Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time-and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H 2 metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H 2 suggests that its role is anaplerotic. Indeed, H 2 via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for. M ethanogenesis is an anaerobic respiration carried out by a phylogenetically related group of Archaea within the phylum Euryarchaeota. Methanogens are divided into two metabolic types, those without and those with cytochromes (1). Methanogens without cytochromes use H 2 as an electron donor and are termed hydrogenotrophic. Some species can substitute H 2 with formate, and a few can use secondary alcohols. CO 2 is the electron acceptor and is reduced to methane. Methanogens with cytochromes reduce certain methyl compounds or the methyl carbon of acetate to methane and are called methylotrophic. Many can also use H 2 and CO 2 , as can hydrogenotrophic methanogens.Although the pathways of methanogenesis have long been known, an understanding of energy conservation has been slower to emerge. Methanogens with and without cytochromes both export Na + when a methyl group is transferred from the carrier tetrahydromethanopterin (H 4 MPT) to coenzyme M (CoM) (Fig. 1). The Na + gradient across the membrane is used directly for ATP synthesis or is converted by an antiporter to a proton gradient. However, for methanogenesis from CO 2 , the initial reduction of CO 2 to a formyl group attached to methanofuran (MFR) is endergonic. How energy is provided to drive this reaction is not well understood. Methanogens with and without cytochromes have membrane-associated energy-converting hydrogenases that couple the reduction of low-potential ferredoxins (Fd) to a chemiosmotic membrane gradient (2). If such a Fd donates electrons for CO 2 reduction, an energy-converting hydrogenase is the conduit of ener...

BackgroundMethanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens.Methodology/Principal FindingsIn order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales.Conclusions/SignificanceOur results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H 2 production by a mutant containing a deletion of the coenzyme F 420 -reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H 2 production comprised Fdh1 and Fru. Because a ⌬fru-⌬frc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F 420 -dependent methylene-H 4 MTP dehydrogenase (Mtd) or the H 2 -forming methylene-H 4 MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H 2 production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H 2 production was not a direct intermediate of methanogenesis. In conclusion, high rates of formatedependent H 2 production demonstrated the potential of M. maripaludis for the microbial production of H 2 from formate.Many hydrogenotrophic methanogens use H 2 or formate for the reduction of CO 2 to obtain energy for growth. Methanococcus maripaludis, the model microorganism in this study, is a hydrogenotrophic, formate-utilizing, mesophilic methanogen. It is common in salt marsh sediments, from which it was isolated (12). An extraordinarily active H 2 consumer, M. maripaludis is exceptionally well equipped with enzymes responsible for H 2 metabolism. M. maripaludis contains genes for seven different hydrogenases, whose expression depends upon the growth conditions (18). It possesses two membrane-bound, energy-converting [Ni-Fe] hydrogenases, designated Eha and Ehb, that are involved in the reduction of low-potential ferredoxins for anabolism (16,26). There are also four cytoplasmic [Ni-Fe] hydrogenases, including two coenzyme F 420 -reducing (Fru and Frc) and two coenzyme F 420 -nonreducing (Vhu and Vhc) hydrogenases. One hydrogenase of each type (Fru and Vhu) contains a selenocysteinyl residue. The other hydrogenases (Frc and Vhc) contain cysteinyl residues at homologous positions (18). The selenocysteine-containing isozymes are abundant during cultivation in medium containing selenium, and the cysteine-containing isozymes (Frc and Vhc) are produced only upon selenium limitation (27). Lastly, the cells contain a cytoplasmic [Fe-S] cluster-free hydrogenase, the H 2 -forming methylenetetrahydromethanopterin (methylene-H 4 MPT) dehydrogenase (Hmd), which in other species has been shown to play an important role at high levels of H 2 or under nickel limitation (1, 2, 29).When formate is the substrate, it is oxidized for the reduction of CO 2 to methane. The key enzyme for formate utilization is formate dehydrogenase, Fdh. The genome of M. maripaludis harbors two sets of genes encoding Fdh, fdhA1B1 and fdhA2B2 (33). Both Fdhs contain selenocysteinyl residues. While fdhA1B1 are found in an appa...

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