Charles Colson

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

SummarySeveral thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: ε-N-trimethyllysine, ε-N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.

DNA restriction and modification systems in Salmonella

SummaryHaploid hybrids between Salmonella typhimurium Hfr and Escherichia coli Fexercise two additive types of restriction and modification (SA and SB) on phage γ. System SA had been detected previously in S. typhimurium with phage L. Independent mutants in the SA and SB systems were isolated. P22- and P1-mediated transductions in S. typhimurium and in hybrids established that the genes governing these systems are independent but linked and situated counter-clockwise of serB on the map, in the order: pyrB-hsdSA-hsdSB-serB.

Sequence of the Salmonella typhimurium StyLT1 restriction-modification genes: homologies with EcoP1 and EcoP15 type-III R-M systems and presence of helicase domains.

The StyLT1 restriction-modification (R-M) system of Salmonella typhimurium has recently been suggested to belong to the type-III R-M systems [De Backer and Colson, Gene 97 (1991) 103-107]. The nucleotide sequences of StyLT1 mod and res have been determined. Two closely adjacent open reading frames were found 12 bp apart with coding capacities of 651 (Mod) and 982 (Res) amino acids (aa), respectively. The genes, lying in the same direction of transcription in the mod-res order, are transcribed as distinct units. The deduced aa sequences reveal homologies with known type-III enzymes from the Escherichia coli P1 prophage, E. coli P15 plasmid and Bacillus cereus chromosome. In addition, the StyLT1 restriction endonuclease (ENase), like other type-I and type-III ENases, contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site.

A Locus Involved in Kanamycin, Chloramphenicol and L-serine Resistance Is Located in the Bgly-galu Region of the Escherichia-coli K12-chromosome

SummarySpontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected. Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min). This positive selection of deletions allowed detection of a new locus located between galU and bglY. Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to l-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.

Purification of Escherichia coli amplifiable plasmids by high-salt Sepharose chromatography

Abstract This new method allows an easy and rapid purification of amplifiable Escherichia coli plasmids such as pBR 322 without the use of cesium chloride centrifugation. After gentle lysis, centrifugation, and phenol extraction, the material is reextracted with acid phenol to remove the bacterial DNA. The high-molecular-weight ribosomal RNA is removed by precipitation with 2 m ammonium sulfate and the tRNA by passage through a small column of Sepharose CL 4B in the presence of 2 m ammonium sulfate.

Host-controlled Restriction Mutants of Salmonella typhimurium

SUMMARY Forty-eight independent restriction-deficient mutations of Salmonella typhimurium LT2 were isolated by using selective and non-selective methods. With phage P 22 it was shown that some mutations affected the restriction capacity only, while others affected both restriction and modification. The host-restriction of S. typhimurium decreased the recovery of F-lac + infected cells and decreased the yield of recombinants in bacterial mating and in phage P 22-mediated transduction.

Partial purification and characterization of the specific protein-lysine N-methyltransferase of YL32, a yeast ribosomal protein.

YL23 and YL32 are two of the three most heavily methylated ribosomal proteins of Saccharomyces cerevisiae. Using an in vitro assay, it was determined that they are methylated by two distinct enzymes. The protein-lysine N-methyltransferase that methylates YL32 was partially purified by affinity and ion-exchange chromatography. Its molecular mass was estimated to be 82 kDa, and its isoelectric point to be 4.45. Optimum activity was expressed at pH 7.5, and the enzyme was irreversibly inactivated at pH lower than 5.0. The Km of the enzyme for AdoMet is 1.7 +/- 0.4 microM, and the Ki toward AdoHcy was 0.71 microM. Formation of epsilon-N-dimethyllysine was observed to occur in two steps via epsilon-N-monomethyllysine. Like other protein-lysine N-methyltransferases, the methylase of YL32 exhibits a high substrate specificity.

Cloning of An Endoglucanase Gene From Pseudomonas-fluorescens Var Cellulosa Into Escherichia-coli and Pseudomonas-fluorescens

SummaryAn endoglucanase chromosomal gene from the cellulolyticPseudomonas fluorescens var.cellulosa (NCIB 10462) was cloned inEscherichia coli. Chromosomal DNA was partially digested with the restriction enzymeEcoRI and ligated into the broad host-range, mobilizable plasmid pSUP104 that had been linearized with the same enzyme. After transformation ofEscherichia coli, and endoglucanase-positive clone was detected in situ by use of the Congo-red assay procedure. The endoglucanase gene on the recombinant plasmid pRUCL 100 was expressed in the non-cellulolyticPseudomonas fluorescens PF41. The DNA fragment carrying the gene was transferred to the plasmid pBR322, generating plasmids pRUCL150 and pRUCL151, and its restriction map was derived.

Characterization and expression in Escherichia coli of an endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa.

An endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa present on plasmid pRUCL150 and expressed in Escherichia coli was subcloned in plasmid pBR322. Plasmid pRUCL153 contained the smallest DNA insert (2.9 kb) with endoglucanase activity. The plasmids directed the synthesis of a mostly periplasmic enzyme in E. coli and the level of enzyme activity was comparable in several strains. Analysis by non-denaturing polyacrylamide gel electrophoresis of the endoglucanase produced with various recombinant plasmids showed that it was unique. The endoglucanase gene on plasmid pRUCL153 was localized by physical mapping of independent transposon Tn5 insertions. Hence, its size was estimated to be approx. 1.3 kb. In vivo radioactive labelling of plasmid-encoded proteins using minicells, followed by denaturing polyacrylamide gel electrophoresis, allowed us to determine the size of the endoglucanase: Mr 40,000 for the precursor and Mr 38,000 for the mature enzyme. It was demonstrated that no cellulase operon, but a single gene, was cloned. The direction of transcription of the gene was determined by placing it under the control of the promoter of the lactose operon.