Deletion of the Escherichia coli crp Gene

Spontaneous crp mutants Escherichia coli were selected from a strain that does not require 3',5'-cyclic adenosine monophosphate for CAP activity. Several deletions of the crp gene were characterized. The crp gene was not essential for growth of E. coli. crp mutations reduced the donor ability of Hfr strains.     

Enhanced acid sensitivity of pressure-damaged Escherichia coli O157 cells

Pressure-damaged Escherichia coli O157 cells were more acid sensitive than native cells and were impaired in pH homeostasis. However differences in acid sensitivity were not related to differences in cytoplasmic pH (pH(i)). Cellular beta-galactosidase was more acid labile in damaged cells. Sensitization to acid may thus involve loss of protective or repair functions.     

Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli

We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.     

Identification of the uvrA6 mutation of Escherichia coli.

The uvrA6 mutation has been cloned on a multicopy plasmid by using a chloramphenicol resistance marker introduced next to the uvrA gene in the Escherichia coli chromosome. The mutation was shown to reside in the N-terminal part of the uvrA gene. Sequencing part of this region of the mutant gene revealed a frameshift mutation at positions 207 to 209, which leads to a stop codon at position 262. A marker rescue experiment showed that this frameshift is the only mutation responsible for the UV-sensitive phenotype of the UvrA6 mutant. The method presented is suitable for the cloning of every chromosomal uvrA mutation and can be useful for the study of the functional domains of the UvrA protein.     

An Rts1-derivative plasmid conferring UV sensitivity on Escherichia coli host

A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on $\text{Escherichia}\text{coli}$ host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, $\text{puv}$, being responsible for the UV sensitivity was located on the kanamycin resistance gene containing $\text{Bam}$H1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of $\text{puv}$.     

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established. Several lines were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein. The expression of uvrA was found to be 50-100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.     

Increased UV sensitivity of Escherichia coli cells after introduction of foreign photolyase genes

High-expression plasmids for photolyase (phr) genes from the bacteria Escherichia coli, Anacystis nidulans, Streptomyces griseus and Halobacterium halobium and the yeast Saccharomyces cerevisiae were constructed and introduced into E. coli phr recA cells. As previously reported, al introduced phr genes provided the host cells with photoreactivation-repair activity and the introduced E. coli phr gene rendered the host cells more UV-resistant in the dark. E. coli cells harboring foreign phr genes, however, were found to be more sensitive to UV light in the dark than cells containing the vector plasmid only. These differences in UV sensitivity in the dark disappeared when the host cells had an additional mutation, uvrA, suggesting that the foreign photolyases inhibited the E. coli excision-repair system.     

The mutator property of uvr502 mutation affecting UV sensitivity of Escherichia coli

Summary The frequency of direct and reverse mutations at several chromosomal loci increased in UV sensitiveuvr502 strains ofE. coli. Both UV sensitivity and mutator (Mut⁻) phenotype are due to the singleuvr502 mutation. Inuvr ⁺/uvr502 merodiploid the Mut⁻ phenotype is recessive.     

Cloning of uvrA, lexC and ssb genes of Escherichia coli.

The L' subunits, phosphorylatable precursors of L-type pyruvate kinase, can be proteolyzed in vivo and in vitro, and transformed into several molecular species.Trypsin induces the appearance of a phosphorylatable form similar to liver L-type enzyme.Subtilisin splits the phosphorylatable site and induces the appearance of a form similar to the minor form occuring with red cell aging.Sensitivity to proteolysis of some pyruvate kinase variants is modified.     

Study of the regulation of crp gene expression in Escherichia coli K12

The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.     

Cloning and sequence of the crp gene of Escherichia coli K 12.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.     

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.     

Photoreactivation of UV damage in Escherichia coli uvrA6: lethality is more effectively reversed than either premutagenic lesions or SOS induction

The effect of cyclobutyl pyrimidine dimers on cytotoxicity, induction of synthesis of the RecA and UmuC proteins, and mutagenesis was studied in Escherichia coli uvrA6 cells possessing excess amounts of photoreactivating enzyme. Exposure of 254 nm ultraviolet-irradiated (10 J/m2) cells to radiation from daylight fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence-dependent manner, up to about 90% reduction at 5 min exposure. Of the lethal ultraviolet damage, 85% was photoreactivable (i.e. cyclobutyl pyrimidine dimers) and 15% was non-photoreactivable. An incident fluence of 1 J/m2 resulted in approximately a 5-fold increase in the synthesis of the RecA and UmuC proteins, as compared to the spontaneous level. If the UV-irradiated cell suspensions were illuminated with a fluorescent lamp at a dose which resulted in the full photoreactivation of viability, the yields of both proteins were reduced to 60% of the non-photoreactivated control cells. Furthermore, photoreactivation was shown to be more effective in the repair of lethal damage than in the repair of premutational damage. These experiments suggest that, among lethal damages, non-photoreactivable damage plays a more important role in both induction of the SOS functions and mutagenesis in uvrA6 cells than do cyclobutyl pyrimidine dimers.     

Enhanced cytidine production by a recombinant Escherichia coli strain using genetic manipulation strategies

Cytidine is a nucleoside molecule that is widely used as a precursor for antiviral drugs. In this study, a cytidine-producing strain Cyt18 was developed from Escherichia coli K-12 through 3-step genetic manipulation strategies. Cytidine deaminase gene (cdd) was firstly deleted from the E. coli K-12 strain to develop Cyt10. Furthermore, homoserine dehydrogenase gene (thrA) was inactivated from the Cyt10 strain to develop Cyt12, in which the intracellular aspartate concentration was expected to be increased. The recombinant plasmid pMG1105 containing an pyrB-pyrA operon from Bacillus amyloliquefaciens CYTI was constructed and was introduced into Cyt12 to obtain the Cyt18 strain. Compared to the Cyt12 strain, the cytidine production by the recombinant strain Cyt18 was increased by ~3-fold (722.9 mg/l vs. 249.3 mg/l).     

Ultraviolet light sensitivity of Escherichia coli K-12 strains carrying ruv mutations in combination with uvrA or lon mutant alleles.

Strains of Escherichia coli K12 have been prepared that carry the ruv mutation in combination with lon or uvrA mutant alleles. The ruv minus uvrA minus double mutant is more sensitive to ultraviolet light than the urvA minus single mutant, whereas the strain with ruv and ion mutations shows the same sensitivity to ultraviolet light as the ruv minus single mutant.     

Enhanced sensitivity of Escherichia coli umuC to photodynamic inactivation by angelicin (isopsoralen).

Escherichia coli umuC cells were inactivated four times more rapidly than umuC+ cells by angelicin (a monofunctional psoralen) plus near-UV irradiation. With other DNA-damaging treatments, either no or much smaller differences in sensitivity were observed. These results show that functions associated with the UmuC+ phenotype contribute to the repair (or tolerance) of some categories of DNA damage more efficiently than others.