We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

http://compbio.korea.ac.kr/wiki/images/8/83/Mmbr.1996.expression.pdf

Strategies for achieving high-level expression of genes in Escherichia coli.

Barophilic bacteria inhabit the deep oceans, and the specific functional modifications and regulatory mechanisms which govern adaptation to hydrostatic pressure are beginning to be understood. For example, the rate of production of several proteins by some hydrothermal vent archaebacteria and the degree of saturation of membrane lipids in other deep-sea bacteria have been found to change as a result of cultivation at high pressure. We report here the cloning of gene, ompH, which encodes a major pressure-inducible protein of strain SS9, a gram-negative eubacterium isolated from a depth of 2.5 kilometres in the Sulu Sea. Messenger RNA encoded by ompH is expressed when cells are grown at 280 atm but not at 1 atm, indicating that transcription of the ompH gene is controlled by hydrostatic pressure. The function of the OmpH protein in adaptation to high pressure and the use of the ompH gene in studying how bacteria sense and respond to pressure is discussed.

Isolation of a gene regulated by hydrostatic pressure in a deep-sea bacterium

Specificity of restriction endonucleases and DNA modification methyltransferases — a review (edition 3)

Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently. About two dozen potential restriction–modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes. Here we describe a functional analysis of the 16 putative Type II R-M systems in the H.pylori J99 genome. To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements. To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine. We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities. Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial ‘immune’ system, by alternatively turning on/off a subset of numerous R-M systems.

/pdf/functional-analysis-of-putative-restriction-modification-1j6dpiwlly.pdf

Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome.

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase Transformants harboring this construct were only viable when both repression controls were operational

C D O'Connor

Papers

Highly repressible expression system for cloning genes that specify potentially toxic proteins.