Characterization of pyridoxine auxotrophs of Escherichia coli: chromosomal position of linkage group I

The chromosomal location of Group I pyridoxine mutations in Escherichia coli is shown to be adjacent to dsdA,aroC, and purF (old purC) in E. coli B x K-12 hybrids. All mutants previously classified into Group I by nutrition tests and transduction frequency tests are shown to be linked to dsdA.     

Isolation and characterization of pyridoxine auxotrophs of Escherichia coli

Dempsey, Walter B. (University of Florida, Gainesville), and P. F. Pachler. Isolation and characterization of pyridoxine auxotrophs of Escherichia coli. J. Bacteriol. 91:642-645. 1966.-Pyridoxine auxotrophs of Escherichia coli B have been consistently produced by a modified penicillin enrichment method. This modified penicillin technique included a 6-hr growth period in the absence of any pyridoxine followed by a 2-hr treatment with penicillin at 1,000 units per ml. Penicillin was removed from these cultures by membrane filtration and the process was repeated. A minimum of three cycles was found necessary to isolate auxotrophs. Different phenotypes within the group of pyridoxine auxotrophs were distinguished by their responses to feeding with the various forms of pyridoxine, as well as by cross-feeding tests. Two auxotrophs were also differentiated by their frequency of reversion to prototrophy. Cross-feeding tests indicated that seven of the phenotypes fed in a linear sequence. These phenotypes had a very low frequency of reversion. The auxotrophs with a high frequency of reversion cross-fed in a linear sequence and fed the first five of the other seven auxotrophs. One of the auxotrophs isolated was a pyridoxal auxotroph.     

Escherichia coli mutants tht require either pyridoxine or alanine

Some pyridoxineless mutants of genetic group V grow to normal cell yields in glucose-salts medium containing 0.11 mm d- or l-alanine as the sole supplement.     

Apparent pyridoxine transport mutants of Escherichia coli with pyridoxal kinase deficiency

By nitrosoguanidine treatment of a vitamin B-6 auxotroph (KG980) of Escherichia coli, mutants were isolated that require for growth markedly higher concentrations of pyridoxine than the parent strain. One of the mutants, strain HN1, exhibited a severely reduced ability to take up extracellular pyridoxine. Besides, cell-free extracts of HN1 showed an extremely low activity to phosphorylate pyridoxine compared to that of KG980. These findings together with other results suggest that phosphorylation of pyridoxine is essential for the concentration uptake of the vitamin.     

Riboflavin auxotrophs of Escherichia coli]

Escherichia coli riboflavin auxotrophs having a different level of riboflavin requirement were isolated. This auxotrophic mutations are located near cysB93 and trpA62 markers. The complementary effect of Bacillus subtilis riboflavin operon linked with pPR1 hybrid plasmid with rib8-1 and rib1-1 mutations was obtained.     

Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.     

Massive accumulation of phosphatidic acid in conditionally lethal CDP-diglyceride synthetase mutants and cytidine auxotrophs of Escherichia coli

Escherichia coli mutants partially defective in CTP: phosphatidic acid cytidylyltransferase (CDP-diglyceride synthetase) are more resistant to the antibiotic erythromycin than are isogenic wild type strains. When 100 micrograms/ml erythromycin is added to nutrient agar plates, it is possible to obtain a 30-fold enrichment for cds mutants from a mutagen-treated stock, as judged by colony autoradiography (Ganong, B. R., Leonard, J. M., and Raetz, C. R. H. (1980) J. Biol. Chem. 255, 1623-1629). Using this approach, we have isolated 38 new cds mutants, nine of which are unable to grow at a culture pH greater than 8. A typical conditionally lethal mutant like GN80 contains a 3 to 5% phosphatidic acid below pH 7. Above pH 8, GN80 accumulates phosphatidic acid to about 30% of the total membrane lipid, while the de novo syntheses of phosphatidylethanolamine and phosphatidylglycerol are abruptly inhibited by over 10-fold. GN80 loses viability after 60 min at pH 8.5, and the liponucleotide pool of GN80 is about one-seventh that of an isogenic wild type, GN85, under these conditions. The pH optimum of the residual CDP-diglyceride synthetase present in extracts of GN80 is 0.5 pH units lower than normal. Twenty-one of 26 spontaneous pH-resistant revertants of GN80 concomitantly regain parental levels of the enzyme. Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. Independent evidence for this is provided by the observation that cytidine auxotrophs, which are defective in the conversion of UTP to CTP, also accumulate very high levels of phosphatidic acid after 1 h of cytidine starvation.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis

