Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.     

Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2.

We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2. The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with lambda bacteriophage carrying a metB-lacZ gene fusion (lambda JBlac) in which beta-galactosidase production is dependent upon metB gene expression. The mutant lysogens show elevated, poorly regulated beta-galactosidase production. The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor. The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter. We propose that these mutations alter the repressor binding site and define the metB operator sequence. In addition, we discuss a highly conserved, nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA, metC, metE, metF, metG, and metJB genes of both S. typhimurium and E. coli.     

Nucleotide sequence of Klebsiella pneumoniae lac genes.

The nucleotide sequences of the Klebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and oriented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherichia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a common ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comparison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lacI genes and lac repressor proteins each are related to the galR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the galR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes.     

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme

The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector. The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level. Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.