fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2427-2433 ◽  
Author(s):  
Martin Stancek ◽  
Leif A. Isaksson ◽  
Monica Rydén-Aulin
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Temperature Sensitivity ◽  
Minimal Medium ◽  
Synthetic Medium ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Atp Binding Site ◽  
Pleiotropic Phenotype

Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.

ISOLATION AND INVESTIGATION OF INDUCED ASPOROGENIC MUTANTS

1960 ◽  
Vol 6 (2) ◽  
pp. 135-151 ◽  
Author(s):  
D. G. Lundgren ◽  
G. Beskid
Keyword(s):  
Minimal Medium ◽  
Synthetic Medium ◽  
Normal Growth ◽  
Building Blocks ◽  
Culture Technique ◽  
Temperature Sensitive ◽  
Good Growth ◽  
Active Growth ◽  
Casein Hydrolyzate ◽  
Weight Protein

A simple chemically defined (minimal) medium was developed which supported good growth and sporulation of B. cereus var. lacticola. Normal growth and sporulation habits in the medium were determined employing an active growth-culture technique. Induced asporogenic mutants were isolated employing ultraviolet light as the mutagen. Mutants differed in their sporulation habits from the normal culture when grown at 37 °C under the standardized conditions in a minimal synthetic medium. Three mutant cultures appeared to be temperature-sensitive mutants as regards sporulation, that is, in the minimal medium mutant cultures were able to sporulate when grown at 28 °C but were non-sporulating at 37 °C. Sporulation blocks were partially reversed when casein hydrolyzate and some low molecular weight protein building blocks were added to the synthetic medium. The pH changes of the medium during growth and sporulation of normal and mutant cultures were compared.

scholarly journals RHO AND RIBOSOME MUTATION INTERACTION: LETHALITY OF rho-15 IN rpsL or rpsE STRAINS, AND rho-15 METHIONINE AUXOTROPHY IN rps  + STRAINS OF ESCHERICHIA COLI

Genetics ◽  
1979 ◽  
Vol 93 (2) ◽  
pp. 353-360
Author(s):  
S K Guterman ◽  
C L Howitt
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Genetic Background ◽  
Minimal Medium ◽  
Complex Medium ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
K 12

ABSTRACT The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ: :IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42° on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.—Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42° in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.

Temperature-sensitive mutants of MscL mechanosensitive channel

2019 ◽  
Vol 166 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Naoto Owada ◽  
Megumi Yoshida ◽  
Kohei Morita ◽  
Kenjiro Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Structural Change ◽  
Cell Growth ◽  
Thermodynamic Stability ◽  
Transmembrane Helix ◽  
Mechanosensitive Channel ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Membrane Stretching

Abstract MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified. The L86P mutation occurred in the second transmembrane helix, TM2. Substitution of residues neighbouring L86 with proline also led to a Ts mutation, but the substitution of L86 with other amino acids did not result in a Ts phenotype, indicating that the Ts phenotype was due to a structural change of TM2 helix by the introduction of a proline residue. The D127V mutation was localized in the electrostatic belt of the bundle of cytoplasmic helices, indicating that stability of the pentameric bundle of the cytoplasmic helix affects MscL structure. Together, this study described a novel class of MscL mutations that were correlated with the thermodynamic stability of the MscL structure.

scholarly journals Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Sarah C. Pulvermacher ◽  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Minimal Medium ◽  
Luria Bertani ◽  
Cell Physiology ◽  
Rt Pcr ◽  
Luria Bertani Broth ◽  
Increased Sensitivity ◽  
Transcriptional Fusions

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

2000 ◽  
Vol 46 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Takashi Kubo ◽  
Toshiko Aiso ◽  
Reiko Ohki
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Lacz Gene ◽  
Permissive Temperature ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Genes ◽  
Beta Galactosidase ◽  
Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

scholarly journals The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

1974 ◽  
Vol 140 (3) ◽  
pp. 443-450 ◽  
Author(s):  
John R. Dean ◽  
John Sykes
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Maximum Activity ◽  
Exponential Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Experimental Conditions ◽  
Inhibition Of Growth ◽  
Ribosomal Ribonucleic Acid

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

Temperature Sensitivity of Cell Growth in Escherichia coli associated with the Temperature Sensitive R(KM) Factor

Nature ◽  
1968 ◽  
Vol 219 (5151) ◽  
pp. 284-285 ◽  
Author(s):  
YOSHIRO TERAWAKI ◽  
YOSHIHIRO KAKIZAWA ◽  
HISAO TAKAYASU ◽  
MASANOSUKE YOSHIKAWA
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Temperature Sensitivity ◽  
Temperature Sensitive

THE INFLUENCE OF TIME AND MEDIA CHANGES ON THE INDUCTION AND SUPPRESSION OF β-GALACTOSIDASE SYNTHESIS IN ESCHERICHIA COLI B

1967 ◽  
Vol 13 (3) ◽  
pp. 257-269
Author(s):  
S. J. Webb ◽  
Janet L. Walker
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Minimal Medium ◽  
Sole Source ◽  
Enzyme Synthesis ◽  
Induction Medium ◽  
Nucleic Acid Bases ◽  
Apparent Effect ◽  
Lag Period ◽  
Escherichia Coli B

When cells of Escherichia coli B were grown in a glucose – amino acid medium and then transferred to a minimal medium containing lactose or isopropyl-β-D-thiogalactopyranoside as a sole source of carbon, no induction of β-galactosidase occurred unless one or several amino acids were supplied. Of the amino acids tested, aspartic acid was the most effective and its ability to initiate the synthesis of the enzyme was increased by the addition of arginine. In the presence of these two, or all of the amino acids, there was a lag period of 10 min before enzyme synthesis occurred. The duration of the lag period was unaffected by the addition of nucleic acid bases or succinate to the induction medium. Succinate or glutamate partially inhibited the synthesis of the enzyme, whereas glucose, inositol, or chloramphenicol completely suppressed it. With the exception of that produced by chloramphenicol, inhibition was dependent on the time at which the inhibitor was added. If inhibitors were added after the 10-min lag period, they had no apparent effect until 45 min had elapsed. Cells transferred after 15 min from one induction medium to another displayed for 30 min the induction characteristics of the first medium. It appears that a process occurring during the early 15-min period determines the rate at which enzymes will be synthesized for the next 30 min and that the action of inhibitors is to prevent this process. The process seems to require intact DNA and amino acids and it is suggested that it determines the specificity and quantity of mRNA manufactured.

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