Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Mutant of Escherichia coli with Anomalous Cell Division and Ability to Decrease Episomally and Chromosomally Mediated Resistance to Ampicillin and Several Other Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain, we isolated two smooth mutants which were both sensitive to ampicillin and carried defects in the cell envelope. One of the strains (with the envA gene) is hindered in its completion of septa and forms chains of cells. The envA gene has been mapped to a position between leu and proB, at 2 to 4 min. The envA gene decreased the resistance mediated by both episomal and chromosomal genes for resistance to several antibiotics. During growth the envA mutant was characterized by abnormal ratios between viable count or cell count and optical density. The ratio between viable count and optical density was affected during shift-up and shift-down experiments. When compared to the parent strain, the envA mutant was found to be more resistant to ultraviolet irradiation on plates. Prestarvation for tryptophan had a protective effect against irradiation both on the parent strain and the envA mutant.     

Escherichia coli mutants deficient in exonuclease VII.

Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.     

Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

Isolation and characterization of a mutant of Salmonella typhimurium deficient in a major deoxyribonucleic acid polymerase activity

A mutant of Salmonella typhimurium strain LT2 that is deficient in a major deoxyribonucleic acid (DNA) polymerase activity has been isolated and characterized. This mutant resembles the pol mutants of E. coli in that it has low DNA polymerase activity and it is sensitive to methyl methane sulfonate as well as ultraviolet irradiation. Revertants selected for methyl methane sulfonate resistance are no longer sensitive to ultraviolet irradiation and contain normal DNA polymerase levels. No direct role in replication can be ascribed to this polymerase activity since cells grow well in its absence. In addition, the LT2 plasmid has been shown to exist in the mutant strain.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Bacterial Cell Division Regulation: Characterization of the dnaH Locus of Escherichia coli

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli. A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH(+) is carried on the F'(13) episome and is dominant over the dnaH(-) mutation.     

Molecular cloning and characterization of the genes (pbpA and rodA) responsible for the rod shape of Escherichia coli K-12: analysis of gene expression with transposon Tn5 mutagenesis and protein synthesis directed by constructed plasmids.

Two cell shape-determining genes of Escherichia coli K-12, pbpA, the structural gene for penicillin-binding protein 2, and rodA, whose protein is unknown, were subcloned into plasmid vectors from the transducing phage lambda MAd lip24, which carries the lip-leuS region of the E. coli chromosome. Plasmids with restriction enzyme-created deletions or transposon Tn5 insertions were isolated, and studies of genetic complementation of these plasmids with chromosomal mutations were carried out. Thus, a physical and genetic map of the rodA-pbpA region was established. The genes rodA and pbpA lie side by side within a 4.4-kilobase-pair region. The size of the rodA gene has been shown to be between 0.86 and 1.6 kilobase pairs; such DNA would encode a protein with a molecular weight between 32,000 and 59,000. Since Tn5 mutagenesis of the rodA gene did not affect the expression of the pbpA gene and vice versa, the genes rodA and pbpA seem to have independent promoters. Analysis of the proteins synthesized from the constructed plasmids in maxicells revealed that the plasmid carrying the pbpA gene encoded penicillin-binding protein 2 and amplification of the protein occurred. The product of the rodA gene was not identified.     

Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation.

A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.     

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Autolysins and shape change in rodA mutants of Bacillus subtilis.

The biochemical phenotype of rodA mutants was not affected by the simultaneous presence in double mutants of the lyt gene which makes them 90 to 95% deficient in autolysin action. The only morphological effect of this deficiency on the expression of the rod gene was that both the rod and the coccal forms of the mutant failed to separate and grew as long chains of cells. Inhibition of protein synthesis stopped the increase in peptidoglycan that occurred when the growth temperature for the mutants was raised to 45 degrees C. These observations support the idea that a derepression of peptidoglycan synthesis occurs at this temperature. The increased amount of cellular peptidoglycan at the higher growth temperature is not likely to be the result of the concomitant switching off of autolytic enzyme action.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.     

Control of cell shape and elongation by the rodA gene in Bacillus subtilis

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli , the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA , encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible P_spac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.     

Escherichia coli mutants deficient in guanine-xanthine phosphoribosyltransferase.

We studied the purine phosphoribosyltransferases (PRTases) of Escherichia coli and were able to isolate a mutant that is defective in its ability to convert guanine and xanthine to their respective ribonucleotides. The affected gene (gpt) lies between metD and proA and is 78.6% co-transducible with proA. Both this point mutant and a strain with a pro-lac deletion contain less than 2% of wild-type xanthine PRTase activity, yet still contain about 30% of wild-type guanine PRTase activity. Thus, the gpt gene is only one of at least two genes responsible for guanine PRTase activity in E. coli.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

Biochemistry and genetics of Klebsiella pneumoniae mutant strains unable to fix N2.

Selected mutant strains of Klebsiella pneumoniae that are unable to fix nitrogen have been characterized according to nitrogenase component activity as well as antigenic cross-reacting material. The lesions in these strains have been mapped by transduction, and the results indicate that there are at least five genes specifically responsible for nitrogen fixation in vivo. Besides genes that specify the structure of the two nitrogenase components, there is a gene for a factor that is required for component I activity and a gene that codes for a factor possibly involved in electron transport to component II. A mutation in another site does not allow the organism to produce either of the nitrogenase components. All of these genes are co-transducible with the gene that specifics the structure of histidinol dehydrogenase.     

Saccharomyces Mutants with Invertase Formation Resistant to Repression by Hexoses

Production of invertase by many strains of yeast is repressed in the presence of hexoses. This phenomenon interferes with studies on the secretion of invertase and with the preparation of large quantities of the enzyme for examination of its chemical and physical characteristics. Saccharomyces strain 303-67, a diploid carrying the single gene SUC-2 for (hexose repressible) invertase production, was subjected to ultraviolet irradiation. No single-step mutations to high level resistance were detected. By a two-step irradiation process mutants were obtained with differing degrees of resistance. The biochemical and genetic characteristics of these mutants are summarized with particular emphasis on FH4C (the most resistant). Although the steady state level of cyclic 3', 5'-adenosine monophosphate (cyclic AMP) was usually slightly higher in cells grown in low- rather than in high-glucose media, the level of cyclic AMP was not correlated with the sensitivity of invertase synthesis to glucose repression. In mutant FH4C, 1 to 2% of the total cell protein is present as invertase; synthesis of alpha-glucosidase is also resistant to repression by hexoses. This mutant does not sporulate and is probably a haploid of a-mating type with low frequency of conjugation and poor viability of conjugants. Mutants 1016 and 1710 are substantially resistant to hexose repression and still sporulate well. They may be useful for genetic analysis of hexose resistance.     

Similarity in properties and mapping of three Rec mutants of Haemophilus influenzae.

Three Rec- mutants of Haemophilus influenzae have been studied with respect to their transformability, ultraviolet and mitomycin C sensitivities, spontaneous and ultraviolet-induced deoxyribonucleic acid breakdown, inducibility of lysogens, and the linkage of the three mutations to a streptomycin resistance marker. The data indicate that the three mutations cause the same phenotypic changes, and that they are all on the same gene. Transformability of the mutants is different when two different media are used for competence development, although transformability with the two competence methods is not different in a Rec- strain that is mutant at another gene.