Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258.

The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.     

Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA.

We have determined a nucleotide seuqence of 87 residues surrounding a ribonuclease III (endoribonuclease III; EC 3.1.4.24) processing site in the bacteriophage T7 intercistronic region between early genes 0.3 and 0.7. The structural requirements necessary for proper recognition and cleavage by RNase III are discussed. In addition, other structural features characteristic of this intercistronic boundary are described.     

Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway.

The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.     

Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322.

I have determined the nucleotide sequence of the ampicillin resistance gene of pBR322, an Escherichia coli plasmid that encodes a penicillin beta-lactamase. This gene codes for a protein of 286 amino acid residues. The first 23 amino acids presumably form a signal for secretion, because they do not appear in the mature enzyme, whose partial amino acid sequence has been determined independently.     

Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid.

The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.     

Nucleotide sequence of the recognition site for the restriction-modification enzyme of Escherichia coli B.

The nucleotide sequence of the recognition site for the restriction-modification enzyme of Escherichia coli B (SB site) has been determined. The recognition site is a 15-nucleotide sequence consisting of the trimer 5'TGA3', followed by an 8-nucleotide domain of variable sequence, which in turn is followed by tetramer 5'TGCT3'. The sequence has no 2-fold rotational symmetry. Single base changes in the constant nucleotide domains result in the loss of sensitivity to both restriction and modification. Our data are also consistent with modification occurring by methylation of two adenine residues per SB site: one on the adenine of the trimer 5'TGA3' and the other on the complementary strand on the adenine complementary to the first thymine of the tetramer 5'TGCT3'. All nine independently isolated spontaneous mutants at the SB1 site of bacteriophage f1 are caused by a G-to-T transversion. Mutations at the SB2 site are caused by various single base changes.