Chimeric chemoreceptors in Escherichia coli: signaling properties of Tar-Tap and Tap-Tar hybrids.

The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of Escherichia coli together mediate chemotactic responses to dipeptides. Tap is a low-abundance receptor. It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr. Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run). To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains. The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator. Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain. Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap. The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability. The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation. However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody. Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg. The hybrid, truncated, and extended receptors exhibit various levels of methylation. However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected. We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.

Construction of shuttle plasmids which can be efficiently mobilized from Escherichia coli into the chromatically adapting cyanobacterium, Fremyella diplosiphon.

In some strains of cyanobacteria the composition of the light-harvesting antennae is determined by the color of available light. The mechanism of this chromatic adaptation involves the regulation of gene expression by red and green light and has been most studied in Fremyella diplosiphon (Calothrix sp. PCC 7601), a filamentous cyanobacterium for which there has been no reported means of genetic manipulation. We have constructed shuttle plasmids which can be efficiently mobilized by RP4 from Escherichia coli into F. diplosiphon and which can be recovered from transconjugant F. diplosiphon and returned to E. coli by transformation. The ability of these plasmids to replicate in F. diplosiphon is conferred by an 8.0-kb DNA fragment isolated from pFDA, a plasmid native to F. diplosiphon. To create these shuttle plasmids the 8.0-kb fragment was cloned into pJCF22, a mobilizable plasmid constructed from oriV and bom from pBR322, cat from pACYC184 and aphA from pACYC177.pJCF22 lacks sites for the restriction enzymes FdiI and II. Transconjugant F. diplosiphon containing shuttle plasmid pJCF62 are resistant to chloramphenicol and highly resistant to the aminoglycosides, G418 and neomycin. When aadA from the omega interposon was incorporated into a shuttle plasmid transconjugant F. diplosiphon could also be selected with streptomycin or spectinomycin. In F. diplosiphon shuttle plasmid pJCF62 replicates with a minimum copy number of seven. The oriV for replication in F. diplosiphon was localized to a 2.8-kb region within the cyanobacterial part of pJCF62. The presence on a shuttle plasmid of a single recognition site for FdiI reduced the efficiency of mobilization into F. diplosiphon by 5- to 10-fold. Restriction at this site was prevented when the E. coli donor strain in the mating contained the enzyme Eco47II methylase.