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Newly cultured bacteria with broad diversity isolated from eight-week continuous culture enrichments of cow feces on complex polysaccharides.

ABSTRACT: One of the functions of the mammalian large intestinal microbiota is the fermentation of plant cell wall components. In ruminant animals, the majority of their nutrients are obtained via pregastric fermentation; however, up to 20% can be recovered from microbial fermentation in the large intestine. Eight-week continuous culture enrichments of cattle feces with cellulose and xylan-pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented: Firmicutes (51.9%), Bacteroidetes (30.9%), Proteobacteria (11.1%), Actinobacteria (3.5%), Synergistetes (1.5%), and Fusobacteria (1.1%). The majority of bacterial isolates had <98.5% identity to cultured bacteria with sequences in the Ribosomal Database Project and thus represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, B. ovatus, and B. xylanisolvens and members of the Porphyromonadaceae family. Many of the Firmicutes and Bacteroidetes isolates were related to species demonstrated to possess enzymes which ferment plant cell wall components; the others were hypothesized to cross-feed these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 98% of the isolates not represented as previously cultured, there are new opportunities to study the genomic and metabolic capacities of these members of the complex intestinal microbiota.

SUBMITTER: Ziemer CJ

PROVIDER: S-EPMC3911107 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Newly cultured bacteria with broad diversity isolated from eight-week continuous culture enrichments of cow feces on complex polysaccharides.

Ziemer Cherie J CJ

Applied and environmental microbiology 20131108 2

One of the functions of the mammalian large intestinal microbiota is the fermentation of plant cell wall components. In ruminant animals, the majority of their nutrients are obtained via pregastric fermentation; however, up to 20% can be recovered from microbial fermentation in the large intestine. Eight-week continuous culture enrichments of cattle feces with cellulose and xylan-pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were classified phylogene ...[more]

PMID: 24212576

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Project description:Although development of next generation sequencing (NGS) has substantially improved our understanding of the microbial ecology of animal feces, previous studies have mostly focused on freshly excreted feces. There is still limited understanding of the aging process dynamics of fecal microbiomes in intact cowpats exposed to natural environments. Fresh cowpats were sampled at multiple time points for 57 days under field conditions; half the samples were exposed to sunlight (unshaded) while the other half was protected from sunlight (shaded). The 16SRNA hypervariable region 4 was amplified from each sample and sequenced on an Illumina MiSeq Platform. While Clostridia, Bacteroidia, and Sphingobacteria were dominant classes of bacteria in fresh cowpats, Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Bacilli were the dominant classes by the end of the study, indicating a general shift from anaerobic to aerobic bacterial populations. This change was most likely influenced by the shift from cattle gut (anaerobic) to pasture ground (aerobic). Reduced moisture in cowpats may also contribute to the community shift since air can penetrate the dryer cowpat more easily. Twelve genera consisting pathogenic bacteria were detected, with Mycobacterium, Bacillus, and Clostridium being the most abundant; their combined abundance accounts for 90% of the total pathogenic genera. Taxonomic richness and diversity increased throughout the study for most samples, which could be due to bacteria regrowth and colonization of bacteria from the environment. In contrast to the high taxonomic diversity, the changes of PICRUSt inferred function profile were minimal for all cowpats throughout the study, which suggest that core functions predicted by PICRUSt may be too conserved to distinguish differences between aerobe and anaerobe. To the best of our knowledge, this is the first study demonstrating that cowpat exposure to air and sunlight can cause drastic microbiome changes soon after deposition in natural environments. Our findings offer important insights for future research characterizing the microbiome of feces collected in natural environments and the impact of cattle fecal contamination on water resources.

Project description:Resistant starch (RS) enrichments were made using chemostats inoculated with human feces from two individuals at two dilution rates (D = 0.03 h(-1) and D = 0.30 h(-1)) to select for slow- and fast-growing amylolytic communities. The fermentations were studied by analysis of short-chain fatty acids, amylase and alpha-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide probes. Considerable butyrate was produced at D = 0. 30 h(-1), which corresponded with reduced branched-chain fatty acid formation. At both dilution rates, high levels of extracellular amylase activity were produced, while alpha-glucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia became more important at D = 0.30 h(-1). Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominated in RS enrichments at D = 0.30 h(-1). This organism attached apically to RS granules and formed rosette-like structures which, with glycocalyx formation, agglomerated to form biofilm networks in the planktonic phase. Attempts to isolate this bacterium in pure culture were repeatedly unsuccessful, although a single colony was eventually obtained. On the basis of its 16S rDNA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I Clostridium sp. A 16S rRNA-targeted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at D = 0.03 and 0.30 h(-1), respectively. This study indicates that bacterial populations with significant metabolic potential can be overlooked using culture-based methodologies. This may provide a paradigm for explaining the discrepancy between the low numbers of butyrate-producing bacteria that are isolated from fecal samples and the actual production of butyrate.

Project description:BackgroundClostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation.ResultsWe cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant.ConclusionIn this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

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Newly cultured bacteria with broad diversity isolated from eight-week continuous culture enrichments of cow feces on complex polysaccharides.

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Newly cultured bacteria with broad diversity isolated from eight-week continuous culture enrichments of cow feces on complex polysaccharides.

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