A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Characterization and antifungal activity of chitosanase produced by Pedobacter sp. PR-M6.

To investigate the temperature requirements of chitosanase activity, as well as the degradation patterns generated by enzyme-induced chitosan oligomer hydrolysis, Pedobacter sp. PR-M6 was inoculated onto 0.5% colloidal chitosan medium agar plates. Cell growth was higher at 30 °C than at 20 °C during the initial 2 days of incubation. The protein content rapidly increased on day 1 at both temperatures and then it slowly increased at 20 °C and slowly decreased at 30 °C during the following 5 days of incubation. In order to characterize the electrophoretic pattern, Pedobacter sp. PR-M6 was cultured in 1% powder chitosan medium at 20 °C and 30 °C for 5 days after incubation and analyzed by SDS-PAGE. Four bands were visible, corresponding to ct1 (25 kDa), ct2 (17 kDa), ct3 (15 kDa), and ct4 (14 kDa), at both 20 °C and 30 °C. The optimal conditions for the activity of chitosanase produced from Pedobacter sp. PR-M6 were 60 °C and 1.81 enzyme units/mg protein. Two major isozyme bands (ct3 and ct4) exhibited their strongest chitosanase activity at 50 °C in SDS-PAGE gel. The reaction products generated from (GlcN)2-(GlcN)5 substrates at 60 °C after a 1 h incubation were investigated by thin-layer chromatography. Low-molecular weight chitosan and oligochitosan (LCOC) and soluble chitosan showed antifungal activity against A. brassicicola, B. cinerea, F. solani, and R. solani. LCOC exhibited higher antifungal activity than soluble chitosan. Moreover, LCOC treatments (500 ppm and 1000 ppm) inhibited conidia germination in A. brassicicola.

Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.

Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro.

In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 °C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures.

Development of a semidefined growth medium for Pedobacter cryoconitis BG5 using statistical experimental design.

Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K2HPO4 along with three additional nutrients (yeast extract, MgSO4, and NH4Cl) were identified to form the basal medium which was further analyzed by Plackett-Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH4Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600 nm) cell density of 7.5 using 23.7 g/L of tryptone, 8.8 g/L of yeast extract, and 0.7 g/L of NH4Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.

Single-cell genomics reveals metabolic strategies for microbial growth and survival in an oligotrophic aquifer.

Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site (a largely decommissioned nuclear production complex) in eastern Washington state, USA, and have been shown to change significantly in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single-cell genomics techniques to shed light on the physiological niche of these micro-organisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide range of both intra- and extracellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors, which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic micro-organisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area (in the south-eastern corner of the site) subsurface and biogeochemical shifts associated with Columbia River water intrusion.