Pond sediment magnetite grains show a distinctive microbial community.

Formation of magnetite in anaerobic sediments is thought to be enhanced by the activities of iron-reducing bacteria. Geobacter has been implicated as playing a major role, as in culture its cells are often associated with extracellular magnetite grains. We studied the bacterial community associated with magnetite grains in sediment of a freshwater pond in South Korea. Magnetite was isolated from the sediment using a magnet. The magnetite-depleted fraction of sediment was also taken for comparison. DNA was extracted from each set of samples, followed by PCR for 16S bacterial ribosomal RNA (rRNA) gene and HiSeq sequencing. The bacterial communities of the magnetite-enriched and magnetite-depleted fractions were significantly different. The enrichment of three abundant operational taxonomic units (OTUs) suggests that they may either be dependent upon the magnetite grain environment or may be playing a role in magnetite formation. The most abundant OTU in magnetite-enriched fractions was Geobacter, bolstering the case that this genus is important in magnetite formation in natural systems. Other major OTUs strongly associated with the magnetite-enriched fraction, rather than the magnetite-depleted fraction, include a Sulfuricella and a novel member of the Betaproteobacteria. The existence of distinct bacterial communities associated with particular mineral grain types may also be an example of niche separation and coexistence in sediments and soils, which cannot usually be detected due to difficulties in separating and concentrating minerals.

The kinetic basis of a general method for the investigation of active site content of enzymes and catalytic antibodies: first-order behaviour under single-turnover and cycling conditions.

The theoretical foundation has been laid for the investigation of catalytic systems using first-order kinetics and for a general kinetic method of investigation of the active site content, E(a), of enzymes, catalytic antibodies, and other enzyme-like catalysts. The method involves a combination of steady-state and single-turnover kinetics to provide Vmax and Km and k(lim)(obs) and K(app)(m), respectively. The validity of the method is shown to remain valid for two extensions of the simple two-step enzyme catalysis model (a) when the catalyst preparation contains molecules (Eb) that bind substrate but fail to catalyse product formation and (b) when the catalyst itself binds substrate non-productively as well as productively. The former is a particularly serious complication for polyclonal catalytic antibodies and the latter a potential complication for all catalysts. For the simple model and for (b) Vmax/k(lim)(obs) provides the value of [Ea]T and for (a) its upper limit. This can be refined by consideration of the relative values of Km and the equilibrium dissociation constant of EbS. For the polyclonal catalytic antibody preparation investigated, the fact that K(app/m) > Km demonstrates for the first time the presence of a substrate-binding but non-catalytic component in a polyclonal preparation. First-order behaviour in catalytic systems occurs not only with a large excess of catalyst over substrate but also with lower catalyst/substrate ratios, including the equimolar condition, when K(app)(m) >> [S]0, a phenomenon that is not widely appreciated.

A general kinetic approach to investigation of active-site availability in macromolecular catalysts.

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (E(a)) and inert (i.e. non-binding, non-catalytic) material (E(i)); (b) an extension of the conventional model (a) involving only E(a) and E(i), but with non-productive binding to E(a) (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (E(b)), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters V(max) and K(m) obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (klim/obs, the limiting value of the first-order rate constant, k(obs), at saturating concentrations of catalyst; and Kapp/m) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack E(b), such as the non-productive binding model (b), may be calculated using [E(a)](T)=V(max)/klim/obs. This was validated by showing that, for alpha-chymotrypsin, identical values of [E(a)](T) were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known 'all-or-none' spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain E(b), such as polyclonal catalytic antibody preparations, V(max)/klim/obs is more complex, but provides an upper limit to [E(a)](T). Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [E(a)](T) is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].