Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Heterothermal acclimation: An experimental paradigm for studying the control of thermal acclimation in crabs

A method for the study of the control of the attainment of thermal acclimation has been applied to the crabs, Cancer pagurus and Carcinus maenas. Crabs were heterothermally acclimated by using an anterior–posterior partition between two compartments, one at 8°C and the other at 22°C. One compartment held a three-quarter section of the crab including the central nervous system (CNS), eye stalks, and ipsilateral legs; the other held a quarter section including the contralateral legs. Criteria used to assess the acclimation responses were comparisons of muscle plasma membrane fatty acid composition and “fluidity.” In both species, the major fatty acids of phosphatidylcholine were 16:0, 18:1, 20:5, and 22:6, whereas phosphatidylethanolamine contained significantly less 16:0 but more 18:0; these fatty acids comprised 80% of the total. Differences in fatty acid composition were demonstrated between fractions obtained from the ipsilateral and contralateral legs from the same heterothermally acclimated individual. In all acclimation states (except 22CNS, phosphatidylcholine fraction), membrane lipid saturation was significantly increased with acclimation at 22° as compared with 8°C. Membrane fluidity was determined by using 1,3-diphenyl-1,3,5 hexatriene (DPH) fluorescence polarization. In both species, membranes from legs held at 8° were more fluid than from legs held at 22°C irrespective of the acclimation temperature of the CNS. Heterothermal acclimation demonstrated that leg muscle membrane composition and fluidity respond primarily to local temperature and were not predominately under central direction. The responses between 8°C- and 22°C-acclimated legs were more pronounced when the CNS was cold-acclimated, so a central influence cannot be excluded.

Studying Early Nodulin Gene ENOD40 Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa1

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40–1 and MsENOD40–2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40–1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40–2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40–1 promoter construct, whereas pseudonodules from plants containing the MsENOD40–2 promoter construct stained blue. A 616-bp region at the distal 5′ end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.