Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics

Proteolysis and modulation of the activity of the cell division inhibitor SulA in Escherichia coli lon mutants.

Intracellular accumulation of the inducible cell division inhibitor SulA is modulated by proteases that ensure its degradation, namely, the Lon protease and another ATP-dependent protease(s). Lon- cells are UV sensitive because SulA is stable. We asked whether these ATP-dependent proteases are more active when lon cells are grown at high temperature or in synthetic medium since these conditions decrease the UV sensitivity of lon cells. We found that these growth conditions have no direct effect on Lon-independent degradation of SulA. They may, instead, decrease the SulA-FtsZ interaction

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage