The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system.

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites.

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.

Characterization of the tyrosine recombinase MrpA encoded by the Streptomyces coelicolor A3(2) plasmid SCP2*.

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required. Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination.

Dynamic response of the expression of hxt1, hxt5 and hxt7 transport proteins in Saccharomyces cerevisiae to perturbations in the extracellular glucose concentration.

Glucose transport in Saccharomyces cerevisiae relies on a multi-factorial uptake system. The modulation of its efficiency depends on the differential expression of various sets of hexose transport-related proteins whose glucose affinity differs considerably. The expression of three different glucose transport proteins (HXT1, HXT5 and HXT6/7 with low-, intermediate- and high-affinity, respectively) was monitored as a result of modified extracellular glucose concentrations. Cultivation at glucose-limited (continuous) conditions was instantly replaced by a batch (and thus, non-limited) mode. Further, to mimic concentration gradients in large-scale production bioreactors, multiple and rapid transient glucose pulses were applied to chemostat cultivation. Antibodies against the HXT-proteins were used to monitor the proteins' expression levels prior to and after perturbing the external glucose concentrations. HXT5 and HXT6/7 were either expressed during the starvation-like steady-state phases in the chemostat cultivations, whereas HXT1 could not be detected at all. HXT1, however, is subsequently expressed during the excess of glucose in the batch mode, while the HXT5 and HXT6/7 transporters were at least found to decline. These findings coincide well with the transporters' affinity profiles. As a result of repeated and rapid transient glucose pulses during continuous fermentation, especially HXT6/7 pointed out to alter the protein expression pattern.