A Flexible System for Cultivation of Methanococcus and Other Formate-Utilizing Methanogens.

Many hydrogenotrophic methanogens use either H2 or formate as the major electron donor to reduce CO2 for methane production. The conventional cultivation of these organisms uses H2 and CO2 as the substrate with frequent replenishment of gas during growth. H2 is explosive and requires an expensive gassing system to handle safely. Formate is as an ideal alternative substrate from the standpoints of both economy and safety but leads to large changes in the culture pH during growth. Here, we report that glycylglycine is an inexpensive and nontoxic buffer suitable for growth of Methanococcus maripaludis and Methanothermococcus okinawensis. This cultivation system is suitable for growth on liquid as well as solid medium in serum bottles. Moreover, it allows cultivation of liter scale cultures without expensive fermentation equipment. This formate cultivation system provides an inexpensive and flexible alternative for the growth of formate-utilizing, hydrogenotrophic methanogens.

Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis.

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.

Formate-dependent H2 production by the mesophilic methanogen Methanococcus maripaludis.

Methanococcus maripaludis, an H(2)- and formate-utilizing methanogen, produced H(2) at high rates from formate. The rates and kinetics of H(2) production depended upon the growth conditions, and H(2) availability during growth was a major factor. Specific activities of resting cells grown with formate or H(2) were 0.4 to 1.4 U mg(-1) (dry weight). H(2) production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (K(f)) was 3.6 mM. In contrast, in H(2)-grown cells this process followed sigmoidal kinetics, and the K(f) was 9 mM. A key enzyme for formate-dependent H(2) production was formate dehydrogenase, Fdh. H(2) production and growth were severely reduced in a mutant containing a deletion of the gene encoding the 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 Deltafru-Deltafrc 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 formate-dependent H(2) production demonstrated the potential of M. maripaludis for the microbial production of H(2) from formate.

Proteocatella sphenisci gen. nov., sp. nov., a psychrotolerant, spore-forming anaerobe isolated from penguin guano.

A novel, obligately anaerobic, psychrotolerant bacterium, designated strain PPP2T, was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Chilean Patagonia. Cells were Gram-stain-positive, spore-forming, straight rods (0.7-0.8x3.0-5.0 microm) that were motile by means of peritrichous flagella. Growth was observed at pH 6.7-9.7 (optimum pH 8.3) and 2-37 degrees C (optimum 29 degrees C). Growth was observed between 0 and 4% (w/v) NaCl with optimum growth at 0.5% (w/v). Strain PPP2T was a catalase-negative chemo-organoheterotroph that was capable of fermentative metabolism. Peptone, bacto-tryptone, Casamino acids, oxalate, starch, chitin and yeast extract were utilized as substrates. The major metabolic products were acetate, butyrate and ethanol. Strain PPP2T was resistant to ampicillin, but sensitive to tetracycline, chloramphenicol, rifampicin, kanamycin, vancomycin and gentamicin. The DNA G+C content of strain PPP2T was 39.5 mol%. Phylogenetic analysis revealed that strain PPP2T was related most closely to Clostridium sticklandii SR (approximately 90% 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic characteristics, strain PPP2T is considered to represent a novel species of a new genus, for which the name Proteocatella sphenisci gen. nov., sp. nov. is proposed. The type strain of Proteocatella sphenisci is PPP2T (=ATCC BAA-755T=JCM 12175T=CIP 108034T).

Genomic characterization of methanomicrobiales reveals three classes of methanogens.

BACKGROUND: Methanomicrobiales 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 FINDINGS: In 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/SIGNIFICANCE: Our 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).

Properties of the reversible nonoxidative vanillate/4-hydroxybenzoate decarboxylase from Bacillus subtilis.

Bacillus subtilis (ATCC 6051) reversibly decarboxylates vanillate and 4-hydroxybenzoate under both aerobic and anoxic conditions. Thus, we have identified on the basis of gene sequence homology with Sedimentibacter hydroxybenzoicus and Streptomyces sp. strain D7, a putative B. subtilis hydroxybenzoate decarboxylase. The native form of this enzyme is encoded by 3 genes yclBCD (GI Sequence Identification Nos.: 2632649, 2632650, 2632651) that we have renamed during this research as bsdBCD to align with existing nomenclature. The bsdD gene is reported in the database to be 690 bp; however, our sequence analysis revealed that the size of this gene is in fact 228 bp, an observation that results in a shortening of YclD (i.e., BsdD) from 229 to 75 aa. The corresponding bsdBCD genes were cloned into Escherichia coli, and the heterologously expressed enzyme was assayed for activity. The decarboxylase exhibited a narrow substrate range, with only 2 of the tested substrates, vanillate (Kmapp = 4 mmol.L-1) and 4-hydroxybenzoate (Kmapp = ~1 mmol.L-1), being decarboxylated. The recombinant enzyme had properties similar to that of the native enzyme in respect to specific activity, kinetic properties, bidirectional decarboxylase-carboxylase activity, oxygen insensitivity, and substrate specificity.

Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases.

Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous ( approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.

Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases.

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.