A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum.

Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.

MamO Is a Repurposed Serine Protease that Promotes Magnetite Biomineralization through Direct Transition Metal Binding in Magnetotactic Bacteria.

Many living organisms transform inorganic atoms into highly ordered crystalline materials. An elegant example of such biomineralization processes is the production of nano-scale magnetic crystals in magnetotactic bacteria. Previous studies implicated the involvement of two putative serine proteases, MamE and MamO, during the early stages of magnetite formation in Magnetospirillum magneticum AMB-1. Here, using genetic analysis and X-ray crystallography, we show that MamO has a degenerate active site, rendering it incapable of protease activity. Instead, MamO promotes magnetosome formation through two genetically distinct, noncatalytic activities: activation of MamE-dependent proteolysis of biomineralization factors and direct binding to transition metal ions. By solving the structure of the protease domain bound to a metal ion, we identify a surface-exposed di-histidine motif in MamO that contributes to metal binding and show that it is required to initiate biomineralization in vivo. Finally, we find that pseudoproteases are widespread in magnetotactic bacteria and that they have evolved independently in three separate taxa. Our results highlight the versatility of protein scaffolds in accommodating new biochemical activities and provide unprecedented insight into the earliest stages of biomineralization.

Magnetite Biomineralization in Magnetospirillum magneticum Is Regulated by a Switch-like Behavior in the HtrA Protease MamE.

Magnetotactic bacteria are aquatic organisms that produce subcellular magnetic particles in order to orient in the earth's geomagnetic field. MamE, a predicted HtrA protease required to produce magnetite crystals in the magnetotactic bacterium Magnetospirillum magneticum AMB-1, was recently shown to promote the proteolytic processing of itself and two other biomineralization factors in vivo Here, we have analyzed the in vivo processing patterns of three proteolytic targets and used this information to reconstitute proteolysis with a purified form of MamE. MamE cleaves a custom peptide substrate with positive cooperativity, and its autoproteolysis can be stimulated with exogenous substrates or peptides that bind to either of its PDZ domains. A misregulated form of the protease that circumvents specific genetic requirements for proteolysis causes biomineralization defects, showing that proper regulation of its activity is required during magnetite biosynthesis in vivo Our results represent the first reconstitution of the proteolytic activity of MamE and show that its behavior is consistent with the previously proposed checkpoint model for biomineralization.

Comparison of biosurfactant detection methods reveals hydrophobic surfactants and contact-regulated production.

Biosurfactants are diverse molecules with numerous biological functions and industrial applications. A variety of environments were examined for biosurfactant-producing bacteria including soil, water and leaf surfaces. Biosurfactant production was assessed with an atomized oil assay for a large number of bacterial isolates and compared with a commonly used drop collapse assay from broth and plate cultures. The atomized oil assay detected every strain that produced a biosurfactant detectable by the drop collapse test, and also identified additional strains that were not detected with the drop collapse assay because they produced low levels of surfactant or hydrophobic (low water solubility) surfactants such as pumilacidins. Not all strains that produced a biosurfactant detectable by the drop collapse when cultured on agar surfaces produced surfactants detectable by drop collapse when cultured in broth, and vice versa. Many bacterial strains exhibited preferential production of surfactants when grown on an agar surface compared with broth cultures, and such surface enhancement of production could also be stimulated by increasing the viscosity of liquid culture media. Surface induction of surfactant production in the epiphyte Pseudomonas syringae was regulated at the transcriptional level.

Novel high-throughput detection method to assess bacterial surfactant production.

A novel biosurfactant detection assay was developed for the observation of surfactants on agar plates. By using an airbrush to apply a fine mist of oil droplets, surfactants can be observed instantaneously as halos around biosurfactant-producing colonies. This atomized oil assay can detect a wide range of different synthetic and bacterially produced surfactants. This method could detect much lower concentrations of many surfactants than a commonly used water drop collapse method. It is semiquantitative and therefore has broad applicability for uses such as high-throughput mutagenesis screens of biosurfactant-producing bacterial strains. The atomized oil assay was used to screen for mutants of the plant pathogen Pseudomonas syringae pv. syringae B728a that were altered in the production of biosurfactants. Transposon mutants displaying significantly altered surfactant halos were identified and further analyzed. All mutants identified displayed altered swarming motility, as would be expected of surfactant mutants. Additionally, measurements of the transcription of the syringafactin biosynthetic cluster in the mutants, the principal biosurfactant known to be produced by B728a, revealed novel regulators of this pathway.