Insights into environmental bioremediation by microorganisms through functional genomics and proteomics

Environmental pollutants in the soil are a major concern worldwide. Bioremediation mediated by microorganisms is a highly promising technology that is environmentally friendly, safe, and effective. However, incomplete biological information regarding the cellular responses in many microbial communities restricts progress in the site-specific mineralization process. The application of proteomics in environmental bioremediation research provides a global view of the protein compositions of the microbial cells and offers a promising approach to address the molecular mechanisms of bioremediation. With the combination of proteomics, functional genomics provide an insight into global metabolic and regulatory networks that can enhance the understanding of gene functions. This article deals with the applications of functional genomics and proteomics to dissect the cellular responses to environmental stimuli, such as stress response, induction and expressions of regulatory proteins/enzymes in response to aromatic hydrocarbons and heavy metals. An understanding of the growth conditions governing the expression of the proteome (for example, enzymes and regulatory proteins of aromatic hydrocarbon degradation, energy generation pathways, transport and stress-related proteins) in a specific environment is essential for developing rational strategies for successful bioremediation.     

Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain

Comamonas testosteroni P15 and its mutant strain E23 can tolerate and utilize phenol as the sole source of carbon and energy at up to 15 mM and 20 mM, respectively. Compared to the wild type P15, mutant E23 showed higher values of K _s and K _i but a lower μ_max value, and had lower phenol hydroxylase and catechol 2,3-dioxygenase activities. Without phenol exposure, mutant E23 demonstrated a two-fold greater amount of cardiolipin than the wild type P15. Upon exposure to phenol, an increase in cardiolipin at the expense of phosphatidylethanolamine was observed in the wild type P15. However, there was no significant difference in major phospholipid contents between mutant E23 cells grown in the presence or absence of phenol. It was noted that the ratio of trans/cis fatty acids of phosphatidylethanolamine and cardiolipin in mutant E23 was 65–70% higher than that in the wild type P15. In the absence of phenol, the degree of saturation of cardiolipin in mutant E23 was 33% higher than that in wild type P15. In contrast to earlier findings, an increase in C16:1 9trans with a simultaneous decrease in C18:1 11cis instead of C16:1 9cis was observed in specific classes of phospholipids. Received: 30 July 1998 / Received last revision: 16 November 1998 / Accepted: 12 December 1998     

Upregulation of the Transient Receptor Potential Ankyrin 1 Ion Channel in the Inflamed Human and Mouse Colon and Its Protective Roles

Transient Receptor Potential Ankyrin 1 (TRPA1) channels are localized on sensory nerves and several non-neural cells, but data on their functional significance are contradictory. We analysed the presence and alterations of TRPA1 in comparison with TRP Vanilloid 1 (TRPV1) at mRNA and protein levels in human and mouse intact and inflamed colons. The role of TRPA1 in a colitis model was investigated using gene-deficient mice. TRPA1 and TRPV1 expressions were investigated in human colon biopsies of healthy subjects and patients with inflammatory bowel diseases (IBD: ulcerative colitis, Crohn''s disease) with quantitative PCR and immunohistochemistry. Mouse colitis was induced by oral 2% dextran-sulphate (DSS) for 10 days. For investigating the functions of TRPA1, Disease Activity Index (weight loss, stool consistency, blood content) was determined in C57BL/6-based Trpa1-deficient (knockout: KO) and wildtype (WT) mice. Sensory neuropeptides, their receptors, and inflammatory cytokines/chemokines were determined with qPCR or Luminex. In human and mouse colons TRPA1 and TRPV1 are located on epithelial cells, macrophages, enteric ganglia. Significant upregulation of TRPA1 mRNA was detected in inflamed samples. In Trpa1 KO mice, Disease Activity Index was significantly higher compared to WTs. It could be explained by the greater levels of substance P, neurokinins A and B, neurokinin 1 receptor, pituitary adenylate-cyclase activating polypeptide, vasoactive intestinal polypeptide, and also interleukin-1beta, macrophage chemoattractant protein-1, monokine induced by gamma interferon-1, tumor necrosis factor-alpha and B-lymphocyte chemoattractant in the distal colon. TRPA1 is upregulated in colitis and its activation exerts protective roles by decreasing the expressions of several proinflammatory neuropeptides, cytokines and chemokines.     

香菇的学名及其在当代真菌分类中的位置

本文综述了香菇(Lentinula edodes)的分类历史,确认其在蘑菇目(Agaricales)Tricholomataceae科下的分类地位,并证实了它与多孔菌目(Poriales)Lentinaceae科的Lentinus属没有联系。根据《真菌、地衣汉语学名命名法规》,作者讨论了译为“香菇属”的Lentinus和“小香菇属”的Lentinellus两属的汉语学名问题,提出Lentinus的汉语学名应订正为“韧伞属”,Lentinellus为“螺壳菌属”。香菇所在的Lentinula属的汉语学名建议为“木菇属”。     

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens)

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment- length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist.      

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.     

Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities

xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k_cat/K_m) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.     

Characterization of the hemodynamic wall shear stresses in human umbilical vessels from normal and intrauterine growth restricted pregnancies

Significant reductions in blood flow and umbilical diameters were reported in pregnancies affected by intrauterine growth restriction (IUGR) from placental insufficiency. However, it is not known if IUGR umbilical blood vessels experience different hemodynamic wall shear stresses (WSS) compared to normal umbilical vessels. As WSS is known to influence vasoactivity and vascular growth and remodeling, which can regulate flow rates, it is important to study this parameter. In this study, we aim to characterize umbilical vascular WSS environment in normal and IUGR pregnancies, and evaluate correlation between WSS and vascular diameter, and gestational age. Twenty-two normal and 21 IUGR pregnancies were assessed via ultrasound between the 27th and 39th gestational week. IUGR was defined as estimated fetal weight and/or abdominal circumference below the 10th centile, with no improvement during the remainder of the pregnancy. Vascular diameter was determined by 3D ultrasound scans and image segmentation. Umbilical artery (UA) WSS was computed via computational flow simulations, while umbilical vein (UV) WSS was computed via the Poiseuille equation. Univariate multiple regression analysis was used to test for the differences between normal and IUGR cohort. UV volumetric flow rate, UA and UV diameters were significantly lower in IUGR fetuses, but flow velocities and WSS trends in UA and UV were very similar between normal and IUGR groups. In both groups, UV WSS showed a significant negative correlation with diameter, but UA WSS had no correlation with diameter, suggesting a constancy of WSS environment and the existence of WSS homeostasis in UA, but not in UV. Despite having reduced flow rate and vascular sizes, IUGR UAs had hemodynamic mechanical stress environments and trends that were similar to those in normal pregnancies. This suggested that endothelial dysfunction or abnormal mechanosensing was unlikely to be the cause of small vessels in IUGR umbilical cords.