Chan Yu

An integrated approach of bioassay and molecular docking to study the dihydroxylation mechanism of pyrene by naphthalene dioxygenase in Rhodococcus sp. ustb-1.

Naphthalene dioxygenase (NDO) is the initial enzyme catalyzing the biodegradation of aromatic compounds, and it plays a key role in microbial remediation of polluting sites. In this study, Rhodococcus sp. ustb-1 derived from crude oil was selected to investigate the biodegradation characters and dihydroxylation mechanism of pyrene by an integrated approach of bioassay and molecular docking. The biodegradation experiment proved that the strain ustb-1 shows high effective biodegradability to pyrene with a 70.8% degradation on the 28th day and the metabolite pyrene cis-4,5-dihydrodiol was found. The results of molecular docking indicated that the regions surrounding pyrene are defined by hydrophobic amino acids which are favorable for the binding of dioxygen molecule at C4 and C5 positions of pyrene in a side-on mode. The binding positions of dioxygen are in agreement with the mass spectral analysis of the metabolite pyrene cis-4,5-dihydrodiol. In summary, this study provides a promising explanation for the possible binding behavior between pyrene and active site of NDO.

Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4

Significance Conventional thought holds that the stable isotopic composition (O and H) of water inside the cells of microorganisms and most aquatic macroorganisms is identical to that of the surrounding water. This assumption is widely applied in studies of environmental reconstruction. Results presented here, based on techniques of multilabeled water isotope probing and measurement of 18O/16O ratios in PO4 moieties of intracellular biomolecules, which allows direct sampling of intracellular environments, demonstrate a significant metabolic water component in intracellular water that is consistent across multiple strains of bacteria when grown under similar conditions. The intracellular water probe presented here, based on PO4 δ18O in DNA/biomass, may be extended to other biomolecules and organisms. Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the 18O/16O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30–40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

Functional gene expression of oil-degrading bacteria resistant to hexadecane toxicity

Contamination with oil poses a threat to the environment and to human health worldwide. Biological methodologies have proved to be economical, versatile and efficient for the remediation of pollutants. In this paper, a highly efficient oil-degrading bacterial strain USTB-2 was isolated from an oil production well of Dagang oil field in Tianjin, China. The 16S rRNA sequence of USTB-2 showed 100% similarity with that of Bacillus subtilis BSn5. Hexadecane is one of the most important components in petroleum. The half inhibitory ratio (IC₅₀) of hexadecane inhibited organisms, determined by microcalorimetry, was lower in USTB-2 than in B. BSn5. The results indicate that the strain USTB-2 degrades hexadecane to make it less toxic compared with the normal strain. RT-PCR was used to evaluate the expression of oil-degrading enzymes, specifically 4-hydroxyphenylacetate 3-monooxygenase genes (HPMO). A sharp increase in the expression of HPMO genes was observed for USTB-2, while the expression of HPMO genes in reference strain B. BSn5 remained relatively stable. These methods can be used to study the metabolic potential of microorganisms for in situ oil decontamination.