Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.     

Cloning and characterization of the promoters of temperate mycobacteriophage L1

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the beta-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli sigma70-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early Pleft promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the Pleft equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli sigma70 promoters.     

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.     

Identification and characterization of novel and potent transcription promoters of Francisella tularensis

Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli σ(70)-related promoter motifs. These motifs include the -10 (TATAAT) and -35 [TTGA(C/T)A] domains and an AT-rich region upstream from -35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.     

Regulation of promoter activity through electrostatic interactions with RNA polymerase

Electrostatic potential profiles were calculated for σ⁷⁰-specific phage T4 promoters and four consensus-like synthetic promoters. Promoters could be classified according to the presence of specific elements of the electrostatic profile in the farther upstream region (?60 to ?100). A correlation was found between the kind of such specific element and the functional behavior of the promoter. A general scheme is proposed whereby the promoter activity is regulated through electrostatic interactions between the upstream region of promoter DNA and the RNA polymerase α-subunit.