A mutant of Escherichia coli which accumulates large amounts of coproporphyrin

A mutant of Escherichia coli which accumulates a large amount of coproporphyrin, presumably because of a block in heme biosynthesis, has been isolated after nitrosogunidine mutagenesis. On rich media, the mutant forms colonies which give bright orange fluorescence when illuminated with ultraviolet light. The mutant appears to be similar to a Salmonella typhimurium mutant, deficient in uroporphyrinogen III cosynthase, described by Sasarman and Desrochers ((1976) J. Bacteriol. 128, 717–721). A striking property of the mutant is that coproporphyrin is retained within the cells in rich media but is almost totally excreted out of cells in minimal glucose medium.     

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.     

A yeast mutant which accumulates precursor tRNAs.

It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969). We have examined ts136 to determine whether it is defective in tRNA biosynthesis. At the restrictive temperature, the mutant accumulates twelve new species of RNA. These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs. Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs. Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E. coli plasmid containing a yeast tRNA gene. All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA. Three of these species were tested and were found to hybridize to tRNA genes. Ribosomal RNA synthesis is also defective in ts136. It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport.     

Construction of an Escherichia coli strain which excretes abnormally large amounts of adenosine 3',5'-cyclic monophosphate.

It has previously been shown that an Escherichia coli CRP- strain 5333 accumulates abnormally large amounts of adenosine 3',5'-cyclic monophosphate (cAMP). Using P1 transduction, the CRP- character was transferred to E. coli Crookes strain which is deficient for cAMP phophodiesterase (CPD-). The resulting strain HY22 (CRP-, CPD-) accumulates greater amounts of cAMP both intracellularly and extracellularly than does 5333. In glucose minimal medium, an HY22 cell accumulates 100 times more cAMP intracellularly and excretes cAMP 150 times faster than does a wild-type E. coli cell.     

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.     

A spectinomycin dependent mutant of Escherichia coli.

Summary A mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence. The mutant, termed RD, needs spectinomycin to grow at temperatures of 37° or below; it is unable to grow at 42° in either the presence or absence of spectinomycin. Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10^-6. Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1. Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked.Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E. coli chromosome and was represented by the mutation in S5. The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.     

Escherichia coli mutant containing a large deletion from relA to argA.

A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.     

Characterization of a cytochrome oxidase-deficient yeast mutant which accumulates porphyrins

A cytochrome oxidase-deficient mutant (pop4) of Saccharomycescerevisiae which accumulates porphyrins has been characterized. The pop4 mutation is recessive and affects a single nuclear gene. The bulk of the accumulated porphyrins consists of uroporphyrin and its partial decar?ylation products, suggesting that the mutation causes a block at the level of uroporphyrinogen decar?ylase. However, pop4 is not allelic to hem6 or pop3, putative decar?ylase structural-gene mutants. These results suggest that there may be a uroporphyrinogen decar?ylase isoenzyme specifically involved in heme a production.     

Escherichia coli mutant which restricts T7 bacteriophage has an altered RNA polymerase.

We have previously described an Escherichia coli K-12 mutant, Y49, which restricts the growth of bacteriophage T7 and causes the accumulation of short DNA molecules and head-related particles during infection. We now show that the basis for these effects is the inability of the T7 gene 2 product to inactivate the Y49 RNA polymerase during infection, similar to what has been shown by DeWyngaert and Hinkle (J. Biol. Chem. 254:11247--11253, 1979) for the BR3 and tsnB strains of E. coli.     

Septum formation-defective mutant of Escherichia coli.

Mutants of Escherichia coli defective in septum initiation, as well as in septum formation were obtained spontaneously, without mutagenic treatment, by selection of rifampin-tolerant mutants of an antibiotic-permeable strain carrying the envA mutation. The disturbed phenotype was in all mutants aggrevated the low incubation temperatures. One allele, sefA1, was studied in detail. Septum initiation, as well as septum formation, was promoted by high cell densities or by the addition of low concentrations of certain antibiotics, e.g., rifampin and chloramphenicol, to low-density cultures. The observed rifampicin depencence was studied in detail. These experiments indicated that a very modest shift-down situation suppressed the phenotype and enabled constrictions to proceed to cell separation. The rifampicin sensitivity of the partially purified deoxyribonucleic acid polymerase was not affected by the sefA1 allele, which is located close to proA and is thus distinct from envA. Growth parameters during the shift to 25 degrees C were followed in a transductant carrying HE SEFA1 allele. This constriction was characteristically blunt and did not lead to cell separation. At the time of formation of these frozen constrictions, clear zones representing a separation of wall from cytoplasmic membrane appeared. These polar tips did not inhibit expansion of the cell envelope. The phenotype of cells carrying the sefA1 allele suggests a disturbed relationship among protoplasm expansion, envelope growth, and septum formation. It is thought that the blunt constrictions observed are caused by an inability of the two septal peptidoglycan layers to fuse during an early stage of septation.     

Evidence that Escherichia coli accumulates glycine betaine from marine sediments.

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

Characterization of the Arabidopsis thaliana mutant pcb2 which accumulates divinyl chlorophylls

We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that the pcb2 mutant accumulated divinyl chlorophylls, and the relative amount of divinyl chlorophyll b was remarkably less than that of divinyl chlorophyll a. The responsible gene was mapped in an area of 190 kb length at the upper arm of the 5th chromosome, and comparison of DNA sequences revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis confirmed that the wild-type of this gene suppressed the phenotypes of the mutation. Antisense transformants of the gene also accumulated divinyl chlorophylls. The genes homologous to At5g18660 are conserved in a broad range of species in the plant kingdom, and have similarity to reductases. Our results suggest that the PCB2 product is divinyl protochlorophyllide 8-vinyl reductase.     

D-Alanine-requiring cell wall mutant of Escherichia coli.

A mutant of Escherichia coli is described whose cells show a spherical or irregular morphology, associated with leakage of beta-galactosidase and other intracellular proteins. The expression of the morphologic abnormality is most marked when the mutant is grown in rich media and is suppressed by D-alamine, D-serine, D-glutamate, or glycine supplementation. D-Alanine is the most effective amino acid supplement, half maximally supressing this anomalous property at a concentration of 75 mug/ml, as measured by the reduction in beta-galactosidase released from the cells. The mutant is more sensitive to penicillin G, D-methionine, and D-valine and it is relatively resistant to lysozyme. These phenotypic abnormalities are likewise corrected by the above supplementations. The relative rates of peptidoglycan synthesis in mutant and parent, grown under restrictive conditions, were measured both in vivo and in vitro by rates of incorporation of L-[14-D]alanine and uridine-5'-diphosphate-N-acetyl-D-[1-15C-A1-glucosamine, respectively. There is not metabolic block in the biosynthesis of uridine-5'-diphosphate-N-acetyl-muramyl-pentapeptide as shown by enzymic analysis and the lack of accumulation of uridine-5'-diphosphate-N-acetylmuramyl-peptide precursors. These preliminary studies suggest that the mutant possesses a defect in the biosynthesis of peptidoglycan although the exact lesion has not yet been established.     

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.