A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced, which was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of the linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method were demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high-impact research at small liberal arts colleges with limited resources. However, in-frame fusion proteins were obtained from 8 different targets suggesting that the method is broadly applicable in any research setting.

Expression phenotypes suggest that Der participates in a specific, high affinity interaction with membranes.

The GTPase Der is universally conserved in bacteria and is structurally unique as it consists of two GTP-binding domains in tandem (G-domain 1 and G-domain 2) whereas all the other GTPases posses a single GTPase domain. In order to assess the function of Der we have fractionated whole cell lysates containing over expressed Der. This analysis indicated that Der was present in sucrose gradient fractions containing membrane proteins. The interaction with the membrane fraction was specific for Der, since the related GTPase, Era, did not form the membrane complex. In addition, three independent criteria suggested a high affinity interaction; (1) the interaction can be detected under partially denaturing conditions using a gel electrophoresis co-migration assay, (2) the interaction survived 16 h sucrose gradient centrifugation, and (3) the complex could be efficiently reconstituted from purified components. Microscopic examination of cells containing over expressed Der showed that the cell wall structure was disrupted at both cell poles. This phenotype required Der domain three since domain deletion mutations showed no affect on cell wall structure. Surprisingly point mutations that ablate nucleotide binding of either GTP binding domain result in a defect in cell wall structure at only a single cell pole. The data reported here were considered together with results presented previously to suggest that Der may engage in a functional cyclic interaction between ribosomes and the membrane in Escherichia coli.

Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners.

RNase E is an essential enzyme that catalyses RNA processing. Microdomains which mediate interactions between RNase E and other members of the degradosome have been defined. To further elucidate the role of these microdomains in molecular interactions, we studied RNase E from Vibrio angustum S14. Protein sequence analysis revealed that its C-terminal half is less conserved and structured than its N-terminal half. Within this structural disorder, however, exist five small regions of predicted structural propensity. Four are similar to interaction-mediating microdomains identified in other RNase E proteins; the fifth did not correspond to any known functional motif. The function of the V. angustum S14 enolase-binding microdomain was confirmed using bacterial two-hybrid analysis, demonstrating the conserved function of this microdomain for the first time in a species other than Escherichia coli. Further, PNPase in V. angustum S14 was shown to interact with the last 80 amino acids of the C-terminal region of RNase E. This raises the possibility that PNPase interacts with the small ordered region at residues 1026-1041. The role of RNase E as a hub protein and the implications of microdomain-mediated interactions in relation to specificity and function are discussed.

Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb.

Modifying the Site-directed, Ligase-Independent Mutagenesis (SLIM) protocol from a single reaction mode to a two-reaction mode enables highly efficient mutagenesis of plasmid constructs that exceed 8kb. This modified approach reduces the complexity of the PCR step and is optimised for generation of heteroduplexes from long PCR products. The two-reaction mode SLIM has 92% efficiency.

Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase.

Processivity of T7 DNA polymerase relies on the coupling of its cofactor Escherichia coli thioredoxin (Trx) to gene 5 protein (gp5) at 1:1 stoichiometry. We designed a coexpression system for gp5 and Trx that allows in vivo reconstitution of subunits into a functional enzyme. The properties of this enzyme were compared with the activity of commercial T7 DNA polymerase. Examination of purified enzymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the thioredoxin subunit of the two enzymes did not comigrate. To our surprise, we identified a mutation (Phe102 to Ser) in the Trx component from the commercial T7 DNA polymerase (gp5/TrxS102) that was not in the enzyme from the coexpression system (wild type gp5/Trx). A comparison of polymerase activity of the T7 DNA polymerases shows that both enzymes possessed similar specific activity but they were different in their residual activity at 37 degrees C. The half-life of gp5/TrxS102 was 7 min at 37 degrees C and 12 min for gp5/Trx. gp5/TrxS102 polymerase activity was reduced by fourfold with 3'-5' exonuclease activity as the prominent activity detected after 10 min of heat inactivation at 37 degrees C. Supplementation of reaction mixtures containing gp5/TrxS102 with exogenous nonmutant thioredoxin restored the enzyme activity levels. Pulse proteolysis was used to demonstrate that TrxS102 unfolded at lower urea concentrations than wild type thioredoxin. Thus, Ser substitution at position 102 affected the structural stability of thioredoxin resulting in a reduced binding affinity for gp5 and loss of processivity.

Coexpression of the subunits of T7 DNA polymerase from an artificial operon allows one-step purification of active gp5/Trx complex.

T7 DNA polymerase expression was performed from an artificial operon by cloning its cofactor, thioredoxin, downstream of a N-terminal 9xHis-tagged T7 gene 5 (gp5). Up to 90% of gp5 was soluble in the presence, but not in the absence of thioredoxin coexpression suggesting that free-form thioredoxin assisted solubilization of gp5. Expression and single-step nickel-agarose affinity purification resulted in recovery of an enzyme that was 97% pure. Copurification of thioredoxin was observed and the estimated molar ratio of thioredoxin to gp5 was 1:1 in the purified DNA polymerase complex. Purified T7 DNA polymerase exhibited full polymerase activity compared to the commercial enzyme and required no exogenous thioredoxin for activity.

Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h.

Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.

Thiostrepton-resistant mutants of Thermus thermophilus.

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.

Function of the universally conserved bacterial GTPases.

The GTPase superfamily of cellular regulators is well represented in bacteria. A small number are universally conserved over the entire range of bacterial species. Such a pervasive taxonomic distribution suggests that these enzymes play important roles in bacterial cellular systems. Recent advances have demonstrated that bacterial GTPases are important regulators of ribosome function, and important for the distribution of DNA to daughter cells following cell division. In addition, the atomic structure of a unique GTPase, EngA, has recently been established. Unlike any other GTPase, EngA contains tandem GTP-binding domains. This structural study suggests that the GTPase cycles of the domains are regulated differentially in a manner that remains to be elucidated.

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.