Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides.

Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca(2+). PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, K(M) and k(cat), were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection.

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium.

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C1 unit carrier where N(5) -formyl-tetrahydromethanopterin is converted to methenyl-tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N-terminal domain of six α-helices encompassing a series of four ß-sheets and a predominantly anti-parallel ß-sheet at the C-terminus flanked on one side by α-helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation.