA gene. WhenS. typhimurium strain Q was plated in the dark on nutrient agar containing nalidixic acid (20 μg/ml) as a photosensitizer and briefly exposed to white light or near VU light prior to incubation at 42°C, nalidixic acid-resistant mutants arose in about 16 h at frequencies of 5×10−8 for white light and 1×10−6 for near UV light. About 10% of these nalidixic acid-resistant mutants derived from photodynamic mutagenesis exhibited the thermotolerant characteristic.

Anaerobic expression of the gyrA gene

Abstract E. coli C600Δ lac was infected with a Mu- lac defective phage. Lysogens resistant to ampicillin (25 μg/ml) and nalidixic acid (20 μg/ml) were unable to grow anaerobically and insensitive to nalidixic acid (150 μg/ml), indicating formation of gyrA ::Mu- lac fusions. Mapping of the Mu- lac by P1 phage cotransduction with two different gyrA linked Tn 10 transposon loci suggests that the insertion is located in the gyrA gene. These fusion derivatives form small uncolored colonies on lactose MacConkey agar plates aerobically. When these plates are placed in an anaerobic jar for about 4 h, the colonies turn red indicating that the inserted lac gene is expressed only under anaerobic environment. Thus the gyrA promoter is activated under the anaerobic environment and loss of gyrA activity prevents anaerobic cell growth.

High Frequency Transduction by Phage Hybrids Between Coliphage ø80 and Salmonella Phage P22

phi 80immP22dis, a hybrid between phi 80 and P22, carries all the late genes of phi 80 and most of the P22 early region including the immC and immI bipartite immunity loci. The presence of the immI region allows this hybrid to grow on lysogens of phi 80immP22 hybrids which have the immC locus, but not the immI locus. In addition to these P22 immunity regions, phi 80immP22dis contains the P22 att marker so that the prophage can be inserted into the chromosomal P22 attachment site adjacent to the proA-proB region of the host. Unlike its phi 80 parent which performs specialized transduction of the trp region, phi 80immP22dis transduces markers located adjacent to its attachment site to Escherichia coli K12 recipients at high frequencies (0.3% for argF and 0.18% for proA). Induction of phi 80immP22dis lysogens yields new hybrid phage clones which have incorporated E. coli K12 chromosomal segments in place of the P22 immI to att segment. Having lost the immI region, the new hybrids no longer grow in phi 80immP22 lysogens. These new hybrids, termed phi 80immP22dis-, possess specialized transducing properties, transferring the argF and proA markers at higher frequencies (21% for argF and 12% for proA) than previously obtained with the phi 80immP22dis phage.

Analysis of Proteins Induced by the Salmonella typhimurium Phage P221, a Hybrid between Serologically and Morphologically Unrelated Phages P22 and Fels 1

Summary The Salmonella phage P221 is a hybrid between the morphologically and serologically unrelated Salmonella phages P22 and Fels 1. P221 carries about 30% of P22 DNA containing a number of the early genes. Head and tail proteins are morphologically and serologically indistinguishable from those of Fels 1. The early proteins synthesized in Salmonella typhimurium strain Q1 after P221 infection were pulse-labelled with 35S and analysed by gel electrophoresis and autoradiography. These P221 early proteins were compared with early P22 proteins in an effort to detect proteins specified by the homologous region between P22 and P221. This analysis showed that nine P221 protein bands correspond to P22 protein bands. Seven of these protein bands appeared early, about 6 min after infection, and two additional protein bands appeared about 18 min after infection. The origin of these nine protein bands was also determined by protein synthesis patterns of P22 amber mutants. A large number of previously isolated P22 amber mutants were subdivided as to their location within the homologous or non-homologous region by complementation and marker rescue experiments. Four P22 amber mutants located within the homologous region were chosen for analysis of their early proteins. When the non-permissive host Q1 was infected with a P22 amber mutant in gene 24 (the gene which initiates the transcription of the P22 phage genome) no phage protein bands were observed. Similarly, when two P22 amber mutants located in gene 12 and gene 23 infected the non-permissive host Q1 no phage protein bands were detected. Mutants of the gene for endolysin showed that one specific protein band was lacking in the non-permissive host. When the amber suppressor host Qsu + was infected with any of the above amber mutants all protein bands were found. By the use of an amber mutant in gene 3 of the P22 late gene region located outside the P22–P221 homologous region, we showed that mutations in the non-homologous region did not affect the synthesis of these nine proteins. The genetic origin of proteins found in mature P221 particles was determined by subjecting proteins of purified P221, P22 and Fels 1 phage particles to analysis by SDS-gel electrophoresis. It was concluded that P221 and Fels 1 share the same head, tail and internal proteins in mature phage particles, whereas none of the protein components of these mature phage particles is the same as those of P22 particles.

The leucine operon carrying theleu-500 promoter mutation is expressed under anaerobic conditions

TheSalmonella typhimurium leu-500 auxotrophic mutant grew when cultivated in minimal medium anaerobically, but not aerobically. This mutant carries an AT → CG mutation in the Pribnow box of the promoter region of the leucine operon and was found to be suppressible by anaerobic conditions. Analysis of the anaerobic gases revealed that hydrogen in the anaerobic gas mixture (85% N2, 10% CO2, 5% H2) is essential for the suppression of theleu-500 mutation. Whenleu-500 mutant cells were incubated in the presence of the hydrogen gas, the synthetic rates for the first and last gene products of theleu-500 operon were similar to those of the wild-type cells. It was concluded that the entire leucine operon was efficiently expressed inleu-500 when the cells were grown under the hydrogen gas-containing anaerobic environment. Thus, theleu-500 promoter mutant is a model system for regulation of gene expression by a specific atmospheric environment, i.e., hydrogen gas found in the anaerobic environment.