Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase]

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.     

Positive selection of mutants with cell envelope defects of a Salmonells typhimurium strain hypersensitive to the products of genes hisF and hisH.

Strain SB564 and its derivative DA78 are hypersensitive to the inhibitory action of the proteins coded for by genes hisF and hisH on cell division. Transduction of hisO1243, a regulatory mutation that elicits a very high level of expression of the histidine operon, into these strains resulted in the production of long filamentous cells carrying large "balloons" and in growth failure. Forty-one hisO1242 derivatives that escaped inhibition were isolated. These strains showed a large variety of alterations, many of which were related to the cell envelope. The more-frequent alterations included: changes in cell shape, increased sensitivity to one or more of several drugs (deoxycholate, cycloserine, penicillin, novobiocin, acridine orange), increased autolytic activity in alkaline buffer, anomalous fermentation of maltose on eosin--methylene blue plates, and temperature-conditional cell division. The alterations are produced, in some of the strains, by pleiotropic mutations in gene envB (Antón, Mol, Gen. Genet. 160:277--286, 1978) or envD (Antón and Orce, Mol. Gen. Genet. 144:97--105, 1976). Strains affected in divC, divD, and rodA loci have also been identified. Genetic analysis has shown that several strains carry more than one envelope mutation. It is assumed that envelope mutations are positively selected because they somehow alleviate the particularly severe inhibition of cell division caused, in strains SB564 and DA78, by the excessive synthesis of hisF and hisH gene products.     

Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation.

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.     

A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.     

Construction of Escherichia coli K12 phr deletion and insertion mutants by gene replacement

We replaced an Escherichia coli phr gene by a 1.4-kb fragment of DNA coding for resistance to chloramphenicol. Characterization of 2 deletions (phr-19 and phr-36) and 1 insertion (phr-34) in the phr gene revealed no photoreactivation. Photoreactivation-deficient strains of either recA56 or lexA1(ind-) were more sensitive to UV radiation in the dark than phr-proficient counterparts. The presence of the phr defect in uvrA6 strains increased by 1.5-2-fold his-4(Ochre) to His+ mutation induced by ultraviolet light compared to uvrA6 phr+ strains, although there was no difference in UV sensitivity between uvrA6 phr+ and uvrA6 phr- strains. 30-35% of the His+ mutations thus induced were suppressor mutations in uvrA6 phr+ and 49-55% in uvrA6 phr- strains. The UV mutagenesis results are consistent with the previous observations that suppressor mutations targeted by a thymine-cytosine pyrimidine dimer are reduced in the dark in cells with amplified DNA photolyase.     

Procedure for Identifying Nonsense Mutations

A method has been devised for the rapid identification of nonsense mutations (UAG, UAA, UGA codons) in Salmonella. The mutations to be tested are reverted, and the revertants are replica-printed onto lactose plates spread with lawns of tester strains. These tester strains contain F' lac episomes with nonsense mutations in the episomal Z gene. The revertants are infected with the episome from the tester strain lawn. Because S. typhimurium is unable to ferment lactose, only those revertants which have nonsense suppressors are able to grow on lactose. If colonies appear on the lactose plate, it may be concluded that the original strain carries a nonsense mutation, since nonsense suppressors suppress the mutant phenotype.     

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.     

Structure and function in a Bacillus subtilis sporulation-specific sigma factor: molecular nature of mutations in spoIIAC

The spoIIAC gene was cloned from chromosomal DNA of seven spoIIAC mutants of Bacillus subtilis, and the complete sequence of the gene was determined for each mutant. Three of the mutants proved to have chain-terminating mutations (one of which, previously shown to be suppressible by sup-3, was identified as amber); these led in every case to complete failure either to manufacture spores or to synthesize two enzymes normally associated with stage II of sporulation. The four remaining mutations were missense, and these corresponded to a phenotype in which a few spores are formed and about half the wild-type quantities of the two enzymes are made. Of the four missense mutations, two were near the promoter-distal end of the gene, in a region believed to correspond to the DNA-binding domain of the sigma factor that spoIIAC encodes. The remaining two mutations were in the region of the gene that is thought to correspond to the domain of the protein that interacts with core RNA polymerase.     

An encoded N-terminal extension results in low levels of heterologous protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form" (using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium.     

Genetic locus, distant from ptsM, affecting enzyme IIA/IIB function in Escherichia coli K-12.

Most strains of Escherichia coli K-12 are unable to use the enzyme IIA/IIB (enzyme IIMan) complex of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in anaerobic growth and therefore cannot utilize glucosamine anaerobically. Introduction into these strains of a ptsG mutation, which eliminates activity of the enzyme IIIGlc/IIB' complex of the PTS, resulted in inability to grow anaerobically on glucose and mannose. Derivative strains able to grow anaerobically on glucosamine had mutations at a locus close to man, the gene coding for phosphomannose isomerase, and had higher enzyme IIA/IIB activities during anaerobic growth than did the parental strain. These results establish a locus affecting function of enzyme IIA/IIB that maps distant from ptsM, the probable structural gene for enzyme IIB.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Regulatory mutations of the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk+ strains. These revertants showed altered responses to normal inducers of alkA+, alkB+, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 X 10(-8). Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity, and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.     

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.     

Phenylalanine Biosynthesis in Escherichia coli K-12: Mutants Derepressed for Chorismate Mutase P-Prephenate Dehydratase

Mutants were isolated which are derepressed for the synthesis of chorismate mutase P-prephenate dehydratase. No other enzymes involved in the synthesis of phenylalanine are derepressed in these strains. These mutants are able to grow in concentrations of o- and p-fluorophenylalanine that inhibit the growth of AB3259, the strain from which they were derived. They also excrete phenylalanine. Genetic analysis shows that the mutations causing this derepression are closely linked to the structural gene for this enzyme (cotransduction frequency of 95% or more with pheA). The gene in which they occur has been designated pheO since this gene has all of the properties predicted for an operator gene controlling the pheA structural gene. Finally, the pheO mutant alleles have been shown to be dominant in diploids.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.     

Breeding of yeast strains able to grow at 42 degrees C

Saccharomyces cerevisiae strain 2-39/10A is able to ferment alcohol at 42 degrees C. The ability of various yeast strains, including 2-39/10A, to grow at high temperatures was compared. The strain 2-39/10A was able to grow at 42 degrees C and the high temperature growth was found to be governed by more than one gene. The yeast strains that can grow at 42 degrees C were bred by crossing the haploid strains, which are inherently unable to grow at high temperatures.