Members of the serpin (serine proteinase inhibitor) superfamily have been identified in higher multicellular eukaryotes (plants and animals) and viruses but not in bacteria, archaea, or fungi. Thus, the ancestral serpin and the origin of the serpin inhibitory mechanism remain obscure. In this study we characterize 12 serpin-like sequences in the genomes of prokaryotic organisms, extending this protein family to all major branches of life. Notably, these organisms live in dramatically different environments and some are evolutionarily distantly related. A sequence-based analysis suggests that all 12 serpins are inhibitory. Despite considerable sequence divergence between the proteins, in four of the 12 sequences the region of the serpin that determines proteinase specificity is highly conserved, indicating that these inhibitors are likely to share a common target. Inhibitory serpins are typically prone to polymerization upon heating; thus, the existence of serpins in the moderate thermophilic bacterium Thermobifida fusca, the thermophilic bacterium Thermoanaerobacter tengcongensis, and the hyperthermophilic archaeon Pyrobaculum aerophilum is of particular interest. Using molecular modeling, we predict the means by which heat stability in the latter protein may be achieved without compromising inhibitory activity.

NO, a free radical gas, is the signal for Nitrosomonas europaea cells to switch between different growth modes. At an NO concentration of more than 30 ppm, biofilm formation by N. europaea was induced. NO concentrations below 5 ppm led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the proteins expressed by N. europaea were identified. Comparison studies of the protein patterns of motile-planktonic and attached (biofilm) cells revealed several clear differences. Eleven proteins were found to be up or down regulated. Concentrations of other compounds such as ammonium, nitrite, and oxygen as well as different temperatures and pH values had no significant effect on the growth mode of and the proteins expressed by N. europaea.

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMSdegrading methanogen M. hollandica in freshwater sediments.The cycling of dimethyl sulfide (DMS) and methanethiol (MT) has been intensively studied due to the impact the oxidation products of these compounds (e.g., methanesulfonic acid and SO 2 ) have on the processes of global warming, acid precipitation, and the global sulfur cycle (1,3,24). Previous research revealed that MT and DMS were the dominant volatile organic sulfur compounds (VOSC) in freshwater sediments and water columns (20). Fluxes of MT and DMS to the atmosphere depend on the steady-state concentrations of these compounds in the sediment and water surface layers. These steady-state concentrations are the result of biological (and chemical) production and degradation. Various studies reported that microbial production and degradation of these VOSC in freshwater, marine, estuarine, and salt lake sediments are relatively well balanced (15-18, 20-23; B. P. Lomans, J.-J. Wesselink, P. Bakkes, A. Pol, C. van der Drift, and H. J. M. Op den Camp, submitted for publication). In anaerobic freshwater sediments, formation of MT and DMS has been demonstrated to occur mainly by methylation of sulfide (7,(20)(21)(22)(23) Lomans et al., submitted) and to a lesser extent by the degradation of sulfur-containing amino acids (14...

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteomewide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N 5 -methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.