The human gut is colonized by a complex microbiota with multiple benefits. Although the surfaceattached, mucosal microbiota has a unique composition and potential to influence human health, it remains difficult to study in vivo. Therefore, we performed an in-depth microbial characterization (human intestinal tract chip (HITChip)) of a recently developed dynamic in vitro gut model, which simulates both luminal and mucosal gut microbes (mucosal-simulator of human intestinal microbial ecosystem (M-SHIME)). Inter-individual differences among human subjects were confirmed and microbial patterns unique for each individual were preserved in vitro. Furthermore, in correspondence with in vivo studies, Bacteroidetes and Proteobacteria were enriched in the luminal content while Firmicutes rather colonized the mucin layer, with Clostridium cluster XIVa accounting for almost 60% of the mucin-adhered microbiota. Of the many acetate and/or lactate-converting butyrate producers within this cluster, Roseburia intestinalis and Eubacterium rectale most specifically colonized mucins. These 16S rRNA gene-based results were confirmed at a functional level as butyryl-CoA:acetate-CoA transferase gene sequences belonged to different species in the luminal as opposed to the mucin-adhered microbiota, with Roseburia species governing the mucosal butyrate production. Correspondingly, the simulated mucosal environment induced a shift from acetate towards butyrate. As not only inter-individual differences were preserved but also because compared with conventional models, washout of relevant mucin-adhered microbes was avoided, simulating the mucosal gut microbiota represents a breakthrough in modeling and mechanistically studying the human intestinal microbiome in health and disease. Finally, as mucosal butyrate producers produce butyrate close to the epithelium, they may enhance butyrate bioavailability, which could be useful in treating diseases, such as inflammatory bowel disease.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.

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A syntrophic propionate-oxidizing bacterium, strain MPOBT, was isolated from a culture enriched from anaerobic granular sludge. It oxidized propionate syntrophically in co-culture with the hydrogen-and formate-utilizing Methanospirillurn hungateii, and was able to oxidize propionate and other organic compounds in pure culture with sulfate or fumarate as the electron acceptor. Additionally, it fermented f umarate. 16s rRNA sequence analysis revealed a relationship with Syn trophobacter wolinii and Syn trophobacter pfennigii. The G+C content of i t s DNA was 60.6 mol%, which is in the same range as that of other Syntrophobacter species. DNA-DNA hybridization studies showed less than 26% hybridization among the different genomes of Syntrophobacter species and strain MPOBT. This justifies the assignment of strain MPOBT to the genus Syntrophobacter as a new species. The name Syntrophobacter fumaroxidans is proposed; strain MPOBT (= DSM 10017T) is the type strain. INTRODUCTIONFor a long time, Syntrophobacter niolinii was the only described bacterium which could oxidize propionate syntrophically in co-culture with the hydrogen-consuming Desulfovibrio G11 (1). Several methanogenic syntrophic co-cultures were enriched, but obtaining defined co-cultures remained difficult. S. wolinii was only recently obtained in pure culture, and found to be able to grow on pyruvate or on propionate and sulfate (1 7). Two related syntrophic propionate-oxidizing bacteria, Sjwtrophobacter pfennigii, previously known as KOPROPl, and strain HP1.l were isolated with propionate and sulfate (1 8,19). A mesophilic bacterium (strain MPOB') enriched by us on propionate was able to ferment fumarate to succinate and carbon dioxide without a syntrophic partner (12). This strain could oxidize propionate by using fumarate or sulfate as electron acceptors (12,15 pionate by the use of HS-CoA transferase (1 1). 16s rRNA sequence analysis of S. wolinii, S. pfennigii, strain HP1.l and strain MPOBT revealed that these syntrophic bacteria are closely related and belong to the delta subclass of Proteobacteria (3,4,19). Remarkably, it was observed that another bacterium was related to this group : Desulforhabdus amnigenus, a sulfate-reducing bacterium which is not able to grow syntrophically on propionate (10).Recently, we obtained a pure culture of strain MPOBT. Its morphological and physiological characterization are presented here, and its taxonomic position within the genus Syntrophobacter is discussed. University, The Netherlands). A previously described bicarbonate-buffered medium was used for isolation and cultivation (1 2). For isolation of strain MPOB' the roll-tube-dilution method ( 5 ) and direct dilution series in liquid media with fumarate as carbon and energy sources were used. Purity was checked by growth in Wilkins-Chalgren anaerobe broth (Oxoid), and in media containing 1 % yeast extract and 20 mM glucose, and by microscopy. METHODSPhylogeny and DNA analysis. Phylogenetic analysis of the strain has been described previously (4, 10). The nucleo...

An aerobic Lactobacillus plantarum culture displayed growth stagnation during early growth. Transcriptome analysis revealed that resumption of growth after stagnation correlated with activation of CO 2 -producing pathways, suggesting that a limiting CO 2 concentration induced the stagnation. Analogously, increasing the CO 2 gas partial pressure during aerobic fermentation prevented the temporal growth stagnation.Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium used worldwide in production of fermented food and feed products, and its natural habitats are anaerobic or microaerobic. Nevertheless, the responses of L. plantarum to aerobic growth conditions and the corresponding oxidative stresses are relevant for a variety of industrial processing conditions (10). Moreover, a potential ability to respire and increase the biomass yield during aerobic growth has been demonstrated for several lactic acid bacteria, including Lactococcus lactis (2, 5). L. plantarum is able to utilize oxygen under certain circumstances, including glucose limitation conditions (6), and the absence of the ubiquitous defensive reaction catalyzed by superoxide dismutase in L. plantarum was found to be compensated for by the capacity of this bacterium to accumulate very high concentrations of intracellular Mn(II) ions (up to 35 mM), which act as a scavenger system for superoxide (1,8).In this work an aerobically grown culture showed consistent temporary growth stagnation during the early logarithmic phase. Transcriptome analyses revealed genes that were differentially expressed before and after the growth stagnation, and comprehensive analysis of these differentially expressed genes revealed that resumption of growth after the observed growth stagnation corresponded with the activation of a range of CO 2 -producing metabolic pathways, suggesting that the growth stagnation was due to CO 2 limitation. Correspondingly, it could be shown that modifying the gas supplementation regimen by increasing the CO 2 partial pressure relieved growth stagnation, confirming the CO 2 limitation hypothesis.Growth of L. plantarum under aerobic conditions. L. plantarum strain WCFS1 (9) was grown aerobically and anaerobically at 37°C in MRS medium (3), and growth was monitored for 12 h (Fig. 1). L. plantarum displayed two phases of logarithmic growth (Fig. 1), which is in agreement with previous observations (4). The final cell density was higher in the aerobic culture (optical density at 600 nm [OD 600 ], 5.5) than in the anaerobic culture (OD 600 , 4.5). Interestingly, temporary growth stagnation occurred during aerobic fermentation after approximately 2 h, a feature not observed in the anaerobic culture (Fig. 1). Following this stagnation, growth resumed, and the maximum growth rate was comparable to that observed in anaerobic cultures (Fig. 1).The high growth rates and rapid acidification of the medium in the early growth phase, apparently irrespective of the presence of oxygen, confirmed previously described results (6, 7). More...