In Escherichia coli, taxis to certain chemoeffectors is mediated through an intrinsic membrane protein called methyl-accepting chemotaxis protein I (MCP I), which is the product of the tsr gene. Mutants were selected that are defective in taxis toward all MCP I-mediated attractants (alpha-aminoisobutyrate, L-alanine, glycine, and L-serine) but are normal to MCP I-mediated repellents and to chemoeffectors mediated by other MCPs. The mutants could be divided into two classes based on their ability to respond to various concentrations of L-serine. Two MCP I-mediated L-serine systems appear to function in the wild type: one of high and one of lower affinity. The mutations responsible for the serine taxis defects map at about 99 min on the E. coli chromosome and are not complemented by episomes carrying mutations in the tsr gene; this suggests that they are defective in tsr function. Low concentrations of L-[14C]serine specifically bound to wild-type membranes with a Km of 5 microM; in contrast, there was greatly decreased binding to vesicles prepared from the new mutants or from the tsr mutant AW518. Binding of labeled serine to wild-type vesicles was inhibited by MCP I-mediated attractants, but not by MCP II-mediated attractants. The data suggest that MCP I may function as the L-serine chemoreceptor in E. coli.     

3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12.

Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine.     

Characterization of a membrane-associated serine protease in Escherichia coli.

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M. Pacaud, J. Bacteriol. 149:6-14, 1982). The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. We termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.     

Thermosensing properties of Escherichia coli tsr mutants defective in serine chemoreception.

Tsr, a chemoreceptor for serine and repellents in Escherichia coli, also functions as a thermoreceptor. The relationship between the chemoreceptor and thermoreceptor functions of Tsr was examined in five tsr mutants with altered serine detection thresholds. The thermosensing abilities of the mutant Tsr proteins were not affected by the alterations in their affinities to serine. In contrast, the ability of serine to inactivate thermoreceptor function was altered in these mutants. The minimal serine concentration required for thermoreceptor inactivation was directly related to the decreased affinity of the mutant Tsr for serine. The amino acid replacements in the mutant receptors were deduced from DNA sequence analyses and occurred at two different locations in the presumed periplasmic domain of Tsr. Two mutations caused histidine or cysteine replacements at arginine 64, whereas three others caused isoleucine or proline replacements at threonine 156.     

Escherichia coli thymine auxotrophs suppressible by carbon dioxide

Two independently-isolated thymine-requiring mutant strains of Escherichia coli were found to possess an unusual phenotype: in the presence of CO2, growth was independent of thymine. The two strains showed different profiles of temperature sensitivity. Glycine, serine and methionine were unable to relieve the thymine-dependence. Both strains were susceptible to trimethoprim under conditions where they were thymine-independent. The results are consistent with the occurrence of partial defects in thymidylate synthase.     

Characterization and generation of Escherichia coli adenylate cyclase deletion mutants.

Escherichia coli delta cya-283 is a 75-bp in-frame deletion overlapping the 5' end of delta cya-854; delta cya-201 is a 41-bp frameshift deletion overlapping the 3' end of delta cya-854. Sequence repeats were found at the boundaries of delta cya-283 and delta cya-201, suggesting a mechanism for deletion formation. Recombinant DNA procedures were used to construct a strain in which the total cya structural gene in the chromosome was replaced by the kanamycin resistance gene.     

Characterization of Escherichia coli adenylate cyclase mutants with modified regulation

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.     

Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes

Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Characterization of Escherichia coli null mutants for glutaredoxin 2.

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.