Optimization of a small laccase by active-site redesign.

Small but faster: A small laccase from Streptomyces coelicolor (SLAC) has been engineered by structure-based design and site-directed mutagenesis to improve the activity on commercially relevant substrates. The variants generated showed up to 40-fold increased efficiency on 2,6-dimethoxyphenol and the ability to use mediators with considerably higher redox potentials (methylsyringate and TEMPO).

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library.

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences.

Chemically modified oligonucleotides with efficient RNase H response.

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex.

LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes.

Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity.

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps.

Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E.

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase.

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils.

Identification of the C-signal, a contact-dependent morphogen coordinating multiple developmental responses in Myxococcus xanthus.

The regulated accumulation of the contact-dependent extracellular C-signal morphogen in the bacterium Myxococcus xanthus ensures the temporal and spatial coordination of multicellular morphogenesis and cellular differentiation during fruiting body formation. Synthesis of the C-signal depends on the csgA gene. The CsgA protein exists in two forms, the full-length 25-kD protein (p25), which is homologous to short-chain alcohol dehydrogenases, and a 17-kD protein (p17).

Coupling gene expression and multicellular morphogenesis during fruiting body formation in Myxococcus xanthus.

A recurring theme in morphogenesis is the coupling of the expression of genes that drive morphogenesis and the morphogenetic process per se. This coupling ensures that gene expression and morphogenesis are carried out in synchrony. Morphogenesis of the spore-filled fruiting bodies in Myxococcus xanthus illustrates this coupling in the construction of a multicellular structure.