Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.     

Cloning of Beneckea genes in Escherichia coli.

Genes from Beneckea harveyi, a luminescent marine bacterium, were cloned in Escherichia coli. This was done by producing randomly sheared fragments of Beneckea DNA and inserting them into the EcoRI site of plasmid pMB9 by the adenine-thymine joining procedure. The hybrid plasmids were used to transform E. coli C600 SF8. Among the transformants selected for tetracycline resistance, one clone that appeared to complement a leucine tb mutation was identified. The transformants were screened for the presence of Beneckea 5S genes. Four of these clones were analyzed in detail by hybridization with 16S, 23S, and 4S Beneckea RNA. The observations suggest that the ribosomal genes in Beneckea are linked, but are present in a different order than those in E. coli.     

Use of Transposons in Cloning Poorly Selectable Genes of Escherichia coli: Cloning of uvrA and Adjacent Genes

A transposon was introduced close to a poorly selectable gene. This gene could be cloned by using selection for the antibiotic resistance marker of the transposon.     

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.     

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein.

Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein.     

Cloning and characterization of the xyl genes from Escherichia coli

Summary Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase (xylA), xylulokinase (xylB), and regulatory (xylR) or transport (xylT) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10–15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp BglII fragment and a 2.6 kbp HindIII-SalI fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.     

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.     

Cloning of genes for proline biosynthesis in Escherichia coli

Restriction map of Escherichia coli chromosome fragment (7.4 MD) carrying proAB genes was constructed. Localization of proA and proB genes on the cloned chromosome fragment was determined by complementation test and the measuring of glutamylkinase activity (proB gene product). ProA and proB genes were cloned separately on multicopy plasmids of alternative orientation and their expression being, probably, under the control of their own regulatory regions, studied.     

Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli.

We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background. Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells. No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells. The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation. Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites.     

Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli.

A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated. Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels.     

Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.