Carbohydrate-active enzymes identified by metagenomic analysis of deep-sea sediment bacteria.

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding ß-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three ß-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.

Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

Occurrence of Cryptosporidium in a wastewater treatment plant in North Germany.

Cryptosporidium parvum is one of the most common human parasitic protozoa and is responsible for many waterborne outbreaks in several industrialized countries. The oocyst, which is the infective form, is known to be highly resistant to wastewater treatment procedures and represents a potential hazard to human populations through contaminated raw or treated wastewater. In this investigation, the occurrence of Cryptosporidium in wastewater samples was monitored and removal efficiency was assessed. Treated (effluent) and untreated (influent) wastewater samples were collected seasonally over a period of 2 years. Oocysts were repeatedly detected in influent and effluent samples collected from the treatment plant during all sampling seasons, with a mean concentration of 782 oocysts/L. The seasonal distribution showed that oocysts are predominant during autumn and winter. Molecular analyses via the small (18S) subunit of rRNA amplification and subsequent sequencing with an objective of characterizing the oocysts revealed that Cryptosporidium parvum was the dominant Cryptosporidium parasite present in wastewater.

Complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, polysaccharide-degrading members of the family Flavobacteriaceae.

Two members of the family Flavobacteriaceae were isolated from subseafloor sediments using artificial seawater with cellulose, xylan, and chitin as the sole carbon and energy sources. Here, we present the complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, which both encode putatively novel enzymes involved in cellulose, hemicellulose, and chitin metabolism.

Complete genome sequence of the marine cellulose- and xylan-degrading bacterium Glaciecola sp. strain 4H-3-7+YE-5.

Glaciecola sp. strain 4H-3-7+YE-5 was isolated from subseafloor sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecola sp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases, offering a high potential for plant biomass degradation.